Hung-h?-
..unufir-H-

pvt... .tpn. nil)

"-

f’l‘tt

 

.u

. If!»

...!- .
. 2.3!)..-
. .. . . . “Hi-x.
... ..I.---.:. .. . . .. .... .. u .
2.... s13}: . . -

.-_. “'
gr

. :4 12..££§.r:.ourwa
- I ... ... art-E. iii-Mr.“
. .l I.‘ 6 I n .
- 01») it“. 'I.ol’t|o lfr‘ 'IIIO’
(ti-43.. . \- ... .\:
. It I.

21-" '"

1!;

h. .
€53

g

\vh¢\ . .
..I‘tr‘ ‘13....-
_I\..:. .i-rln.‘ . .
:ihs. ...I\
.ss. ;.

I2}.

I

I

I

1:
1‘
4?- ‘
35?.

.
I:

,r

\

'.
g.
‘l
a

u}. '

 

. ‘g
a":
\“
..,. .
l ‘5‘
.h‘ sz.‘

2}: -j
‘o‘l'd'r'
t

c I ....l. ...-...... .. 4...1.-..-t..a 1...: 3.5.5. v . .
.-.f. -. .---l. I .1.-.
..‘IV 1 0‘7!‘ '- ..l :‘Qll it... C. .. ‘73.. .III?‘ ’I’txi.hl
. \ L - .. . ...-$3 .- .. 1 «r...

's'wlw‘

I
§

3:: u

')
L.
«r.»-
6’

3i.

. s" -'
- Hg.
I"

..rpllu..!.t; .5? . ..r.~wra-.OD\ 9...: .l.
. M A 5!. (fiiégv-‘a ' ..
tl ff). ft}...- .( r)...
C .(..'r.ttsl. . L. .

‘1
;:\:
Jr

.';
.3 *-

 

.OJI
11.1%.; .H ....LIE.....2-$-.M£- .u-
. . :l6 LII-a...
y at)“. t.~bfl4.ll;.§flv-X .1.“
El. .Al-\£ 4 '|.. J‘

:
....
V
38‘.» x
w, I

2-

5*

.d
J
lull

. u {4.
g «I \ ... if: -

|. - n
o)’:..).rl%.\..l f5.-

" A
g?»
F
.
J

 

I .. .
.. a.» n i b .. . ..
A151! Ill..kl)rx;. . .7 t!
. 1v- ..1::.?. ft! .7 nit-l toklfvazn»!
.-|‘|.’v‘.tboal> . ( 1’ Dbl...‘ I! -H‘” i_’l..b ...)...HJ;
v11. {.75. . v I ..l; (I! 9 .
59.1 '1: .. . r... ii.- rztlg ’-
blo vt .‘I- .. it‘fF

.'
1.

£-
"

. .ai-‘\
3’2?sz
' \R-IL.

 

 

«(IE3 I

it- (in? . .
\\t.»LJ..'av-££- -c
. ; v t ... A): ..

I“
‘E-iv

.... '07“ no“ I l.
.ttl ...
. "Ft-lu-fiuutn
. n-. [...va his“...
t. - cc. )1. .

2.-

.
ll.

ll"

‘ IQVV‘V.
.. .. .64.. .
.uk »- b. . .
«...-....Vtkauur.‘ la

 

\
..xbléu

.-.. a 0.:
{...-..Lm-rn:
. 2-1,. ...)-
..vl--.-. I2! 2.
... . .

- .

 

.H.
... r

  

\J‘n

,

.01 it... . {I

it. .iuo)|"450|frl
OIOIpll-{ob

. . .. I}; DI

.\ n N
.unr..1....n .
...:gs}

. . . I): {\a I:

. . - . .....ll:l]\ .3!- av\ fulfil}? I

. . , I t. I145!) . . . . . tut-L.) iiilllll“. ‘f'vn

.. f ..- fit... ..\..-..-.:?. . . .lI-l. .2 . .

.4... ..C. \V «Ii! .llf‘o-.rlért.tuv ‘1. . . . . . . 11%..
.. 11.31. i}. ..n1. 51v:l..p no. . . .

{til-......- ‘rl. f»‘.:u (Q431-A v‘ «I t . .

 

 

  

. _ . . . nllrv‘iu‘lzvi.
«:5 c. .L. F\ ‘31)»! .. vyia. . ... , - N .. . . . ... . II-Illlnl IIOIII‘IIIIII.
.32)....- P‘Sw. .. vhf-7 Fun-u... ...é . . . ,. .. . ..
"-'.Q\ (’3, 17",3ol if C fis‘ . . . . n
. . ; . .. . .N . .. ...-{555.53}
l . . . . . c ,, . . . . . :l. i... ...-... ...
...l ‘.h...-.fi..f....a .. . . . . : . ... . . . HQFIullkuah.
. - .1 .11... L;..\i'n.u.-.. . - . . . . .. . ... .. . . . .. .. . . . if: v \‘n l 11.!»
. . . . . . ... . .
.. n.1,. .II .21
I. - . v . . I I: . . - .5
, , . c - .
Ruuufidhhwrv no. EAR-vi 1 run-... N!...§s_+.§-S.WK 1
. .. 1. . s....n.-L.o.-..«H-Rvub. 1.. 1 E. . - - .

  

 

.I'

”ll!

 

 

 

 

 

 

\ll‘llllll-l“. uMTllWilli CA 3 01 e 0 c, -, 4

1293 00606

 

 

 

 

LIBRARY
Michigan State

University

 

 

 

This is to certify that the

dissertation entitled

NATURE AND NUTRITIONAL SIGNIFICANCE
OF ACIDOGENIC AND METHANOGENIC BACTERIA

IN CUTS 0F TERMITES AND COCKROACHES
presented by

MATTHEW DAVID KANE

has been accepted towards fulfillment
of the requirements for

 

Ph . D. degree in MICROBIOLOGY AND
PUBLIC HEALTH

 

W
fl Ma joflr

FEBRUARY 8 , 1 990
Date

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN BOX to remove We checkout from your record.
TO AVOID FINES return on or before due due.

 

DATE DUE DATE DUE DATE DUE

JWI Mggwr

__J
TIL-fl
l

_________;_J
—_ll———_|

MSU Is An Alfirmelive Adlai/Equal Opportunity Inflation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NATURE AND NUTRITIONAL SIGNIFICANCE
OF ACIDOGENIC AND HETHANOGENIC BACTERIA

IN CUTS 0F TERHITES AND COCKROACHES

3?

Matthew David Kane

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health
1990

activj
anaerc
termit
acetat
methant

Which (

bacteri
“P to

could b(
acetoger
emitted

rates 0f

CultUre
phySiolog
iSolates -

ABSTRACT

NATURE AND NUTRITIONAL SIGNIFICANCE
OF ACIDOGENIC AND NETHANOGENIC BACTERIA
IN CUTS OF TERHITES AND COCKROACHES

By

Matthew David Kane

Research described herein investigated the occurrence,
activities and specific identities of members of two groups of strictly

anaerobic, hydrogenotrophic bacteria which compete for H in the guts of

2

termites: Hz/CO2 acetogenic bacteria, which use H2 to reduce 002 to

acetate (3 major source of carbon and energy for termites); and Hz/CO2

methanogenic bacteria, which use H2 to reduce 602 to methane (a compound

which cannot be utilized by termites).

Hz/CO2 acetogenic bacteria outcompeted H2/CO methanogenic

2
bacteria for H2 in the hindguts of wood- and grass-feeding termites, and
up to one-third of the respiratory requirement of some termite species
could be met by oxidation of acetate derived from bacterial Hz/CO2
acetogenesis. By contrast, fungus-cultivating, or soil-feeding termites
emitted significantly more methane, and exhibited significantly lower

rates of H2/C0 acetogenesis than their wood-feeding counterparts.

2

Two novel H2/602 acetogenic bacteria were isolated in pure
culture from termite guts, and the general morphological and
physiological characteristics, nutrition, and molecular phylogeny of the
isolates were studied in detail. Results have led to the description of
Acetonema elongata, gen. nov., sp. nov., a Gram negative, endospore-

forming rod isolated from gut contents of the phylogenetically lower,

wood-feeding termite, Pterotermes occidentis (Kalotermitidae), and

 

 

6105
1501;

Cubil

ameri
speci
For e
cockr-
fiber
bactex
diet 1
guts o
functi

may ma}

Clostridium mayombei, sp. nov., a Gram positive, endospore forming rod
isolated from gut contents of the higher, soil-feeding termite,
Cubitermes speciosus (Termitidae).

Additional studies with the omnivorous cockroach Periplaneta
americana (Blattidae) demonstrated that numbers and activities of
specific gut bacteria depended upon the insect's diet and life stage.
For example, immature cockroaches emitted more methane than adult
cockroaches, particularly when insects were fed diets high in plant
fiber. By contrast, lactic and acetic acid production by lactic acid
bacteria in the foregut was a significant process in cockroaches fed a
diet low in plant fiber. Results of these studies demonstrated that, in
guts of termites and cockroaches, microbial community structure and
function is related to the developmental stage and diet of the host, and

may make a significant contribution to the insect's nutrition.

Copyright by
MATTHEW DAVID KANE

1990

With love -
For my parents and my grandmothers,

and in memory of my grandfathers.

 

 

 

 

suppr

for a

Steir
under
commi
your

envir

and i

Stahl

Uane]

Dr. ;
both c

and Di

made

well 1
living
"Onderj
and abc

.shpilk

ACKNOWLEDGEMENTS

My deepest gratitude to my family and friends for your love,
support and encouragement. And, of course, heartfelt thanks to my dogs
for always being there when I needed them.

I owe much to Dave Odelson, Jody Switzer, Patty Murray
Steinberger and Sheridan Kidd Haack for your ideas, patience and
understanding. I also am indepted to the members of my guidance
committee, Drs. Mike Klug, Mike Martin, Jim Tiedje and Bob Uffen for
your many helpful suggestions, and for creating an outstanding
environment in which to learn biology and make discoveries (both here,
and in Ann Arbor).

I was very fortunate to do collaborative work with Dr. David
Stahl of the University of Illinois, and Dr. David White of the
University of Tenessee. Thank you for your help now, and in the future.

A special note of thanks to my friend, colleague and sidekick,
Dr. Alain Brauman, and to my friend and colleague, Dr. Marc Labat (and
both of your families) for everything in Marseille, Paris, Brazzaville
and Dimonika. I hope there will be a sequel!

My associations with all of the scientists listed above were
made possible only because I worked in the laboratory of a universally
well liked and greatly admired microbiologist, Dr. John Breznak. He is
living proof that a great scholar, researcher and teacher can also be a
wonderful human being. For all that you have taught me about science
and about life, and for accepting with grace and good humor all of those

”shpilkes" I gave you, I thank you, John.

vi

TABLE OF CONTENTS

List of Tables ........................................... viii
List of Figures ........................................... x
Introduction .............................................. 1

Chapter 1 Relationship between termite diet

and acetogenesis and methanogenesis

by gut bacteria ................................ 30
Chapter 2 H2/602 acetogenic bacteria from termite guts:

I. Acetonema elongata, gen. nov., sp. nov.,

a gram negative endospore-forming bacterium

from Pterotermes occidentis .................... 44
acetogenic bacteria from termite guts:

2
II. Clostridium mayombei, sp. nov.,

Chapter 3 Hz/CO

from guts of the African soil-feeding termite,

Cubitermes speciosus ........................... 77
Chapter 4 Effect of host diet on the production

of organic acids and methane

by cockroach gut bacteria ...................... 99
Appendix Analysis of 168 rRNA sequences of HZ/CO2

acetogenic bacteria isolated from

termite guts ................................... 127

vii

LIST OF TABLES

Introduction

Table 1

Chapter 1

Table 1

Chapter 2
Table 1

Table 2

Tabel 3

Table 4

Chapter 3

Table 1

Table 2

List of H2/002 acetogenic bacteria

described previous to this study,

and some of their properties .................

Reduction of 14C02 to 14C-acetate

by termite gut contents,

and CH emission by live termites ............

h

Substrates used for growth by strain APO-l

Fermentation products formed during growth

of strain APO-l with various substrates ......

Evolutionary distance between strain APO-1

and other selected eubacteria ................

Characteristics useful

for distinguishing strain APO-l

from members of the genus Sporomusa ..........

Substrates used for growth by strain SFC-S
Molar growth yields and fermentation balances

for strain SFC-S grown with

various substrates ...........................

viii

.17

.36

..63

.65

.66

.69

..89

.90

 

 

 

 

LIST

LIST OF TABLES (continued)
Chapter 3
Table 3 Substrates useful for distinguishing
strain SFC-S from other mesophilic,
homoacetogenic clostridia ..................... 94
Table 4 Characteristics useful for distinguishing
112/602 acetogenic bacteria isolated
from termite guts ............................. 95
Chapter 4

Table 1 Protein and fiber content

of P. americana diets ......................... 104
Table 2 Effect of diet on Hz-dependent fixation

of 14C02 into organic acids by adult

P. americana whole-gut homogenates ............ 109

Table 3 Acetate and lactate content of gut fluid
of adult P. americana ......................... 113
Table 4 Effect of diet on populations of
lactic acid bacteria in adult
P. americana foreguts ......................... 120
Appendix
Table 1 Nearly complete sequences of 168 rRNA from
HZ/CO2 acetogenic bacteria isolated

from termite guts .............................. 127

ix

LIST OF FIGURES

Introduction

Figure 1 The Odelson-Breznak model for symbiotic

Chapter 2
Figure

Figure
Figure
Figure
Chapter 3
Figure

Figure

Chapter 4

Figure

degradation of wood polysaccharides
(principally cellulose) by anaerobic protozoa
and bacteria in the hindgut region

of the alimentary tract of R. flavipes ........ 8

Morphology of strain APO-l .................... 53
Cell length and endospore formation

during growth of strain APO-1 in AC-K3 medium

with H2 + 602 as substrate .................... 56
Utilization of H2 by strain APO-l
for growth and acetogenesis ................... 58

Effect of teperature on Hz/COZ-grown (A)

and glucose grown (B) cells of strain APO-1 ...61

Morphology of strain SFC-S .................... 84
Utilization of H2 by strain SFC-S

for growth acetogenesis ....................... 87

Effect of diet on methane emission

from P. americana ............................. lll

 

 

 

 

LIST C

LIST OF FIGURES (continued)

Chapter 4

Figure 2

Figure 3

Appendix

Figure 1

Scanning electron micrographs

of the luminal surface of the foregut

of P. americana (A) and of bacteria attached

to the surface of food particles

in the foregut (3.0); (D) is a

higher magnification of the

enclosed area in (B) ........................... 115
Effect of diet on acetate and lactate

production by foregut homogenates of

adult P. americana ............................ 118

168 rRNA phylogenetic tree of Hz/CO2
acetogenic bacteria from termite guts with
various other eubacteria presented in bush (a)

and bar (b) formats ........................... 132

xi

 

 

 

 

indiV
most

are D

insec

them

accoz
nutr:
adap1
and

nutrj

whic}

INTRODUCTION

If biological success is measured either by numbers of
individuals, or diversity of species, then insects are undoubtedly the
most successful group of animals in the history of life on earth. There
are more species of insects than all other animal species combined (48).
This observation is especially remarkable in light of the fact that
insects do not colonize marine habitats, a limitation which restricts
them to approximately 30 percent of the planet's surface area.

The extreme phylogenetic radiation exhibited by insects has been
accompanied by extensive diversification in dietary habit and
nutritional physiology. Several intriguing behavioral and physiological
adaptions have been employed by insects to assist them in this dietary
and nutritional diversification, including the establishment of
nutritionally based symbiotic relationships with microorganisms, some of
which are described below.

Insects have supplemented the nutritive potential of their food
by establishing mutualistic relationships with fungi (58,80), protozoa
(36,44,98), and/or bacteria (14,17,29). Some of the insects for which
nutritional relationships with microbes have been examined include: ants
(58); beetles (5); cockroaches (29); cranefly larvae (49,83); crickets
(49,87); millipedes (85); termites (l4,lS,l7,96 ) and wood-boring wasps
(52). Many other insect-microbe nutritional interactions probably
remain to be discovered, and even those that have been identified need
to be examined more closely to have a better understanding of the nature

1

 

 

 

 

and d
synop
the p:
nutri1
insecl

cockrc

termit
of ter
of int
first
0f mi
and ‘

bacte]

2
and diversity of this fascinating biological phenomenon. A complete
synopsis of insect-microbe nutritional symbioses is beyond the scope of
the present review, so the following passages deal only with those
nutritional relationships that microorganisms have established with the
insect groups used for this study, specifically, termites and

cockroaches.

I. Termites and Cockroaches, and Their Nutritional Interactions
with Microorganisms

Nutritionally based insect-microbe interactions involving
termites (Isoptera), and cockroaches [Dictyoptera (ancestral relatives
of termites)], with fungi, protozoa and bacteria, have been the subject
of intensive study (summarized in references 14,15,17,29 and 58). The
first section of this review will focus on the nutritional relationships
of microorganisms with termites, and to a lesser extent, cockroaches,
and the second section will discuss in detail certain competing
bacterial processes in the guts of these two insect groups.

There are over 2200 living species of termites, one or more of
which can be found in various habitats over two-thirds of the earth's
land surface (96). They cause considerable damage to agriculture and
architecture, but as decomposers of large amounts of biomass termites
are also important to the recycling of nutrients, particularly in
tropical regions (34,97). Their food consists of living, dead,
decomposing, or highly decomposed plant matter, i.e., substances that
can generally be described as lignocellulosic in composition (95).
Termites are social insects, and in the nest of each species,

morphologically differentiated castes perform different functions. Food

 

 

 

 

 

 

res
the
(95
liv:
thrc

(95.

(tre

stud

basi¢
plant

but I

soil
leaSt

(96)

five

Hodo.
adVar
t°get
temai
thIC
w00d.
and/c

rePre

3
resources are collected by termites belonging to a "worker" caste, which
then feed themselves as well as other members of the termite nest
(95,96). Although a few termites are polyphagous, and eat a variety of
living or dead plant materials, termite biologists recognize at least
three categories of specialization with regard to feeding behavior
(95,96):

1) Termites that eat living, dead or decomposing vegetation
(trees, grass and/or leaves). Wood-feeding species are the most well
studied termites of this group due to their economic impact (34).

2) Termites that cultivate and consume aerobic, cellulolytic
basidomycete fungi of the genus Termitomyces, which, in addition to
plant materials, are consumed by the termite. This habit is limited to,
but pervasive among, members of the subfamily, Macrotermitinae.

3) Termites that eat soil and presumably derive nutrition from
soil organic matter (humus). Although their nutrition is understood the
least, they constitute approximately 45 percent of all termite species
(96) and are especially numerous and active in tropical habitats.

The currently accepted classification of termites recognizes
five families of "lower”, or more primitive, termites (Masto-, Kalo-,
Hodo-, Rhino- and Serritermitidae) and one family of ”higher", or more
advanced, termites (Termitidae) (50,51). The lower termite families
together constitute about 25 percent of all living species, the
remainder belong to the Termitidae. There is some correlation between
phylogenetic classification and diet, in that the lower termites are all
wood-feeders, whereas the higher termites include plant- (wood-, grass-
and/or leaf-) feeding, fungus-cultivating, and soil-feeding

representatives (95).

 

 

 

 

beer
inte
such
term
is
regi¢
extra
anaez
their
perma

One 0

(16,2<
0btai1
Proce:

nicrol

relat:
to be
feede1
Puncct
cellu?
°mniV¢

in th(

4

Only a small fraction of the more than 2200 termite species have
been studied with respect to nutritional ecology and symbiotic
interaction with microorganisms, so it is difficult to generalize about
such relationships. However, one property apparently shared by all
termites, regardless of feeding behavior or phylogenetic classification,
is the presence of an anaerobic microbial community in the hindgut
region of their alimentary tract, a community which can be
extraordinarily diverse (6,14,20,58). Lower termites harbor both
anaerobic bacteria and protozoa, including cellulolytic flagellates, in
their hindguts. By contrast, the hindguts of higher termites lack
permanent populations of protozoa, and contain only bacteria (14,23,96).
One of the most conspicuous groups of bacteria observed in wet mounts of
gut contents from both higher and lower termites are spirochetes
(16,20,30). Unfortunately, no termite gut spirochetes have ever been
obtained in pure culture, so their function(s) with respect to gut
processes is still uncertain (16). The role of other termite gut
microbes in host nutrition is discussed in detail below.

Cockroaches are believed to be termites' closest phylogenetic
relatives (50,51). Although most cockroaches are generally considered
to be omnivorous, members of one genus, Cryptocercus, are strictly wood-
feeders (24). Like the phylogenetically lower termites, Cryptocercus
punctulatus harbors a diverse population of anaerobic bacteria and
cellulolytic protozoa in its hindgut (24,44). Others, such as the
omnivorous American cockroach, Periplaneta americana, have only bacteria
in their hindguts (29).

Our understanding of the overall nutritional relationships that

termites and cockroaches have established with microorganisms is

 

 

 

 

gene
cert
inse
this
micr<

cock)

times
tissuc

activ:

activi
that

enOUgh
Contra
Obtain,
POSsib]
gut Mic

eXCre te

also b1
“00d_ fee

5
generally incomplete. However, the role that microorganisms play in
certain key aspects of the digestive and metabolic activities of these
insects has become increasingly better understood. The remainder of
this section will summarize our current knowledge about the role of
microbes in the nitrogen and carbohydrate metabolism of termites and

cockroaches.

I. 1. Role of microorganisms in the nitrogen metabolism
of termites and cockroaches

Wood-feeding termites digest a resource that contains up to 100
times less N (on a dry weight basis) than that exhibited by termite
tissues (15). Consequently, termites have evolved ways of utilizing the
activites of their gut microbiota to aquire and/or conserve combined N.

One method of termite N aquisition is through N2-fixation
activities of gut-associated bacteria (15,18,38,72,75,79). Results show
that Nz-fixation rates for some wood-feeding species are significant
enough to support up to 50% of the termite's N requirements (15,79). By
contrast, most of the dietary nitrogen of fungus-cultivating termites is
obtained directly from digestion of ingested fungal tissue (26). It is
possible that termites might also aquire N by digestion of part of their
gut microbiota, or through assmiliation of combined N (eg. amino acids)
excreted by gut bacteria (59).

Mechanisms for nitrogen conservation involving gut microbes can
also be important to termite N metabolism. In studies with the lower,

wood-feeding termite, Reticulitermes flavipes, Potrikus and Breznak

found that, although this termite has the ability to synthesize uric

 

 

 

 

 

 

acid
is t
trans
tissx
CO

by hi
alth<

be es

soil«

gut:

the c
bacte
termj
noSt
it 1
PreSe

COckr

Until
natur.

6
acid (a common nitrogenous excretory product of insects), the compound
is not voided in the termite's feces, (73). Instead, uric acid
transported via Malpighian tubules from its site of synthesis (fat body
tissue) to the gut is immediately and completely fermented to acetate,
and NH

CO by anaerobic bacteria (74). Uric acid nitrogen liberated

2’ 3
by hindgut bacteria is ultimately assimilated back into termite tissues,
although the principle mechanism for this last recycling step remains to
be established.

Unfortunately, so little is understood about the nutrition of
soil-feeding termites, that any statement concerning the role of their
gut microbes in nitrogen metabolism would be purely speculative.

A system analagous to that of wood-feeding termites, involving
the conservation of uric acid nitrogen through the activities of
bacterial symbionts, may exist for cockroaches as well. As with
termites, uric acid does not appear to be a major excretory product of
most cockroach species, although they synthesize the purine, and store
it in fat body tissue (62-64). Intracellular bacteria (bacteroids)
present in specialized cells in fat body tissue (mycetocytes) (33) have
been implicated as agents of uric acid degradation and mobilization in
cockroaches (25). However, studies of this system have been obstructed
by the unavailability of authentic mycetocyte bacteria in pure culture.
Until direct evidence for bacterial uricolysis is forthcoming, the

nature of the symbiosis between cockroaches and their mycetocyte

bacteria will remain uncertain.

 

 

 

 

 

 

relati
and e
cellul
symbio

them t

largel
cellul
relati
termop
the hi
Subseq
Cellul
Polyme
ProteE

(68 »1(

stUdi.
Thed

is

7
I. 2. Role of microorganisms in the digestion of carbohydrates
by termites and cockroaches

Termites are faced with the formidable task of digesting
relatively refractory lignocellulosic substances as their major carbon
and energy source. For plant-feeding and fungus-cultivating termites
cellulose is the main component which must be digested, and their
symbiotic interactions with microorganisms, to various degrees, help
them to do this.

Evidence suggests that lower, wood-feeding termites depend
largely on anaerobic, cellulolytic protozoa to hydrolyze ingested
cellulose (17,23). A landmark in the study of lower termites was the
relatively recent isolation by M. A. Yamin of Trichomitopsis
termopsidis and Trichonympha sphaerica, dominant anaerobic protozoa from
the hindgut of the lower termite, Zootermopsis angusticollis, and the
subsequent demonstration that these two protozoa converted crystalline
cellulose to acetate, H2 and CO2 (69,99,100). Cellulase and other
polymer-hydrolyzing enzyme activities (eg. amylase, xylanase and
protease) have also been detected in crude extracts of T. termopsidis
(68,102).

Our understanding of the overall utilization of cellulose by
lower, wood-feeding termites is based largely on Yamin's work and
studies with the gut microbiota of another lower termite, R. flavipes.
The dissimilation of wood-polysaccharides in the hindgut of R. flavipes
is essentially a homoacetic fermentation of cellulose, as depicted in
Fig. 1. This diagram is based on a model proposed by Odelson and
Breznak that has received good experimental support (67). Initially,

‘wood polysaccharides (principally cellulose) are endocytosed by

 

 

 

 

Figure
Polysa
bacter
Thickr
activi

from 1

Figure l. The Odelson-Breznak model for symbiotic degradation of wood
polysaccharides (principally cellulose) by anaerobic protozoa and
bacteria in the hindgut region of the alimentary tract of R. flavipes.
Thickness of arrows signifies the relative contributions of microbial
activities to the digestive process. See text for details. Modified

from reference 67.

 

 

FOLYS

 

“2? mi“

BETH

 

 

 

 

 

 

 

PROTOZOA

S
E
m
R

0mm

00

0C

WA
8
W
0
P

Figure 1.

 

 

 

 

 

 

prot
glucu
are 1
beer:
to me
the
the e
the
schem.
of l
aceto;

discus

obscur
is ext
other
nutrit
hindgt

0V8ra]

diffs:

hindgL

10

protozoa, which then hydrolyze the cellulose and ferment the liberated
glucosyl units to acetate, H2 and 002. H2 and 602 produced by protozoa
are then consumed mainly by H2-oxidizing, COZ-reducing, acetogenic
bacteria to form additional acetate (although some H2/C02 is converted
to methane by methanogenic bacteria) (21,67). Acetate is then used by
the termite as an important source of carbon and energy (65). In fact,
the entire respiratory requirement of R. flavipes termites can be met by
the oxidation of microbially produced acetate. According to this
scheme, roughly two-thirds of hindgut acetate is derived from activities
of protozoa, and one-third is derived from bacterial Hz/CO2
acetogenesis. The novel bacterial component of this model will be
discussed in detail in the second section.

Although this simplified model may be conceptually appealing, it
obscures the fact that the hindgut microbial community of lower termites
is extremely diverse (14,20). The specific contribution of some of the
other abundant organsisms in the gut (eg. spirochetes) to host
nutrition, or to the functional stability and activity of the total
hindgut microbial community, may also be of critical importance to the
overall health and development of the insect.

Cellulose digestion in the higher wood-feeding termites must be
different than that in the lower termites, because of the absence of
hindgut protozoa. Moreover, the origin of enzymes that comprise the
cellulase repertoire in wood-feeding Termitidae appears to include the
termite itself. For example, Nasutitermes species that have been
examined secrete cellulase enzymes in the midgut (43,60,66). Treatment
of Nasutitermes with antibacterial drugs had little or no immediate

deleterious effect on cellulase activity (43). Thus, in the Termitidae,

 

 

 

cell
cell
from
wide.
morpl
feed
been
of h
prod1
exam‘
betwc

clar;

the

inge:
comp]
Data]
the 1

from

nakeS

termi:
the SC
DOSteI

(79).

ll

cellulose hydrolysis may not depend exclusively on the presence of
cellulolytic microorganisms. On the other hand, unpublished results
from our laboratory suggest that cellulolytic gut bacteria may be
widespread among wood-feeding Termitidae. Moreover, presence of a
morphologically diverse bacterial community in the hindgut of wood-
feeding representatives of the Termitidae (30) suggests that extensive
bacterial fermentation of carbohydrates probably occurs in the hindgut
of higher termites. Acetate and other typical bacterial fermentation
products were detected in hindguts of one higher termite species
examined (67). Consequently, details of the nutritional interaction
between higher termites and their gut bacteria still remain to be
clarified.

In the subfamily, Macrotermitinae, a major benefit derived from
the cultivation of Termitomyces fungal gardens is the aquisition, by
ingestion of fungal tissue, of some components of the cellulase enzyme
complex not made by the termite itself (58). For example, Macrotermes
natalensis and Macrotermes subhyallnus each make an endoglucanase, as do
the fungi which they cultivate and consume, but both termites aquire
from the fungi an exoglucanase which they do not make themselves (58).
The reverse situation appears to be true for Macrotermes mulleri, which
makes its own exoglucanse, but aquires an endoglucanase by ingesting its
Termitomyces fungus (77,78).

As with the higher, wood-feeding termites, fungus-cultivating
termites harbor a community of bacteria in their hindguts, as well as in
the so-called mixed-segment region (a region of overlap between the
‘posterior midgut and anterior hindgut that occurs in some termites)

(79). However, almost no information exists concerning the role of

l2
bacteria in the nutrition of Macrotermitinae. Not only is it possible
that hindgut bacterial fermentation could be part of the termite's
digestive strategy, but hindgut or mixed-segment-associated cellulolytic
bacteria could, in theory, also contribute to the initial digestive step
of cellulose hydrolysis.

Soil-feeding termites also have an anaerobic hindgut bacterial
community and harbor bacterial populations in the mixed segment (6,7).
The food of such termites has not been well defined in chemical terms,
but probably consists of residues of lignins and tannins that comprise
the organic component of soil referred to as humus. Some interesting
circumstantial evidence suggests that anaerobic, methanogenic, bacterial
consortia capable of degrading aromatic compounds from ingested soil
could be important to carbohydrate metabolism in the guts of soil-
feeding termites (11). However, fundamental aspects of the nutrition
and digestive processes of these fascinating animals have not been
investigated in detail. Therefore, it is difficult, if not impossible,
to assess the role and quantitative significance of bacteria in their
nutrition.

The relationship between microorganisms and wood-eating
cockroaches belonging to the genus Cryptocercus is similar to that of
the phylogenetically lower, wood-feeding termites (14,24). Knowlege of
the nutritional relationship between microbes and cockroaches other than
Cryptocercus is almost exclusively based on studies with two omnivorous
species, P. americana and Eublaberus posticus. These two cockroaches
have dense, anaerobic bacterial populations in their hindguts (29).
.Moreover, bacterial fermentation products (eg. volatile fatty acids)

occur in hindguts, and can be transported across the hindgut epithelium

 

 

of a

cock]
comp]
reare
cockr
elimi
of a
ameri
immat-
gut b.
cockr.
metro:
or ng
Cockrc

contri

II. a

an

of

gut mi
°°°kroa
flavipe
cellulo1

batter]:E

13

of cockroaches (10,103).

Although many bacteria have been isolated from the hindguts of
P. americana and E. posticus (29), the contribution of such bacteria to
cockroach nutrition is unclear, in part because of the omnivorous and
complex nature of the insects' diet (laboratory specimens are usually
reared on a diet of dog chow and water). In one study, P. americana
cockroaches were administered metronidazole, a drug which essentially
eliminated all anaerobic bacteria from the alimentary tract. The health
of adult cockroaches was unaffected, but the growth of immature P.
americana was retarded. Moreover, the guts of metronidazole-fed
immature specimens were degenerate (9). However, it was not known if
gut bacteria were important to the carbohydrate metabolism of immature
cockroaches. Another intriguing explanation for these results is that
metronidazole eliminates cockroach gut bacteria which produce vitamins
or growth factors that are important to the development of immature
cockroaches. Obviously, more work is needed to better understand the

contribution of cockroach gut bacteria to host nutrituion.

II. 112/602 Acetogenic and Methanogenic Bacteria,

and Their Role as Terminal Consumers of Hz in the Guts

of Termites and Cockroaches

In the previous section, it was evident that the contribution of

gut microorganisms to the carbohydrate metabolism of termites and
cockroaches is best understood for lower, wood-feeding termites such as R.
.flavipes. A novel aspect of the model describing symbiotic degradation of
cellulose by R. flavipes was the role accorded to HZ/CO2 acetogenic

bacteria as consumers of H2 produced by protozoa (Fig. 1). To appreciate

 

 

 

why be
is nec

in the

of aqL
many

intern
alcohc
HZ-COI
in na1
comp 01
inter:
Procee

(32.9:

envirc
or CC
be so,
“Snell
Each

rednet
01' (:02
(or H2
a°°ept.
Soa~2

the de

14

why bacterial H2/CO2 acetogenesis is such a novel aspect of this model, it
is necessary to first discuss the role of HZ-consuming bacterial reactions
in the terminal steps of anaerobic microbial decomposition processes.

Anaerobic decomposition of plant polymers occurs in a wide variety
of aquatic and terrestrial habitats (45), and in the digestive tracts of
many animals, including termites and cockroaches (15,104). During
intermediate steps of this process, fermentative and fatty acid- and
alcohol-oxidizing microorganisms may produce large amounts of H2. However,

H2-consuming bacteria effectively remove H keeping H2 partial pressures

29

in natural environments quite low (92). Removal of H2 is a crucial

component of anaerobic microbial decomposition processes, because the
intermediate, Hz-producing reactions of anaerobic microbial food webs will

proceed less efficiently, or not at all, if H2 is allowed to accumulate

(32,92).

Bacteria responsible for H -consumption in anaerobic

2
environments include those that can reductively dissimilate N03-1, 804-2,

or 002. Bacterial competition for H2 in anoxic habitats is thought to

-2
3 and 804 (C02 is not

usually limiting); 2) the free energy yield associated with reduction
2

be governed by: l) the availability of NO

each of these three electron acceptors [N03.1 reduction > 804'

reduction > co2 reduction (28,86,105)]; 3) the affinity of N03'1-, so '2-

or COz-reducing organisms for H2 and 4) the minimum H2 partial pressure

(or H2 threshold) at which the reduction of each of these three electron
acceptors will proceed (41). In environments that are low in N03'1 and

SO“.2 (such as most animal gastrointestinal tracts), 002 reduction is

the de facto terminal, H -consuming (electron sink) process.

2
Two types of H2-consuming, COz-reducing, energy-yielding, bacterial

 

 

 

 

pIOCE

and I
II.

reduc
consu

secti

that

and pj
Whose
can g1
fOrmal
Past 1
PhyloE

which

Hz/CO2
repres‘
signif:

COckroE

15

processes are known to occur in anaerobic habitats: Hz/CO2 methanogenesis

and H2/602 acetogenesis, each of which is described below.

II. 1. Hé/COZ methanogenesis and Hé/COZ methanogenic bacteria

In most anaerobic environments low in N03.1 and 804-2, 002

reduction to methane, rather than acetate, is the main, terminal, H2-
consuming reaction (Reaction 1). Reasons for this are discussed in

section II. 3.

4112 + co2 ---> CH4 + 2H20 (1 c°' - -135.6 kj/reaction [86]) (1)

There are currently 43 species of bacteria described as of 1988
that can grow via Reaction 1, all of which belong to a physiologically
and phylogenetically coherent group of strictly anaerobic archaebacteria
whose growth is obligately methanogenic (8). Most methanogenic bacteria
can grow on H2 + C02, and some can also produce methane from acetate,
formate, methanol, ethanol, CO and methylamines (8,88,89). During the
past ten years, a great deal has been learned about the biochemistry,
phylogeny, distribution and ecology of methanogenic bacteria, most of
which is summarized in recent reviews (eg. 8,88).

Termites and cockroaches emit detectable amounts of methane, and
H2/002 methanogenic bacteria have been observed in gut contents of
representatives of both insect groups (29,67). The quantitaive

significance of H2/C02 methanogenesis in the guts of termites and

cockroaches is also discussed in section II. 3.

 

 

 

II. 2

aceta

C0
HZ/

aceto;
group
been

and Ac
been C
acetOg
is a
which

fOrmin

60 di
alcoho:
VOOdII:
Product
few 0t}
also

heterot
<32).

acetogel

COCu1 tn]

16
II. 2. H2/COZ acetogenesis and Hé/OOZ acetogenic bacteria
An alternative to CO2 reduction to methane is 002 reduction to
acetate by Hz/CO2 acetogenic bacteria (Reaction 2). The biochemistry of

H2/002

432 + 2co2 ---> cu3coou + 2H20 (1 c°' - -104.6 [86]) (2)
acetogenesis has been thoroughly investigated (see reviews by Wood's
group [93,94] and Fuchs [39]). Although most biochemical studies have
been done by using a few selected species (Clostridium thermoaceticum
and Acetobacterium woodii), many new H2/002 acetogenic bacteria have
been described recently (Table 1). Unlike H2/602 methanogenesis, H2/602
acetogenesis is not restricted to a phylogenetically coherent group, but
is a property of several distantly related taxa of anaerobic bacteria
which include both Gram positive and negative genera, several endospore-
forming representatives, and mesophilic and thermophilic species.

As a group, H2/CO2 acetogenic bacteria can metabolize more than
60 different compounds including sugars, organic and amino acids and
alcohols (56, and references contained therein). Most species (eg. A.
woodii) convert carbohydrates to acetate as the principal fermentation
product and, therefore, have been called ”homoacetogens”. However, a
few others (eg. E. limosum) form both acetate and butyrate (82). It is
also significant that when H2/002 acetogens are growing
heterotrophically, they can be Hz-producers rather than Hz-consumers
(32). For example, Lee and Zinder have demonstrated that one H2/602
acetogen can perform Reaction 2 in the reverse direction when grown in

coculture with an H2-consuming methanogen (54).

 

 

 

 

Table 1

study,

Isolate

 

Acetate
rod (
Acetiec
rumir
Acetoar
note:
Acetobe
carbi
wieri
wood}
Acetoge
kivuj
BUtyrit
math}
Clostri
aceti
strai
thern
theru
EUbacte
1111105
Peptosc
PrOdu
SPOIOmu
acido
maIOn
OVata
Pauci
Sphae.
termi;
~ii‘j“

17
Table 1. List of H2/CO2 acetogenic bacteria described previous to this

study, and some of their properties.

 

 

 

Endo-
Cell spore To t Ref-
Walla form-b OP ' er-

Isolate Original Source Type ation ( C) ence
Acetate-oxidizing c

rod (AOR) Thermal digestor ? - 60 54
Acetiuomaculum

ruminus Bovine rumen + - 38 40
Acetoanaerobium

noterae Oil drilling sediment - - 37 84
Acetobacterium

carbinolicum Freshwater seds./sludge + - 27 35

wieringae Sewage digestor + - 30 12

woodii Marine estuary + - 30 4
Acetogenium

kivui Lake sediment - - 66 55
Butyribacterium d

methylotrophicum Sewage digestor + + 37-40 101
Clostridium

aceticum Mud _e + 30 1,13

strain CV-AAl Sludge _e + 30 2

thermaceticum Horse manure + + 55-60 37

thermautrotrophicum Mud/soil + + 56-60 90
Eubacterlum

limosum Sheep rumen/sludge + - 39 82
Peptostreptococcus

productus Sewage digestor + - 37 57
Sporomusa

acidovorans Distillery effluent - + 35 70

malonica Pond sediments - + 30 31

ovata Sugar beet leaf silage - + 35-37f 61

paucivorans Lake sediments - - n.d. 42

sphaeroides River sediments - + 34 61

termitida Termite guts - + 30 22
8+, Gram positive; -, Gram negative.
b+, forms endospores; -, does not form endospores.

cCell wall of the AOR is not characteristic of either Gram positive or
Gram negative bacteria.

dSpores are atypical.

eCells stain Gram negative, and electron micrographs are inconclusive.
The true cell wall type has not been determined by other methods.

fn.d., not determined.

18

Clostridium formicacetlcum (3) and C. magnum (81) are two
homoacetogenic anaerobes that are unable to grow on H2 + 602, but are
undoubtedly closely related to some of the Clostridium species listed in
Table 1. In addition, H2/CO2 methanogenic bacteria and some sulfate-
reducing bacteria can form acetyl-CoA from H2 + 602 for autotrophic
synthesis of cell carbon (essentially a form of H2/002 acetogenesis),
but such bacteria rely on methanogenesis or sulfate reduction to
generate energy for growth (39).

Despite recent advances in our understanding of the microbiology
and biochemistry of H2/C02 acetogenic bacteria, almost no information
exists concerning their ecology or their role as HZ-consumers in nature.
They have been isolated from a wide variety of anaerobic habitats (Table
1), but it is not yet clear whether H2/CO2 acetogens that have been

isolated using H2 + CO as the carbon and energy source, are, in fact

2

H -consumers (or Hz-producers) in situ.

2
Theoretical considerations suggest that H2/CO2 methanogensis

should always outcompete H2/CO2 acetogenesis for H2 because H2/CO2
methanogenesis (Reaction 1) is thermodynamically more favorable than
Hz/CO2 acetogenesis (Reaction 2) (28). This is especially true under
conditions which involve "typical" natural concentrations of substrates
and products found in anoxic habitats (32,105). Moreover, methanogenic
bacteria have a 10 to lOO-fold lower threshold for H than do their

2
acetogenic counterparts (19,41), despite the fact that the H2-vmax/Km
values of these two groups of hydrogenotrophic bacteria are in the same
range (22). However, there are reports of a few habitats where rates of

H2/602 acetogenesis compare favorably to, or even exceed rates of H2/CO2

methanogenesis. These include certain anaerobic freshwater lakes and

 

 

 

sedi

rode

II.

outcc
sugge
rate

evolv
Moreo
termi:
by 11‘
each

BreZni

Slgnij

SWitz£
IACO
rates

demons

k'l

Condit

1”1311c

l9
sediments (27,46,71), and the gastrointestinal tracts of humans (53),

rodents (76), baleen whales (91) and wood-feeding termites (21).

II. 3. COmpetition for H2 in the guts of

termites and cockroaches

The Odelson-Breznak model of symbiotic cellulose utilization by
R. flavipes termites (Fig. 1) suggested that H2/002 acetogenesis
outcompetes H2/C02 methanogenesis for H2 in termite hindguts. This
suggestion was prompted by the observation that, relative to the overall
rate of cellulose fermentation in the hindgut, R. flavipes termites
evolved quantitatively insignificant amounts of methane and H2 (62).
Moreover, evaluation of the rates of acetate formation in guts and
termite respiration rates, as well as the respiratory quotient exhibited
by live insects, suggested that three acetate molecules were formed from
each glucosyl unit of cellulose fermented (67). Thus, Odelson and
Breznak postulated that H2/002 acetogenesis was the source of a
significant amount (up to one-third) of hindgut acetate.

This speculation was subsequently confirmed by Breznak and
Switzer (21), who also showed that rates of Hz-dependent reduction of

14CO to 14C-acetate by R. flavipes gut contents totally outcompeted

2
14 14
rates of Hz-dependent reduction of 602 to CH4. In addition, they

demonstrated that H2/CO2 acetogenesis in R. flavipes guts was inhibited
by 02 or by feeding termites antibacterial drugs. By contrast,
conditions which resulted in the loss of cellulolytic protozoa from guts
had little effect on HZ/CO2 acetogenesis. These results strongly

implicated anaerobic bacteria as the agents of H2/CO2 acetogenesis. It

was also found that H2/002 acetogenesis outcompeted H2/002 methanogensis

20

in the guts of three other lower, and two higher, wood-feeding termite
species, and in the gut of the wood-feeding cockroach, C. punctulatus,
but not in the gut of the omnivorous cockroach, P. americana (21).

Unfortunately, and for reasons that are not entirely understood,
attempts to isolate HZ/CO2 acetogenic bacteria from gut contents of R.
flavipes, or other lower, wood-feeding termite species were
unsuccessful. However, a new H2/602 acetogenic isolate (Sporomusa
termitida) was recently obtained from gut contents of a higher, wood-

feeding termite, Nasutitermes nigriceps (22).

III. Statement of Purpose

The present study was undertaken in an effort to better
understand the microbiology, ecology and nutritional significance of
bacterial H2/002 acetogenesis in guts of wood-feeding termites such as
R. flavipes. It was hoped that such information might also help clarify
the ability of H2/602 acetogenic bacteria to outcompete H2/CO2
methanogenic bacteria for H2 in this habitat. Chapter 1 examines how
widespread H2/002 acetogenesis and methanogensis are among a diverse
sampling of termite species, and to what extent diet and feeding
behavior are associated with H2 consumption by termite gut bacteria.
Experiments were performed with 23 different species, including fungus-
cultivating and soil-feeding termites, in addition to higher and lower,
wood- and grass-feeding representatives.

Access to a wide variety of termite species also afforded an
opportunity to make further attempts to isolate H2/C02 acetogenic

bacteria from termite gut contents (a project that previously had met

with only limited success). Chapters 2 and 3 describe two novel H2/CO2

21
acetogenic bacteria that were obtained in pure culture. Chapter 2
describes the first H2/002 acetogenic bacterium isolated from gut
contents of a phylogenetically lower, wood-feeding termite; and Chapter
3 describes the first H2/CO2 acetogen isolated from gut contents of a
higher, soil-feeding termite. Morphological, physiological and
molecular phylogenetic characteristics of these two new isolates are
compared to those of Sporomusa termitida, an H2/C02 acetogenic bacterium
previously isolated from gut contents of the higher, wood-feeding
termite, Nasutitermes nigriceps (22).

Unfortunately, many termites are difficult or virtually
impossible to rear in the laboratory, and those that can be maintained
outside of their natural habitat usually have somewhat inflexible
dietary needs. Therefore, to study the effects of dietary changes on
insect gut microbial ecology, and to learn more about the contribution
of gut microorganisms to cockroach carbohydrate metabolism, experiments
were performed with the omnivorous cockroach species, P. americana. The
effect of high fiber diets on the activities and numbers of acidogenic
and methanogenic bacteria in different regions of the alimentary tract
of adult and immature P. americana are described in Chapter 4.

In summary, this study represented an integrated approach to
examine factors (such as diet and developmental stage) that influence
compatible and competitive interactions between acidogenic and

methanogenic bacteria in the guts of termites and cockroaches.

 

 

 

 

 

10,

11,

.Ad

(w

.Ad

Cl

pr
48

.An

CL
di
Mi'

.Ba

Ac.
rel

. Ba‘

na:

. Bi)

Di:
tel
Mic

. Big

Hoc
so.
Prc
Tel

- Boc

arc

Vi]

met
COC
13:

Bra
8nd
Phy

Bra
Net
Hic-
int.

10.

11.

22
LITERATURE CITED

. Adamse, A. D. 1980. New isolation of Clostridium aceticum

(Wieringa). Antonie van Leewenhoek 46:523-531.

. Adamse, A. D. and C. T. M. Velzeboer. 1982. Features of a

Clostridium, strain CV-AAl, an obligatory anaerobic bacterium
producing acetic acid from methanol. Antonie van Leeuwenhoek
48:305-313.

. Andreesen, J. R., G. Gottschalk and H. G. Schlegel. 1970.

Clostridium formicoaceticum nov. spec. isolation, description and
distinction from C. aceticum and C. thermoaceticum. Arch.
Mikrobiol. 72:154-170.

. Balch, W. E., S. Schoberth, R. S. Tanner and R. S. Wolfe. 1977.

Acetobacterium, a new genus of hydrogen-oxidizing, carbon dioxide-
reducing, anaerobic bacteria. Int. J. Syst. Bacteriol. 2:355-361.

. Bayon, C. and J. Mathelin. 1980. Carbohydrate fermentation and

byproduct absorption studied with labeled cellulose in Oryctes
nasicornis (Coleoptera, Scarabeidae). J. Insect Physiol. 26:833-840.

. Bignell, D. E. , H. Oskarsson and J. M. Anderson. 1980.

Distribution and abundance of bacteria in the gut of a soil-feeding
termite, Procubitermes aburiensis (Termitidae, Termitinae). J. Gen.
Microbiol. 117:393-403.

. Bignell, D. E., H. Oskarsson, J. M. Anderson, P. Ineson and T. C.

Wood. 1983. Structure, microbial associations and function of the
so-called "mixed segment" of the gut in two soil-feeding termites,
Procubitermes aburiensis and Cubitermes severus (Termitidae,
Termitinae). J. Zool. Lond. 201:445-480.

. Boone, D. R. and R. A. Mah. 1988. Group I. Methanogenic

archaeobacteria. In J. T. Staley, M. P. Bryant, N. Phennig and J.
G. Holt (eds.) Bergey's manual of systematic bacteriology, vol. 3.
Williams & Wilkins, Baltimore

. Bracke, J. W., D. L. Cruden and A. J. Markovetz. 1978. Effect of

metronidazole on the intestinal microflora of the American
cockroach, Periplaneta americana L. Antimicrob. Agent. Chemother.
13:115-120.

Bracke, J. W. and A. J. Markovetz. 1980. Transport of bacterial

endproducts from the colon of Periplaneta americana. J. Insect
Physiol. 26:85-89.

Brauman, A., M. D. Kane, M. Labat and J. A. Breznak. 1990. Hydrogen
metabolism by termite gut microbes. In J. P. Belaich (ed.),
Microbiology and Biochemistry of strict anaerobes involved in
interspecific hydrogen transfer. Plenum, (in press).

12.

13.

14.

15.

16.

 

17.

18.

19.

20.

 

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

23
Braun, M. and G. Gottschalk. 1981. Acetobacterium wieringae sp.
nov., a new species producing acetic acid from molecular hydrogen
and carbon dioxide. Zbl. Bakt. Hyg., I. Abt. Orig. C 3:368-376.

Braun, M., F. Mayer and G. Gottschalk. 1981. Clostridium aceticum
(Wieringa), a microorganism producing acetic acid from molecular
hydrogen and carbon dioxide. Arch. Microbiol. 128:288-293.

Breznak, J. A. 1982. Intestinal microbiota of termites and other
xylophagous insects. Ann. Rev. Microbiol. 36:323-343.

Breznak, J. A. 1984. Biochemical aspects of symbiosis between
termites and their intestinal microbiota, p.173-203. In J. M.
Anderson, A. D. M. Rayner, and D. W. H. Walton (eds.),
Invertebrate-microbial interactions. Cambridge University Press,
Cambridge.

Breznak, J. A. 1984. Hindgut spirochaets of termites and
Cryptocercus punctulatus, p.68-70. In N. R. Krieg (ed.) Bergey's
manual of systematic bacteriology, vol. 1. Williams & Wilkins,
Baltimore

Breznak, J. A. 1990. Metabolic activities of the microbial flora of
termites. In R. Lesel (ed.) Microbiology of poecilotherms.
Elsevier, Amsterdam (in press).

Breznak, J. A., W. J. Brill, J. W. Mertins, and H. C. Coppel. 1973.
Nitrogen fixation in termites. Nature 244:577-580.

Breznak, J. A. and M. D. Kane. 1990. Microbial H .CO acetogenesis
in animal guts: nature and nutritional signi icance. FEMS
Microbiol. Rev. (In Press).

Breznak, J. A. and H. S. Pankratz. 1977. In situ morphology of the

gut microbiota of wood-eating termites [Reticulitermes flavipes
(Kollar) and Cbptotermes formosanus (Shiraki)]. Appl. Environ.
Microbiol. 33:406-426.

Breznak, J. A. and J. M. Switzer. 1986. Acetate synthesis from H

plus 602
630.

by termite gut microbes. Appl. Environ. Microbiol. 52:623g

Breznak J. A., J. M. Switzer and H.-J. Seitz. 1988. Sporomusa
termitida sp. nov., an Hz/CO -utilizing acetogen isolated from
termites. Arch. Microbiol. 15 :282-288.

Cleveland, L. R. 1924. Symbiosis between termites and their
intestinal protozoa. Proc. Natl. Acad. Sci. USA 19:424-428.

Cleveland, L. R., S. R. Hall, E. P. Sanders and J. Collier. 1934.
The wood-feeding roach Cryptocercus, its protozoa, and the symbiosis
between protozoa and roach. Mem. Am. Acad. Arts Sci. 17:184-342.

Cochran, D. G. 1985. Nitrogen excretion in cockroaches. Ann. Rev.
Entomol. 30:29-49.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

24
Collins, N. M. 1983. The utilisation of nitrogen resources by
termites (Isoptera), p. 381-412. In J. A. Lee, S. McNeill and I. H.
Rorison, Nitrogen as an ecological factor. Blackwell, Oxford.

Conrad, R., F. Bak, H.-J. Seitz, B. Thebrath, H. P. Mayer and H.
Schutz. 1989. Hydrogen turnover by psychrotrophic homoacetogenic
and mesophilic methanogenic bacteria in anoxic paddy soil and lake
sediment. FEMS Microbial. Ecol. 62:285-294.

Card-Ruwisch, R. H.-J. Seitz and R. Conrad. 1988. The capacity of
hydrogenotrophic anaerobic bacteria to compete for traces of
hydrogen depends on the redox potential of the terminal electron
acceptor. Arch. Microbiol. 149:350-357.

Cruden, D. L. and A. J. Markovetz. 1987. Microbial ecology of the
cockroach gut. Ann. Rev. Microbiol. 41:617-643.

Czolij, R., M. Slaytor and R. W. Obrien. 1985. Bacterial flora of
the mixed segment and the hindgut of the higher termite Nasutitermes
exitasis Hill (Termitidae, Nautitermitinae). Appl. Environ.
Microbiol. 49:1226-1236.

Dehning, I., M. Stieb and B. Schink. 1989. Sporomusa malonica sp.
nov., a homoacetogenic bacterium growing by decarboxylation of
malonate or succinate. Arch. Microbiol. 151:421-426.

Dolfing, J. 1988. Acetagenic bacteria, p. 417-468. In A.J.B.
Zehnder (ed.), Biology of anaerobic microorgansisms. John Wiley &
Sons, New York.

Downer, R. G. H. 1982. Fat body and metabolism, p. 151-174. In W.
J. Bell and K. G. Adiyadi (eds.), The American Cockroach. Chapman
and Hall, New York.

Edwards, R. and A. E. Mill. 1986. Termites in buildings. Rentokil
Ltd., W. Sussex. 261 pp.

Eichler, B. and B. Schink. 1984. Oxidation of primary aliphatic
alcohols by Acetabacterium carbinalicum, sp. nov., a homoacetogenic
anaerobe. Arch. Microbiol. 140:147-152.

Fenchel, T. 1987. Ecology of protozoa: The biology of free-living
phagotrophic protists. Science Tech, Inc., Madison.

Fantaine, F. E., W. H. Peterson, E. McCoy, M. J. Johnson and G. J.
Ritter. 1941. A new type of glucose fermentation by Clostridium
thermoaceticum n. sp. J. Bacteriol. 43:701-715.

French, J. R. J., G. L. Turner and J. F. Bradbury. 1976. Nitrogen
fixation by bacteria from the hindgut of termites. J. Gen.
Microbial. 95:202-206.

Fuchs, G. 1986. CO fixation in acetogenic bacteria: variations on
a theme. FEMS Micro 101. Rev. 39:181-213.

 

4O

41

1.2.
43.
44.
45. 1
46.
47. x

48. K

49, K
50- K:
51- K]
52. Ku

53. I-aj

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

25
Greening, R. C. and J. A. Z. Leedle. 1989. Enrichment and isolation
of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria
from the bovine rumen. Arch. Microbial. 151:399-406.

Greening, R. C., W. J. Smalenski, J. A. Z. Leedle and J. A.
Robinson. 1989. Hydrogen threshold determinations for the ruminal
acetogen Acetitomaculum ruminis and other selected hydrogen-
consuming bacteria. FEMS Microbiol. Ecol. (submitted)

Hermann M., M.-R. Papoff and M. Sebald. 1987. Sporamusa paucivorans
sp. nov., a methylatrophic bacterium that forms acetic acid from
hydrogen and carbon dioxide. Int. J. Syst. Bacteriol. 37:93-101.

Hogan, M., P. C. Veivers, M. Slaytor and R. T. Czolij. 1988. The
site of cellulose breakdown in higher termites (Nasutitermes walkeri
and Nasutitermes exitosis). J. Insect Physiol. 34:891-899.

Hanigburg, B. M. 1970. Protozoa associated with termites and their
role in digestion, p. 1-36. In K. Krishna and F. M. Weesner (eds.),
Biology of termites, vol. 1. Acedemic Press, New York.

Hungate, R. E. 1985. Anaerobic biotransformatians of organic
matter, p. 39-95. In E. R. Leadbetter and J. S. Poindexter (eds.),
Bacteria in nature, vol. 1. Plenum Publishing Corp., New York.

Jones, J. G. and B. M. Simon. 1985. Interactions of acetogens and
methanogens in anaerobic freshwater sediments. Appl. Environ.
Microbiol. 49:944-948.

Kaufman, M. G. 1988. The role of anaerobic bacterial metabolism in
the nutrition of crickets (Orthoptera: Gryllidae). Ph.D. Thesis,
Michigan State University, East Lansing.

Keeton, W. 1976. Biological science, third edition. W. W. Norton 8
Co., New York.

Klug, M. J. and S. Kotarski. 1980. Bacteria associated with the gut
tract of larval stages of the aquatic cranefly Tipula abdominalis
(Diptera; Tipulidae). Appl. Environ. Microbiol. 40:408-416.

Krishna, K. 1969. Introduction, p. 1-13. In K. Krishna and F. M.
Weesner (eds.), Biology of termites, vol. 1. Acedemic Press, New
York.

Krishna, K. 1970. Taxonomy, phylogeny, and distribution of
termites, p. 127-152. In Biology of termites, vol. 2. Acedemic
Press, New York.

Kukar, J. J. and M. M. Martin. 1983. Aquisition of digestive
enzymes by siricid woodwasps from their fungal symbiont. Science
220:1161-1163.

Lajoie, S. F., S. Bank, T. L. ller and M. J. Wolin. 1988. Acetate
production from hydrogen and [ C] carbon dioxide by the microflora
of human feces. Appl. Environ. Microbiol. 54:2723-2727.

 

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65,

66

' oBrien

lee,
of a
assoc
-CO
H2
Leigh
new
Micro

 

Ljung
in ac

Larow
strai
Envir

Marti1
funga
Ithic.

Mauldl
synthd
termii

 

McEwe
activ
10:56

Kolle
1984.
inclu
HOV ,

Mulli
cockr

Hulli
Ameri
with
500,

Hulli

Americ
balanc
Bi°Chei

gluCOS

obTIEn
termit
Biochel

 

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

26

Lee, M. J. and S. H. Zinder. 1988. Isolation and characterization
of a thermophilic bacterium.which oxidizes acetate in syntrophic
association with a methanogen and which grows autotropphically on
H2-COZ. Appl. Envrion. Microbial. 54:124-129.

Leigh, J. A., F. Mayer and R. S. Wolfe. 1981. Acetogenium kivui, a
new thermophilic hydrogen-oxidizing, acetogenic bacterium. Arch.
Microbiol. 129:275-280.

Ljungdahl, L. G. 1986. The autotrophic pathway of acetate synthesis
in acetogenic bacteria. Ann. Rev. Microbiol. 40:415-450.

Lorowitz, W. H. and M. P. Bryant. 1984. Peptostreptococcus praductus
strain that grows rapidly with CO as the energy source. Appl.
Environ. Microbiol. 47:961-964.

Martin, M. M. 1987. Invertebrate-microbial interactions: Ingested
fungal enzymes in arthropod biology. Cornell University Press,
Ithica.

Mauldin, J.K., . M. Rich and D. W. Cook. 1978. Amino acid
synthesis from C-acetate by normally and abnormally faunated
termites, Captotermes formosanus. Insect Biochemistry 8:105-109.

McEwen, S. E., M. Slaytor and R. W. Obrien. 1980. Cellobiase
activity in three species of australian termites. Insect Biochem.
10:563-567.

Moller B., R. Ossmer, B. H. Howard, G. Gottschalk and H. Hippe.
1984. Sporomusa, a new genus of gram-negative anaerobic bacteria
including Sporamusa sphaeroides spec. nov. and Sporomusa ovata spec.

nov. Arch. Microbial. 139: 388-396.

Mullins, D. E. and D. G. Cochran. 1972. Nitrogen excretion in
cockroaches: uric acid is not a major product. Science 177:699-701.

Mullins, D. E. and D. G. Cochran. 1975. Nitrogen metabolism in the
American cockroach. I. An examination of positive nitrogen balance
with respect to uric acid stores. Comp. Biochem. Physiol. 50Az489-
500.

Mullins, D. E. and D. G. Cochran. 1975. Nitrogen metabolism in the
American cockroach. II. An examination of negative nitrogen

balance with respect to mobilization of uric acid stores. Comp.
Biochem. Physiol. 50A:501-510.

OBrien, R. W. and J. A. Breznak. 1984. Enzymes of acetate and
glucose metabolism in termites. Insect Biochem. 14:639-643.

OBrien, G. W., P. C. Veivers, S. E. McEwen, M. Slaytor and R. W.
Obrien. 1979. The origin and distribution of cellulase in the

termites, Nasutitermes exitosis and Captotermes lacteus. Insect
Biochem. 9:619-625.

 

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78_

79.

trim

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

27
Odelson, D. A. and J. A. Breznak. 1983. Volatile fatty acid
production by the hindgut microbiota of xylophagous termites. Appl.
Environ. Microbiol. 45:1602-1613.

Odelson, D. A. and J. A. Breznak. 1985. Cellulase and other

polymer-hydrolyzing activites of Trichomitopsis termopsidis, a
symbiotic protozoan from termites. Appl. Environ. Microbiol.
49:622-626.

Odelson, D. A. and J. A. Breznak. 1985. Nutrition and growth
characteristics of TrichomitoPsis termopsidis, a cellulolytic
protozoan from termites. Appl. Environ. Microbiol. 49:614-621.

Ollivier, B., R. Cordruwisch, A. Lombardo and J. L. Garcia. 1985.
Isolation and characterization of Sporomusa acidavorans, sp. nov., a
methylotrophic homoacetogenic bacterium. Arch. Microbiol. 142: 307-
310.

Phelps, T. J. and J. G. Zeikus. 1984. Influence of pH on terminal
carbon metabolism in anoxic sediments from a mildly acidic lake.
Appl. Environ. Microbiol. 48:1088-1095.

Potrikus, C. J. and J. A. Breznak. 1977. Nitrogen-fixing
Enterobacter agglomerans isolated from the guts of wood-eating
termites. Appl. Environ. Microbiol. 33:392-399.

Potrikus, C. J. and J. A. Breznak. 1980. Uric acid in wood-eating
termites. Insect Biochemistry 10:19-27.

Potrikus, C. J. and J. A. Breznak. 1981. Cut bacteria recycle uric
acid nitrogen: a strategy for nutrient conservation. Proc. Natl.
Acad. Sci. USA 78:4601-4605.

Prestwich, G. D. and B. L. Bentley. 1982. Ethylene production by
the fungus comb of Macrotermitines (Isoptera, Termitidae): A caveat

for the use of the acetylene reduction assay fro nitragenase
activity. Sociobiology 7:145-152.

Prins, R. A. and A. Lankhorst. 1977. Synthesis of acetate from CO2
in the cecum of some rodents. FEMS Microbiol. Lett. 1:255-258.
Rouland, C., A. Civas, J. Renoux and F. Petek. 1988. Purification
and properties of cellulases from the termite Macrotermes mulleri
(Termitidae, Macrotermitinae) and its symbiotic fungus Termitomyces
sp. Comp. Biochem. Physiol. 913:449-458.

Rouland, C., A. Civas, J. Renoux and F. Petek. 1988. Synergistic
activities of the enzymes involved in cellulose degradation,
purified from Macrotermes mulleri and from its symbiotic fungus
Termitomyces sp. Comp. Biochem. Physiol. 918:459-465.

Rohrmann, G. F. and A. Y. Rossman. 1980. Nutrient strategies of
Macrotermes ukuzii (Isoptera: Termitidae). Pedobiolagia 20:61-73.

 

80.

81.

82.

83.

84.

 

 

 

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

28
Sands, W. A. 1969. The association of termites and fungi, p.495-
524. In K. Krishna and F. M. Weesner (eds.), Biology of termites,
vol. 1. Acedemic Press, New Yark.

Schink, B. 1984. Clastridium magnum sp. nov., a non-autotraphic
homoacetogenic bacterium. Arch. Microbiol. 137:250-255.

Sharak Genthner, B. R., C. L. Davis and M. P. Bryant. 1981.
Features of rumen and sewage sludge strains of Eubacterium limosum,
a methanol- and H -CO -utilizing species. Appl. Environ. Microbiol.
42:12-19. 2 2

Sinsabaugh, R. L., A. E. Linkins and E. F. Benfield. 1985. Cellulose
digestion and assimilation by three leaf-shredding aquatic insects.
Ecology 66:1464-1471.

Sleat, R., R. A. Mah and R. Robinson. 1985. Acetanaerobium noterae

gen. nov., sp. nov.: an anaerobic bacterium that forms acetate from
H2 and C02. Int. J. Syst. Bacteriol. 35:10-15.

Taylor, E. C. 1982. Role of aerobic microbial populations in

cellulose digestion by desert millipedes. Appl. Environ. Microbiol.
44:281-291.

Thauer, R. K., K. Jungermann and K. Decker. 1977. Energy
conservation in chemolithotrophic bacteria. Bacterial. Rev. 41:100-
180.

Ulrich, R. C., D. A. Buthala and M. J. Klug. 1981. Microbiota
associated with the gastrointestinal tract of the common house
cricket, Acheta domestica. Appl. Environ. Microbiol. 41:246-254.

Whitman, W. B. 1985. Methanogenic bacteria, p.3-84. In C. R. Woese
and R. S. Wolfe (eds.), The bacteria: a treatise on structure and
function, vol. VIIIzArchaebacteria. Academic Press, New York.

Widdel, F. 1986. Growth of methanogenic bacteria in pure culture
with 2-propanol and other alcohols as hydrogen donors. Appl.
Environ. Microbiol. 51:1056-1062.

Wiegel, J., M. Braun and G. Gottschalk. 1981. Clostridium
thermoautotrophicum species novum, a thermophile producing acetate

from molecular hydrogen and carbon dioxide. Curr. Microbial. 5:255-
260.

Wilmarth, K. R., D. R. Boone and R. A. Mah. 1985. Hydrogen
utilizing bacteria in the colon of cetaceans. Abstr. Ann. Mtg.
Amer. Soc. Microbiol., p. 164.

Wolin, M. J. 1982. Hydrogen transfer in microbial communities,
p.323-356. In A. T. Bull and J. H. Slater (eds.), Microbial
interactions and communities. Academic Press, London.

 

93. Wood.
pathv
11:1-

94. Wood,
paths

95. Wood,
Cambr
96. Wood,

97. Wood,

98. Yamid

99. Yamin

101.

 

102,

103,

104,

105,

 

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

29
Wood, H. G, Ragsdale, S. W. and E. Pezacka. 1986. The acetyl-CoA
pathway: a newly discovered pathway of autotrophic growth. TIBS
11:1-5.

Wood, H. G, Ragsdale, S. W. and E. Pezacka. 1986. The acetyl-CoA
pathway of autotrophic growth. FEMS Microbiol. Rev. 39:345-362.

Wood, T. G. 1978. Food and feeding habits of termites, p.55-80. In
M. V. Brian, (ed.), Production ecology of ants and termites.
Cambridge University Press, Cambridge.

Wood, T. G. 1986. The biology, physiology, and ecology of termites,
p.1-68. In S. B. Vinson (ed.), Economic impact and Control of social
insects. Praeger, U.S.A.

Wood, T. G. and W. A. Sands. 1978.. The role of termites in
ecosystems, p.245-293. In M. V. Brian, (ed.), Production ecology of
ants and termites. Cambridge University Press, Cambridge.

Yamin, M. A. 1979. Flagellates of the orders Trichomonadida Kirby,
Oxymonadida Grasse, and Hypermastigida Grassi and Fee reported from
lower termites (Isoptera families Mastotermitidae, Kalotermitidae,
Hodotermitidae, Termopsidae, Rhinotermitidae and Serritermitidae)
and from the wood-feeding roach Cryptocercus (Dictyoptera:
Cryptocercidae). Sociobiol. 4:1-120.

Yamin, M. A. 1980. Cellulose metabolism by the termite flagellate
Trichomltopsis termopsidis. Appl. Environ. Microbiol. 39:859-863.

Yamin, M. A. 1981. Cellulose metabolism by the flagellate
Trichonympha from a termite is independent of endosymbiotic
bacteria. Science 211:58-59.

Zeikus, J. G., L. H. Lynd, T. E. Thompson, J. A. Kryzzcki, P. J.
Weimer and P. W. Hegge. 1980. Isolation and characterization of a

new, methylatrophic, acidogenic anaerobe, the Marburg strain.
Curr. Microbial. 3:381-386.

Yamin, M. A. and W. Trager. 1979. Cellulolytic activity of an
axenically-cultivated termite flagellate, Trichomitopsis
termopsidis. J. Gen Microbiol. 113:417-420.

Hogan, M. E., M. Slaytor and R. W. Obrien. 1985. Transport of
volatile fatty acids across the hindgut of the cockroach, Panesthia
cribrata Saussure and the termite, Mastotermes darwiniensis
Froggatt. J. Insect Physiol. 31:587-591.

Wolin, M. J. 1974. Metabolic interactions among intestinal
microbes. Am. J. Clin. Nutr. 27:1320-1328.

Conrad, R., B. Schink and T. J. Phelps. 1986. Thermodynamics of
H -consuming and H -producing metabolic reactions in diverse

methanogenic environments under in situ conditions. FEMS
Microbiol. Ecol. 38:353-360.

CHAPTER 1

RELATIONSHIP BETWEEN TERMITE DIET

AND ACETOGENESIS AND METHANOGENESIS BY GUT BACTERIA

30

 

E‘
by funda.
In hindgu
bacteria
reducing
was of 1i
feeding t
feeding c
these fi
anaerobic

methane.

 

 

 

31

ABSTRACT

Evolution of different dietary habits in termites is paralleled
by fundamental differences in the activity of their hindgut microbiota.
In hindguts of wood- and grass-feeding termites, COz-reducing acetogenic
bacteria generally outcompeted methanogenic bacteria for available
reducing equivalents (presumably H2). By contrast, 112/002 acetogenesis
was of little significance in hindguts of fungus-cultivating and sail-
feeding termites, which evolved more methane than their wood-and grass-
feeding counterparts. Given the large biomass of termites on Earth,
these findings will help refine global estimates of 002 reduction in
anaerobic habitats and the contribution of termites to atmospheric

methane.

 

1a

(1
sc
fe
ce

it

(1.

32

Termites are unevenly distributed over two-thirds of the earth's
land surface, whereupon they are important decomposers of plant biomass,
but may also incidentally cause extensive damage to dwellings and crops
(1,2). Although perhaps best recognized for their ability to feed on
sound wood, certain termite species obtain most of their nutrition by
feeding on soil (humus) (2,3). Others cultivate and consume
cellulolytic fungi which, when ingested with plant matter, provide
important digestive enzymes for the insect (3,4). Nevertheless, a
property shared by all termites examined thus far is the presence of a
dense and diverse community of microbes in their hindgut (3,5). Indeed,
the digestion of lignocellulosic food resources by termites appears to
involve their gut microbiota, which includes bacteria and anaerobic,
cellulolytic protozoa in phylogenetically "lower" termites, and bacteria
alone in phylogenetically "higher” termites (3,5).

The contribution of gut microbes to termite digestion has
recently been investigated in detail for the North American wood-feeding
lower termite, Reticulitermes flavipes. Odelson and Breznak (6)
demonstrated that acetate was the major product of microbial
fermentation of wood polysaccharides (principally cellulose) in the
hindgut. Subsequent oxidation of microbially produced acetate by
termite tissues (Eq. 1) could support the entire respiratory requirement

of R. flavipes. A model was proposed to describe the major metabolic

CHBCOOH + 2 02 ---> 2 C02 + 2 H20 (1)

interactions among gut microbes of lower termites resulting in acetate

production. This model suggested that cellulolytic protozoa ferment

33

each glucosyl unit of cellulose to acetate, H2 and COZ, [Eq. 2;

demonstrated by using mixed cell suspensions as well as axenic cultures

of termite hindgut protozoa (7)]. The H2 and CO2 were then believed to

C6H1206 ---> ZCH3COOH + 4H2 +2C02 (2)

(Cellulolytic protozoa)

be consumed by H2/002 acetogenic bacteria to form one additional acetate

(Eq. 3; 6). H and CH4 [the later of which is formed via CO reduction

2 2
by methanogenic bacteria (Eq. 4; 6,8,9)] are also emitted by lower
termites, but represent only a minor fate of reducing equivalents (i.e.
H+ + e-) generated during wood polysaccharide fermentation (6). This

model was confirmed by Breznak and Switzer (10) who showed that

4H2 + 2C02 ---> CH3COOH + 2H20 (3)

(HZ/CO2 acetogenic bacteria)

4H2 + 002 ---> CH4 + 2H20 (4)

(Methanogenic bacteria)

H2/002 acetogenesis (Eq. 3) was also the main H2-consuming (i.e.
”electron sink”) reaction in hindguts of three lower and two higher,
wood-feeding termites. Moreover, pure cultures of H2/CO2 acetogenic
bacteria have recently been isolated from termites and studied in detail

(20, Chapters 2 and 3).

To assess the validity of this model for termites in general, we

34

recently examined a variety of tropical species that represented
different patterns of food resource preference. Opportunities to sample
freshly collected specimens of many of these species (especially those
from remote regions) were rare, and therefore our efforts concentrated
on examining a wide variety of species rather than different samples of
one or a few species. Termites used in this study included wood-feeding
members of three lower termite families (Hodo-, Kalo- and
Rhinotermitidae) and wood-feeding, grass-feeding, fungus-cultivating and
soil-feeding representatives of the higher termite family, Termitidae
(3,13). Herein we show that the ability of Hz/CO2 acetogenic bacteria
to outcompete methanogenic bacteria for H2 in termite hindguts is a
widespread phenomenon among wood- and grass-feeding termites, but not
among soil-feeding or fungus-cultivating termites which emit
considerably more methane than their wood-feeding counterparts (11).

Methane production by the gut microbiota was usually estimated
by measuring methane emission from live termites themselves, a technique
which is noninvasive and minimizes disruption of the insects (6).

However, this technique does not distinguish between H -dependent

2

methane production, and methane produced from other substrates (eg.

acetate), so it may overestimate in situ rates of methanogenesis from H2

+ C02. By contrast, because termite tissues readily oxidize acetate
(6), H2/002 acetogenesis rates were determined by measuring the
reduction of 14CO2 to 1[‘C-acetate by anoxic homogenates of termite gut

contents. This was done under two conditions: with exogenously supplied
H2; and with reductant (presumably H2) produced endogenously by microbes
present in the gut homogenates (12). It is important to note that the

latter condition may seriously underestimate in situ rates of H2/602

 

ace'
pro]

mic:

out<
12

espe
(Tat

14

cont
Wher
forn

incl

aCet
tern
time
Cult
term
rate
Cult

Pres

35
acetogenesis, because homogenization and dilution of gut contents
probably disrupt important physical interactions between H2-producing

microbes and HZ-utilizing acetogenic bacteria (15).

H2/002 acetogenesis generally, and sometimes completely,
outcompeted methanogenesis as the main Hz-consuming process in guts of
12 (of 13) wood-feeding termites and one grass-feeding termite,

especially when measured in the presence of exogenously supplied H2

(Table 1). For wood- and grass-feeding termites, the grand mean rate of

14C-acetate formation from ll‘COZ (using endogenously produced H2) by gut

contents was 3-fold greater than the grand mean rate of CH4 emission.

When supplied with exogenous H the grand mean rate of 14C-acetate

2’

formation from 14002 by gut contents of wood- and grass-feeding termites

increased to 10-fold greater than the grand mean rate of CH emission.

4
By contrast, methane emission rates were greater than rates of Hz/CO2
acetogenesis for 3 fungus-cultivating, and 5 (of 6) soil-feeding
termites (Table 1). Grand mean rates of methane emission were 2 to 7
times greater than grand mean rates of 14C-acetate formation for fungus-
cultivating termites and 5 to 12 times greater for soil-feeding
termites. As with wood-feeding termites, individual and grand mean
rates of 14C02 reduction to 14C-acetate by gut contents of fungus-
cultivating or soil-feeding species were usually increased by the
presence of exogenously supplied H2.

Comparison of these hydrogenotrophic bacterial processes between
termite dietary groups indicated that grand mean rates of 14CO2
reduction to 1l‘C-acetate by gut contents from wood- and grass-feeding

termites (with, or without exogenously supplied H2) were significantly

greater than those of fungus-cultivating or soil-feeding termites (Table

36
14 14
Table 1. Reduction of CO to C-acetate by termite gut contents, and

CH4 emission by live termites of different dietary guilds.

 

 

 

 

 

 

 

 

 

 

 

pmol product x g termite.1 x h"1
14C-Acetatea
From From
exogenously endogenously CH4 b
Termite and Diet supplied H2 produced H2 Emitted
wooa-rasnmc TERMITESC:

Cbptotermes formosanus 1.66 0.10 0.01d

Prarhinotermes simplex 1.18 0.57 n.d.

Pterotermes occidentis 2.07 0.48 0.00

Reticulitermes flavipes 0.93 1 0.43 0.09 1 0.06 0.10

Zootermopsis angusticollis 0.33 1 0.25 0.07 1 0.02 1.30

Amitermes sp. 5.16 1.03 0.13

Gnathamitermes perplexus 1.83 0.13 0.21

Microcerotermes parvus 4.96 1 1.34 1.16 1 0.98 0.14

Nasutitermes arborum 2 29 3.00 0.13

Nasutitermes costalis 5.96 0.99 n.d.

Nasutitermes 1ujae 1.91 0.13 0.15

Nasutitermes nigriceps 3 68 0.89 0.24

Tenuirostritermes tenuirostris 0 98 0.05 0.11
GRASS-FEEDING TERMITE:

Trinervitermes rhadesiensis 2.70 2.38 0.18
cm m‘: 2.54 1 0.47 0.79 1 0.24 0.23 1 0.10
FUNGUS-CULTIVATING TERMITES:

Macrotermes mulleri 0.05 0.01 0.25

Pseudacanthotermes militaris 0.23 0.16 0.67

Pseudacanthotermes spiniger 0.17 0.01 0.36
GRAND MEAN: 0.17 1 0.03 0.06 1 0.05 0.43 1 0.13
SOIL-FEEDING TERMITES:

Crenetermes albotarsalis 0.05 0.02 0.93

Cubitermes fungifaber 0.56 0.21 0.48

Cubitermes speciosus 0.02 1 0.01 0.01 1 0.01 0.85

Noditermes sp. 0.03' 0.05 0.64

Procubitermes sp. 0.05 0.03 0.39

Thoracotermes macrothorax 0.07 0.01 1.09

 

 

 

GRAND MEAN: 0.13 1 0.10 0.06 1 0.03 0.73 1 0.11

 

 

Table

37
Table 1 (continued).

 

8Results are mean values of duplicate analyses for n - 1 except for the
following species, which are mean values of duplicate analyses for n as
indicated: R. flavipes, n - 20; Z. angusticolis, n - 3; M. parvus, n -
3; N. lujae, n - 2; C. albotarsalis, n - 2; C. speciasus, n - 3. For
results where n > 3, results are mean values + standard deviation (14).

b Determined essentially as described previously (6), using mean values
of duplicate analyses for n - 3 to 5.

cThe first five species listed are classified as "lower" termites, and
have a hindgut microbiota consisting of bacteria and cellulolytic,
flagellate protozoa. The remaining species are classified as ”higher"
termites, and have a hindgut microbiota consisting only of bacteria (3).

dn.d., not determined.

eGrand means are + standard error (14) and were calculated by using all

of the individual analyses for each dietary group.

38

1; 14). By contrast, grand mean rates of methane emission by soil-
feeding, and, to a lesser extent, fungus-cultivating termites, were
significantly greater than those of wood-feeding termites. It was also
noteworthy that gut contents from all wood- and grass-feeding termites
tested displayed readily detectable levels of H2/CO2 acetogenic
activity, but one fungus-cultivating species, and 5 soil-feeding species
exhibited almost no H2/CO2 acetogenesis, even when supplied with
exogenous H2. Conversely, all fungus-cultivating, and soil-feeding
species examined evolved relatively high amounts of methane, but two
wood-feeding species (P. occidentis, and C. formosanus) evolved little
or none.

It might be argued that the relatively low rate of methane
emission from wood-feeding termites is due to aerobic oxidation of this
gas before it emanates from the insect. However, kinetic analysis of O2
consumption by intact termites suggests that this is not the case (6).
Moreover, we have measured rates of 14CO2 reduction to 14CH4 by anoxic
gut contents from the following wood-feeding species: R. flavipes, Z.
angusticollis, M. parvus, N. Iujae and N. nigriceps; and from the soil-

feeding termite, C. speciosus (10,16). In all cases, rates of 14CO2

reduction to 1(‘CHA by termite gut contents were less than those of

methane emission by live termites, even when supplied with exogenous H2.

In addition, previously published rates of 14CO2 reduction to IACHA by

gut contents from the wood-feeding P. simplex and N. costalis were

considerably less than rates of 14C-acetate formation from 14002 (10).

Unfortunately, due to logistical limitations we were unable to determine

rates of 14CO2 reduction to 14CH4 by gut contents from fungus-

cultivating species or other soil-feeding species.

39

It is not surprising that animals with anaerobic, fermentative
microbial communities in their alimentary tract evolve methane. A
classic example of this is the bovine rumen microbiota which evolves up
to 200 liters CH4 per animal per day (22). However, the ability of
H2/CO2 acetogens to outcompete methanogens for H2 in the guts of wood-
feeding termites [and in certain other habitats including the colon of
some humans (19)], is enigmatic. Thermodynamic and kinetic
considerations suggest that in environments low in sulfate and nitrate,
CO reduction to methane (not acetate) should be the dominant, terminal

2

Hz-consuming process (8,18). Methanogenesis from H2 + 002 yields more
energy (-135.6 kJ/reaction) than does acetogenesis (-104.6 kJ/reaction)
(8), and the threshold of H2/C02 acetogens for H2 is more than 10-fold
greater than that of methanogens (18). Moreover, the apparent K.m for H2
uptake by H2/C02 acetogenic bacteria is in the same range as (not
unusually lower than) that of many methanogens (20). Clearly, factors
other than thermodynamics and affinity for H2 must be important to the
success of H2/CO2 acetogens in ecosystems such as the guts of wood-
feeding termites.

In an effort to understand why H2/002 acetogenesis outcompetes
methanogensis in the guts of wood-feeding termites, we have isolated in
pure culture three strains of H2/002 acetogenic bacteria - one each from
the guts of a higher and lower wood-feeding termite, and one from the
gut of a soil-feeding termite (20, Chapters 2 and 3). Results indicate
that these isolates each represent new and different bacterial species

and we are currently studying various aspects of their nutrition and

physiology which may bear on their ability to compete for H2 in termite

guts (20).

40

Other investigators have suggested that CH4 emission from
termites is an important source of atmospheric methane. Estimates of
termite contributions have ranged from less than 5 percent to greater
than 40 percent of total annual global methane production (21). We feel
that such estimates must be viewed with caution, because of
uncertainties associated with global estimates of termite numbers and
activities. Moreover, previous estimates were made without information
on rates of acetogenesis and methanogenesis for termites of different
dietary groups. From the present study it appears that, due to the
hydrogenotrophic activity of acetogenic bacteria in hindguts, wood- and
grass-feeding termites typically evolve less than 10 percent of the
amount of methane that they might otherwise produce. By contrast,
fungus-cultivating and soil-feeding termites evolve significantly more
CH4 per termite than do termites of the former dietary groups. As
global estimates of populations of specific termite dietary groups
become more reliable, these data will help to refine our understanding

of the contribution of termites and their gut microbes to global methane

production and 602 reduction in anaerobic habitats.

9.

10.

11.

12.

41
REFERENCES AND NOTES

. R. Edwards and A.E. Mill, Termites in Buildings: Their Biology and

Control (Rentokil Ltd., W. Sussex, 1986)

. T.G. Wood, in Production Ecology of Ants and Termites, M.V. Brian,

Ed. (Cambridge University Press, Cambridge, 1978), pp. 55-80; T.G.
Wood and W.A. Sands, ibld, pp.245-292.

. Ch. Noirot and C. Noirot-Timothee, in Biology of Termites, Vol. I, K.

Krishna and F.M. Weesner, Eds. (Academic Press, New York, 1969),
pp.49-88; W. A. Sands, ibid, pp. 495-524.

. M.M. Martin, Invertebrate-Microbial Interactions: Ingested Fungal

Enzymes in Arthropod Biology (Cornell University Press, Ithaca) pp.
1-36.

. J.A. Breznak, Ann. Rev. Microbiol. 36, 323 (1982); ________,

in Invertebrate-Microbial Interactions, J.M. Anderson et al.,
(Cambridge University Press, Cambridge, 1984) pp. 173-203: ,
in Microbiology of Poecilotherms, R. Lesel, Ed. (Elsevier, Amsterdam,
1990), in press.

D.A. Odelson and J.A. Breznak, Appl. Environ. Microbiol. 45, 1602
(1983).

M.A. Yamin, ibid, 39, 859 (1980); , Science 211, 58 (1981);
D.A. Odelson and J.A. Breznak, Appl. Environ. Microbiol. 49, 614
(1985).

. J.Dolfing, in Biology of Anaerobic Microorgansisms, A.J.B. Zehnder,

Ed., (John Wiley & Sons, New York, 1988) pp.4l7-468.
D.A. Odelson M.D. Kane and J.A. Breznak, unpublished.

J.A. Breznak and J.M. Switzer, Appl. Environ. Microbiol. 52, 623,
(1986).

Results for a few individual termite species were previously
published as portions of a separate study ([10], and are included in
two reviews in press: J.A. Breznak and M.D. Kane, FEMS Microbiol.
Rev. [1990]; A. Brauman et al., in Microbiology and Biochemistry of
Strict Anaerobes Involved in Interspecies Hydorgen Transfer, [Plenum
Publishing Corp., 1990].

All experiments were performed in East Lansing, Michigan on freshly
collected or laboratory-maintained specimens (R. flavipes), or
within 48 h of receipt of specimens provided as indicated (13),
except for termites from Peoples' Republic of Congo, all of which
were assayed recently in Brazzaville from freshly collected nests.
The H /CO acetogenesis assay has been described in detail
previously (10) and is only summarized here. Guts from 20 to 60
termites were removed by using an anaerobic chamber and pooled in an
anoxic buffered salts solution prior to homogenization. Reaction
vials (8-ml) had a final liquid volume of 0.5ml, and contained

 

13.

14.

15.

16.

17.

18.

19,

1.2
dpm/
atmo
of
ad
1600
the
prod

Tern
Lake
Gulf

Ami:
area
Nutt

angu

Braz
albc

cost
Less
Niar
Braz
fie]

Sig
com;
Prir
Edit
meax
of <
Whe1
def:

det

13.

14.

15.

l6.

17.

18.

19.

42

1.2 pmol NaHIACO , pH 7.2 (approx. specific activity, 6.5 x 105
dpm/umol) and the contents of 2 to 4 homogenized termite guts. The
atmosphere in reaction vials consisted of 100 N (for determination
of rates of C-acetate formation fro C by endogenously
pgoduced H ) or 100% H (for rates of C-acefate formation from

CO catalyzed by exogenously supplied H ). After termination of
the reaction, the supernatant fluid was analyzed for C-labelled
products by high performance liquid chromatography.

Termites were collected from the fallwing locations : C. formasanus,
Lake Charles, Louisianna, USA (provided by L. Williams, USDA
Gulfport, MS); P. simplex, Coral Gables, Florida, USA (provided by
G. Prestwich, State Univ. New York, Stonybrook); P. occidentis,
Amitermes sp., G. perplexus and T. tenuirostris, Santa Rita Range
area, southwestern Arizona, USA (collected with the help of W.
Nutting, U. Arizona); R. flavipes, Dansville, Michigan, USA; Z.
angusticolis, San Francisco Bay Park, California, USA (provided by
J. Traniello, Boston U.); M. parvus and N. lujae, forest near
Brazzaville, Peoples' Republic of Congo; N. arborum, M. mulleri, C.
albotarsalis, C. speciosus, Naditermes sp., Procubitermes sp. and
T. macrothorax, Mayombe rainforest, Peoples' Republic of Congo; N.
costalis, forest near Frijoles, Panama; N. nigriceps, forest in
Lesser Antilles; T. rhodesiensis and C. fungifaber, savannah near
Niari, Peoples' Republic of Congo; P. militaris, savannah near
Brazzaville, Peoples' Republic of Congo; P. spiniger, sugar cane
fields near Nkayi, Peoples' Republic of Congo.

Significant differences between means were verified by using tests of
comparison for two sample means (R.G.D. Steel and J.H. Torrie
Principles and Practices of Statistics: A Biometrical Approach, 2nd
Edition, [McGraw-Hill, Inc., New York, 1980], pp.86-121). For all
means identified as unequal, a 5 0.05, except for the grand mean rates
of CH emission from wood-feeding termites and soil-feeding termites,
where a 5 0.40. Standard deviation and standard error were used as
defined in ibid, pp. 8-38.

R. Conrad et al., Appl. Environ. Microbiol. 50:595-601.
M.D. Kane, A. Brauman and J.A. Breznak, unpublished.

This mean value is for 20 separate determinations, and is slightly
less than that previously reported (10) which was based on 6
determinations.

R. Cord-Ruwisch et al., Arch. Microbiol. 149, 350 (1988); R. Conrad
et al., FEMS Microbiol. Ecol. 38,353 (1986).

R. A. Prins and A. Lankhorst, FEMS Microbiol. Lett. 1, 255 (1977);
T.J. Phelps and J.G. Zeikus, Appl. Environ. Microbiol. 49, 1088
(1984); J.G. Jones and B.M. Simon, Appl. Environ. Microbiol. 49,
944-948 (1985); S. F. Lajoie et al., Appl. Environ. Microbiol. 54,
2723 (1988); R. Conrad et al., FEMS Microbiol. Ecol. 62, 285 (1989).

JJJM

O
2

PMCN<CEF

1
2

22.

 

43

20. J.A. Breznak et al., Arch. Microbiol. 150, 282 (1988); M.D. Kane and

21.

22.

J.A. Breznak, Abstr. Ann. Meet. Amer. Soc. Microbial. p.234 (1989);
J.A. Breznak and J.M. Switzer, Abstr. Ann. Meet. Amer. Soc.
Microbial. p.160 (1989).

P.R. Zimmerman et al., Science 218, 563 (1982); R.A. Rasmussen and
M.A. Kahill, Nature 301, 700 (1983); P.R.Zimmerman and J.P.
Greenberg, Nature 302, 354 (1983); M.A. Kahill and R.A. Rasmussen,
Nature 302, 355 (1983); N.M. Collins and T.G. Wood, Science 224, 84
(1984); P.R. Zimmerman et al., Science 224, 86 (1984); W. Seiler, in
Current Perspectives in Microbial Ecology, M.J. Klug and C.A. Reddy,
Eds., (American Society for Microbiology, Washington, D.C., 1984)
pp.468-477; P.J. Fraser et a1., J. Atmos. Chem. 4, 295 (1986).

M. J. Wolin, in Advances in Microbial Ecology, Vol. 3, M. Alexander,
Ed., (Plenum, New York, 1973) pp.49-77.

CHAPTER 2
H2/002 ACETOGENIC BACTERIA FROM TERMITE GUTS:
I. Acetonema elongata, GEN. NOV., SP. NOV.,
A GRAM NEGATIVE, ENDOSPORE-FORMING BACTERIUM

FROM Pterotermes occidentis

44

45

Abstract

Strain APO-1, a previously undescribed HZ/CO2 acetogenic
bacterium, was isolated from gut contents of the wood-feeding termite
Pterotermes occidentis. Cells of strain APO-l were anaerobic, Gram
negative, endospore-forming, motile rods which measured 0.30-0.40 x 6-60
pm. Cells were catalase positive, oxidase negative, and had 51.5 mol
percent G + C in their DNA. Optimum conditions for growth on H2 + CO2
were at 330 C and pH 7.8, and under these conditions cells produced
acetate according to the following stoichiometry: 4H + 2C0 --->

2 2
CH COOH + 2H 0. Other substrates supporting good growth of strain APO-1

3 2
included glucose, ribose, and various organic acids. The major
fermentation products were usually acetate and butyrate, but with
certain substrates other products (eg. propionate, succinate, 1,2-
propanediol) were also formed. Based on comparative analysis of 16S
rRNA nucleotide sequences, strain APO-1 was somewthat related to members
of the genus Sporomusa (another genus of Gram negative, endospore-
forming acetogens). However, the morphological and physiological
differences between strain APO-l and the six known species of Sporomusa
were significant. Consequently, it is proposed that a new genus,
Acetonema, be established, with strain APO-1 as the type strain of the
new species, Acetonema elongate. This newly described bacterium could
potentially contribute to the nutrition of P. occidentis termites by

forming volatile fatty acids (eg. acetate, propionate and butyrate)

which are important carbon and energy sources for the insect.

46
Introduction

Microbial fermentation of wood polysaccharides in the hindgut of
phylogenetically "lower" termites such as Reticulitermes flavipes (which
harbor both bacteria and anaerobic, cellulolytic protozoa) has been
described as essentially a homoacetic fermentation of cellulose; i.e., n
C6H1206 ---> n 3CH3COOH (Odelson and Breznak, 1983). The cellulolytic
protozoa ferment each glucosyl unit of cellulose to 2 acetate + 4H2 +
2C02. An additional acetate is formed from each 4 H2 + 2 CO2 by
bacterial H2/C02 acetogenesis. The latter process outcompetes
methanogenesis as the major ”electron sink" reaction in the hindguts of
almost all "lower" termites examined to date, as well as in the hindgut
fermentation of all wood-feeding "higher" termites (which harbor only
bacteria in their hindguts) (Breznak and Switzer, 1986; Chapter 1). In
an effort to understand why this is so, attempts have been made to
isolate H2/CO2 acetogenic bacteria from termite guts for further study.
Recently, the H2/CO2 acetogen Sporomusa termitida was isolated from the
"higher" wood-feeding termite Nasutitermes nigriceps, (Breznak et a1.
1988).

The present report describes the isolation and characterization
of strain APO-1, an H2/002 acetogenic bacterium isolated from the guts
of the "lower" wood-feeding Pterotermes occidentis. Although strain
APO-1 resembled members of the genus Sporomusa in being a Gram negative,
endospore-forming, H2/002 acetogen, its morphology was distinctly
different from known species of Sparomusa, which are more or less
uniform in cell size and shape. Moreover, physiological and molecular

phylogenetic analyses prompted us to propose strain APO-l as the type

strain of a new genus and species, Acetonema elongata.

47
[A preliminary report of these findings has been presented (M.
D. Kane and J. A. Breznak, Abstr. Annu. Meet. Amer. Soc. Microbiol.

1989, 1100, p.234)].

Materials and Methods
Termites

P. occidentis (Walker) (Kalotermitidae) was collected in the
vicinity of the Santa Rita Range area near Tuscan, Arizona, USA. They
were used within 72 hours of collection. Methane emission from live P.
occidentis worker termites was determined as described previously

(Odelson and Breznak, 1983).

Bacteria
Sporomusa termitida strain JSN-2 was described previously
(Breznak, et al., 1988). Clostridium mayombeii strain SFC-S is described

in a companion paper (Chapter 4).

Isolation of strain APO-l

An anoxic homogenate of guts of P. occidentis worker termites
was prepared with a buffered salts solution (BSS) as described
previously (Breznak and Switzer, 1986). Serial ten-fold dilutions of
gut homogenate were made in B88 and then inoculated into l8-mm serum-
stoppered tubes containing 10.0 ml of anoxic AC-Kl or AC-K2 medium. AC-

Kl medium was a COZ/NaHCO -buffered medium containing inorganic salts,

3

vitamins, yeast extract (0.5 g/l) and resazurin. It was identical to

AC-l9 medium (Breznak et al., 1988) except for the addition of

48
trypticase (BBL, Baltimore, MD; 2.0 g/l, and clarified bovine rumen
fluid (50 ml/l). AC-K2 medium was identical to AC-Kl except that the
methanogenesis inhibitor 2-bromoethane-sulfonate (BES; filter sterilized
separately) was included at a final concentration of 50 mM. Tubes were
incubated horizontally on a reciprocal shaker (100 rpm) at 300 C with
1.0 atm of H2/CO2 (80/20, vol/vol) in the headspace. From the highest
dilution tube of AC-K2 medium showing depletion of H2/C02 and production
of acetate, roll tubes were prepared using AC-K2 medium solidified with
2% agar (Hungate, 1969). Isolated colonies were picked from roll tubes
that had consumed H2/C0 and were reinoculated into AC-K2 broth to test

2

for Hz/CO consumption and acetate production. Broth tubes showing the

2
greatest depletion of gas and production of acetate were chosen for
repeated passage in agar roll tubes. Cultures were considered to be
pure after three successive passages in roll tubes, at which time phase
contrast microscopy revealed a single morphological type. They were
then transferred to AC-Kl medium (lacking BES) for routine culture at

O

30 C in 18-mm serum-stoppered tubes containing 10 ml medium and a

headspace of H2/CO2 (80/20, vol/vol).

Growth and nutrition studies

AC-K3 basal medium was used for growth and nutrition studies.
It was identical to AC-Kl medium, except that yeast extract and
clarified rumen fluid were omitted. The medium was prepared under an

0 -free gas phase containing 20% CO and had a final pH of 7.2, except

2 2

where the pH was adjusted by varying the COZ/NaHCO3 ratio (Costilow,

1981) for determination of optimum pH for growth. For studies of

substrate utilization, AC-K3 basal medium was dispensed into sterile,

49

l8-mm screw cap tubes containing a predetermined amount of the substrate
to be tested. Tubes were filled completely with AC-K3 basal medium so
as to leave essentially no gas in the headspace. Molar growth yields of
cells were estimated as described previously (Breznak et al., 1988).

Growth of cells was measured by determining the absorbance of
cultures at 600 nm with a Bausch & Lamb Spectronic 20 calorimeter, or a
Beckman DU spectrophotometer. Cell dry mass determinations were

performed as previously described (Breznak et al., 1988).

Fermentation studies

The fermentation stoichiometry of cells growing with H2/C0 was

2
done by using bottles containing 245 ml AC-K3 basal medium and a 465 m1
gas phase of 112/002 (80/20, vol/vol). Fermentations of glucose,
rhamnose and potassium fumarate were done by growing cells under 100% N2

in basal AC-K3 medium modified by omitting NaHCO3 and including 3-(N-
morpholino) propanesulfonic acid buffer (adjusted to pH 7.4 and filter
sterilized separately ) at a final concentration of 10 mM. Material
balance calculations were corrected for the small amount of products

formed by cells growning in modified AC-K3 medium containing no

additional substrate.

Sequencing of 16S ribosomal RNA

Total nucleic acids were extracted from ca. 200 mg of cells by
using hot phenol and were precipitated from the aqueous phase with
ethanol. Nucleotide sequences were determined by the dideoxynucleotide
- reverse transcriptase method of Lane et a1. (1985). Nearly complete

sequences were obtained by using 16S rRNA as the template and starting

50

reactions with one of seven oligonucleotide primers complimentary to
universally conserved regions of the 168 rRNA (Montgomery et al., 1988).
The sequences for Sporomusa termitida strain JSN-2, Acetonema elongate
strain APO-1 and Clostridium mayombeii strain SFC-5 have been deposited
with GenBank. They can also be obtained by writing us directly.

Sequence similarities and evolutionary distances were determined
in collaboration with D. A. Stahl (Univ. Illinois, Urbana, USA) by the
methods of Olsen et al., (1986) with the modifications of Montgomery et
al., (1988). Unpublished sequences for the 16S rRNA of Megasphaera
elsdenii and Sporomusa paucivorans were kindly made available to us by

C. R. Woese, Univ. Illinois.

Chemical assays
H2 and CH4 were quantified by gas chromatography (Odelson and

Breznak, 1983). In enrichment cultures, nutritional studies and H2/CO2
growth experiments, acetate production was determined by gas
chromatography of culture supernatant fluids (Breznak and Switzer,
1986). For fermentation material balances, acetate, propionate and
butyrate were determined by high performance liquid chromatography
(HPLC) (Breznak and Switzer, 1986). 1,2-propanediol was also quantified
by using the same HPLC system, wherein it displayed a retention time of
16.75 min. Protein was determined by the Folin reaction (Hanson and
Phillips, 1981). Dipicolinic acid (DPA) was extracted from sporulated
cultures and assayed spectrophotometrically as the calcium chelate

(Lewis, 1967).

Lipopolysaccharide was determined by D. C. White (U. Tennessee,

51
Knoxville, USA) using gas chromatography and mass spectrometry (Parker

et al., 1982)

Other procedures

Measurement of H2-dependent reduction of 14CO2 to 14C-acetate by
cell suspensions, and determination of the distribution of 14C in
acetate by Schmidt degradation, were performed as described previously
(Breznak et al., 1988, Breznak and Switzer, 1986).

Polymyxin B sensitivity was assayed by the method of Wiegel and
Quandt (1982).

For determination of cytochromes, crude cell extract

preparations and Na -reduced minus oxidized spectra were performed

28204
as described previously (Breznak et al., 1988) except that a Gilford
ResponseTM spectrophotometer was used.

DNA base composition was determined by B. Mannarelli (USDA-ARS,
Peoria, Illinois, USA) using the buoyant density method (Mannarelli,
1988). Previously described methods were used for electron microscopy
(Breznak and Pankratz, 1977), fluorescence microscopy (Doddema and
Vogels, 1978), catalase and oxidase tests (Potrikus and Breznak, 1977),

nitrate reduction (Smibert and Krieg, 1981) and sulfide production

(Cline, 1969).

Chemicals
All chemicals were reagent grade and were purchased

commercially.

52
Results
Enrichment and isolation of bacteria
Live worker termites of P. occidentis emitted no detectable CH4.

Therefore, it was not too surprising that successful enrichments for

H2/CO acetogenic bacteria were obtained from gut homogenates inoculated

2
into both AC-Kl medium (lacking BES) and AC-K2 medium. After four
weeks, enrichments exhibited turbidity, negative pressure and acetate
production up to 24 mM (with periodic replenishment of H2/C02) out to
the fourth dilution tube suggesting an initial population of at least
106 H2/CO2 acetogens per ml gut fluid. No methane was produced, and no
Fazo-fluorescent cells were observed by microscopy in either medium.
From enrichments in AC-K2 medium, three strains of H2/CO2 acetogenic
bacteria were obtained. All three strains were similar in morphology

and Gram stain reaction, so one of these (strain APO-l) was chosen for

further study.

Colony and cell morphology

Colonies of H2/CO2 grown cells were 1-2 mm in diameter, circular
with uneven edges, and opaque with a slight brown color. Cells of
strain APO-l were straight, thin rods measuring 0.30-0.40 x 6.0-60.0 pM
(Fig. 1a & b). Cells stained Gram negative, and electron micrographs of
thin sections revealed the presence of distinct inner (cytoplasmic) and
outer membranes characteristic of Gram negative bacteria (Fig. 1c). The
cell wall of strain APO-l cells also produced "bleb"-1ike protrusions
when cells were incubated in the presence of polymyxin B (1580

units/ml), a reaction that Wiegel and Quandt (1982) demonstrated to be

53

Figure 1a-c. Morphology of strain APO-l. a. Phase contrast micrograph;
bar - 10.0 pm. b,c. Transmission electron micrographs of thin sections;
bars -1.0 pm (b) and 0.1 pm (c). Note phase-bright, terminal, spherical
endospores in a and b, and the outer membrane of the cell wall (c,

arrow).

54

 

55

specific for Gram negative bacteria. Moreover, there was an appreciable
amount of lipopolysaccharide associated with cell lipid (D. C. White,
personal communication). Cells also formed spherical, terminal
endospores that markedly swelled the sporangium (Fig la & b). Viable
cells could be recovered from sporulated cultures held at 800 C for 10
min, and DPA extracted from sporulated cultures displayed UV absorption
maxima and minima identical to those of authentic DPA.

The average length of strain APO-1 cells during early log phase

growth on H2/C0 was more than twice the average length of cells at the

2
end of log phase, at which time up to 75% of cells had formed endospores
(Fig. 2). Endospores were present only on cells 5 12 pm and, in
general, such cells were more phase dark than cells of greater length
(Fig. la). Cells grown on organic compounds such as glucose (below)
were generally longer (up to 50 pm), less phase dark and formed fewer
endospores than H2/002 grown cells.

In wet mount preparations cells exhibited little or no motility.
However, when allowed to settle on the surface of an agar-covered slide,

cells displayed rapid motility, presumably due to the presence of

peritrichous flagella which were observed in some electron micrographs.

Growth and nutrition studies

Strain APO-1 was a strict anaerobe. The addition of trypticase
(0.2%) or, to a lesser extent, yeast extract (0.2%) stimulated growth,
but the addition of rumen fluid (5%) had little effect. With glucose as
substrate (below), cells grew within a temperature range of 19 to 400 C
and a pH range of 6.4 to 8.6. Optimum growth in AC-K3 medium, with

H2/CO2 as substrate, occurred at 330 C and pH 7.8. At 300 C with H2/CO2

56

Figure 2. Cell length and endospore formation during growth of strain
APO-l in AC-K3 medium with H2 + CO2 as substrate. During growth
(measured by determining O'D'600nm)’ samples of culture fluid were

periodically removed for phase microscopy to measure cell length and to

determine the percentage of cells with endospores.

57

><mw>0m Om...- szmdi A)? 03 fl. Omrrm 52...: MZUvamem AIV

O O O.

1 1 1

 

138 184

92

 

 

 

 

1
0.01

.0. 6.58.0 .0

TIME (H)
Figure 2.

58

Figure 3. Utilization of H by strain APO-l for growth and acetogenesis.

2
Cells were grown on a shaker at 300 C in bottles containing 245 ml AC-K3
medium and 463 ml gas phase at 1 atm. The gas phase was either H2/CO2
(80/20, vol./vol.; (closed symbols, solid lines) or N2/002 (open

symbols, broken lines).

59

a. In .2 Im>omp>0m any Ox 3: >Om._.>._.m APDV

 

 

 

 

 

 

0
0 o 0.
4| 1 1o
4 a d u 4 4 - - q u d a .— d m
A. 0.. A o
AKIN-11W" A? I... 2
1 .a «II 1
\-\\\r ‘/°~~ AA
\I O A $ .. O
.. v/ ..o a 8
I 0 .d A A I
mk WV ,8 .. 0
I / 4
L p c L L _ _ _ c L a . Air 0
o o 1
... ... o.
o 0

TIME (H)

Figure 3.

60

as substrate cells grew with a doubling time of 36h, achieved a final
O.D.600nm of 0.25 to 0.30 (approx. 100 to 125 pg dry mass/ml), and
produced 18 to 23 mM acetate (without replenishment of H2/C02) (Fig. 3).
During growth the pH of the culture dropped from an initial value of 7.8
to a final value of 6.7. However, growth on H2/CO2 could not be
initiated at 370 C, and growth was slightly suppressed when mid-log
phase cells grown on H2/C02 at 300 C were shifted to 370 C (Fig. 4a).
By contrast, growth of cells on glucose was slightly stimulated by a
similar temperature shift (Fig. 4b).

In addition to growth on H2/C02, strain APO-1 grew
heterotrophically on a range of other compounds (Table l). Butyrate and
acetate were the principle acids produced during fermentation of
mannose, glucose, fructose, rhamnose, ribose, mannitol, pyruvate and
oxaloacetate. However, acetate and propionate were the major acids
produced by fermentation of citrate, fumarate and propanol. 1,2-
propanediol was also a significant product from growth on rhamnose.
Acetate was the sole product detected from growth on ethylene glycol and
trimethoxybenzoate. Detailed balances for fermentations of some of
these substrates are presented below. Molar growth yields of strain
APO-l, determined for selected substrates, were as follows (g dry
mass/mol substrate used): H2 (+ C02), 1.0; glucose, 15.1, rhamnose, 4.2
and fumarate, 1.4. Doubling times of cells grown at 300 C with these
substrates were (h): HZ/COZ’ 36; glucose, 8; rhamnose, 15.5 and
fumarate, 40. Neither sulfate or nitrate were used as electron

acceptors.

61

Figure 4. Effect of temperature on Hz/COZ-grown (A) and glucose grown
(B) cells of strain APO-1. Cells were grown in AC-K3 medium on a rotary
shaker at 300 C (closed symbols, solid lines), and then shifted in mid-

log phase (arrows) to 370 C (open symbols, broken lines).

62

 

1.0 I I I I I I

L L l l

 

E 5" ‘
..
8 ./ q].
0 / ’ I,
(O I, I
O I.
o o 1 o . J, . /’ 1 7/ .
o / ,I j
#40 l7. 7
)- /./ . -4
. -

 

 

 

 

 

0
0.01”: L lgk—‘i L 1 I
40 80 1200 40 80 120
TIME(H)

Figure 4.

63

Table 1. Substrates used for growth by strain APO-la.

 

Used by strain APO-l:

. b
Hz/CO glucose, fructose, mannose, rhamnose, ribose, Citrate

2!
pyruvate, oxaloacetate, fumarate, propanolb, mannitol, ethylene glycolb

and 3,4,5-trimethoxybenzoateb.

Tested, but not used:

Melibiose, raffinose, maltose, cellobiose, arabinose, galactose,
lactose, xylose, sucrose, trehalose, starch, L-fucose, formate, lactate,
malate, D-gluconate, acetate, oxalate, succinate, gallate, syringate,
caffeate, 3-hydroxybenzoate, benzoate, pyrogallol, methanol, ethanol,
glycerol, adonitol, sorbitol, erythritol, butanol, isobutanol, dulcitol,
pectin, xanthine, dextrin, betaine, salicin, esculin, N,N

dimethylglycine and Casamino acids.

 

aCompounds were supplied at a final concentration 5 to 10 mM [except for
Casaminoacids (5 g/l final concentration) and HZ/CO2 (80/20)].

bPoor growth on this substrate.

64
Fermentation balances
The stoichiometry of H2/C02 utilization by strain APO-1 was

consistent with that of other H2/CO2 acetogenic bacteria: 4 H + 2 CO

2 2
---> CH3C00H + 2 H20 (Table 2). Moreover, when H2/CO2 grown cell
suspensions were incubated under H2 with 14CO2 as substrate, cells
formed 85.6 nmol 14C-acetate per h per mg cell protein and the 14C-

acetate was labeled in both carbons (54.3% CH3-group and 61.2% COOH-
group). These results confirmed that strain APO-1 could effect a total
synthesis of acetate from H2 + C02' However, strain APO-1 usually
formed other major products in addition to acetate during heterotrophic
growth on organic substrates. For example, fermentation of glucose
yielded primarily butyrate, C02 and H2 (Table 2) and was somewhat
similar to the butyric fermentation carried out by Eubacterium limosum
and certain clostridia (Kluyver and Schnellen, 1937; Gottschalk, 1986).
The major end products of rhamnose fermentaion were 1,2-propanediol,
acetate, butyrate and succinate. The reason for the relatively poor
carbon recovery during analysis of the rhamnose fermentation (75.9%) is
not yet known, however a similar anomaly sometimes accompanies rhamnose
fermentation by clostridia (Ghazvinizadeh et al., 1972). On the other
hand, fumarate fermentation by strain APO-1 was similar to that of
Sporomusa malonica (Dehning et al., 1989) and was consistent with the
theoretical equation 3 fumarate ---> 2 propionate + acetate + 4C02.

Strain APO-l was apparently also able to oxidize propanol to
propionate and use the reducing equivalents liberated to reduce 002 to
acetate. This reaction was first demonstrated with Acetobacterium

carbinolicum (Eichler and Schink, 1984). However, growth of strain APO-l

was poor on pr0panol, so the fermentation was not investigated in detail.

65
Table 2. Fermentation products formed during growth of strain APO-l

with various substrates.

 

 

 

Substrate Product (mmol/100mmol substrate fermented)
H2 CO2 Acet- Propi- Buty- Succi- 1,2-pro- %C
ate onate rate nate panediol recovery

H2 (+ 50

mmol coz)a - n.d.b 22.7 1.3 0.4 0.0 0.0 101.2
Glucose 88.9 137.0 17.3 10.2 98.4 0.0 0.0 99.3
Rhamnose 16.3 27.5 31.4 0.0 18.4 10.4 83.2 75.9
Fumarate 0.0 131.5 17.0 69.8 0.0 0.0 0.0 93.7

 

aAssumed for calculation of material balance.

n.d., not determined.

Analysis of 163 ribosomal RNA sequences

The nearly complete sequences of 168 rRNAs from strain APO-1,
Sporamusa termitida strain JSN-2 (Breznak et al., 1988), and Clostridium
mayombeii strain SFC-5 (Chapter 4) were compared with those of Sporomusa
paucivorans (Olivier et al., 1985), Megasphaera elsdenii (Rogosa, 1971)
(latter two sequences provided by C. R. Woese, U. Illinois) and the
published sequence of Bacillus subtilis (Green et al., 1985) (Table 3).
Of those organisms examined, the closest relatives to strain APO-l were

S. paucivorans and S. termitida. However, the evolutionary distance

66
between the two Species of Sporomusa (0.051) was significantly less than
the evolutionary distance between strain APO-l and S. paucivorans

(0.114) or S. termitida (0.133).

Table 3. Evolutionary distance between strain APO-1 and other selected

 

 

 

eubacteria.
Evolutionary distance8 from:
Organism S. S. M. B. C.
paucivorans termitida elsdenii subtilis mayombeii

Strain APO-1 0.114 0.133 0.152 0.166 0.214
S. paucivorans 0.051 0.143 0.162 0.178
S. termitida 0.145 0.185 0.209
M. elsdenii 0.192 0.216
B. subtilis 0.178

 

a0.1 evolutionary distance unit is equivalent to 0.1-nucleotide changes

per position.

The phylogenetic tree resulting from these comparisons is
presented in Appendix I. The analysis used to construct the tree in

Appendix I included several other unpublished sequences kindly provided

67
by C.R. Woese. Strain APO-1 is related to a broadly defined, but
phylogenetically coherent group that includes 3. termitida, S.
paucivorans and Megashpaera elsdenii. Analysis of 168 rRNA
oligonucleotide catalogues has shown that bacteria in this group (which
also includes Sporomusa ovata, Sporomusa sphaeroides and Selenomonas
ruminantium) are not related to any Gram negative eubacteria, but show a
distinct (although remote) relationship to bacteria in the "Clostridium"

subdivison of Gram positive eubacteria (Stackebrandt et al., 1985).

Other characteristics
Cells were catalase positive, but oxidase negative. Cytochromes
were not detected in crude cell extracts. The DNA base composition of

strain APO-l was 51.5% G + C.

Discussion
Taxonomy of strain APO-l
The ability to obtain energy for growth by formation of acetate

from H + C0 is found among several bacterial genera including:

2 2
Acetitomaculum, Acetoanaerobium, Acetobacterium, Clostridium,
Desulfotomaculum, Eubacterium, Peptostreptococcus and Sporomusa

(Ljungdahl, 1986). Therefore, this distinctive physiological property
is not highly determinative from a taxonomic standpoint. By contrast,
possession of a truly Gram negative cell wall coinciding with the
ability to form endospores is rare in a single bacterial taxon. Aside
from the thermophilic halophile Sporohalobacter (Oren et al., 1987), the

only bacteria currently known to exhibit both of these properties are

68
H2/CO2 acetogens of the genus Sparomusa. However, morphological and
physiological differences between strain APO-l and members of the genus
Sparomusa are significant (Table 4).

Cells of Sparomusa are all curved rods of moderate diameter and
no longer than 8pm. All, except S. paucivorans, form endospores which
can be either subterminal or terminal in each species. By contrast,
cells of strain APO-l are thin, straight rods which are somewhat
flexible when very long (20-60 pm). Endospores of strain APO-l cells
are always terminally located. Wet mounts of Sparomusa cells exhibit
obvious tumbling motility due to the presence of lateral flagella.
However, cells of strain APO-l were only noticably motile when placed on
the surface of agar covered slides.

Formate and methanol are metabolized by all six Sparomusa
species, the latter compound supporting especially good growth, however
neither of these substrates was utilized by strain APO-l (Table 4). In
addition, all sporomusas tested do not use sugars other than fructose
(except S. acidovorans, which also uses ribose), but strain APO-l used
hexoses (fructose, glucose, mannose), ribose, and the methyl pentose
rhamnose. Moreover, when utilizing sugars and certain organic acids,
strain APO-l formed butyrate as a major fermentation product, whereas
the fermentations of sporomusas are almost always homoacetogenic and
never result in the formation of significant amounts of butyrate.

The G + C content in the DNA of strain APO-l was about 3 mol
percent higher than that of the highest Sparomusa. Furthermore, the
evolutionary distance between strain APO-1 and either S. paucivorans or

S. termitida (as determined by comparative rRNA sequencing) was more

69

Table 4. Characteristics useful for distinguishing strain APO-l from

members of the genus Sparomusa.

 

 

Property Strain APO-1 All Sparomusa speciesa
Cell shape: thin, straight rods curved rods
Length (pm): 6-60 2-8

Width (pm): 0.3-0.4 0.4-0.9

Cell wall composition: Gram negative Gram negative
Endospore location: terminal subterminal-terminalb

Growth on:

H2 + CO2 + +
Glucose + -
Mannose + -
Formate - +
Methanol - +
Butanol - +C
Betaine - +c
Mal % G + C in DNA: 51.5 41.3-48.6

 

aInclusive Sparomusa species described to date: S. sphaeroides and S.

ovata (Moller et al., 1984); S. acidovorans (Ollivier et al., 1985); S.

paucivorans (Hermann et al., 1987); S. termitida (Breznak et al., 1988)
b

and S. malonica (Dehning et al., 1989). S. paucivorans does not form

c .
endospores. Not determined for S. ac1dovorans.

70
than twice the evolutionary distance between S. paucivorans and S.
termitida (Table 3). Although strain APO-1 is, in fact, somewhat
related to these two Sparomusa species as determined by 168 rRNA
sequence comparison, there are as yet no specific rules by which
evolutionary distance measurements can be used to delimit different
genera or species. Therefore, in light of the physiological and
distinctive morphological differences between strain APO-1 and members
of the genus Sparomusa, and taking into account the evolutionary
distances inferred by comparative 16S rRNA sequencing, it is herewith
proposed that a new genus, Acetonema, be established, with strain APO-l

as the type strain of the new species Acetonema elongata.

Ecological considerations

Although H2/C02 acetogenesis outcompetes methanogenesis for H2
produced during the hindgut microbial fermentations of wood-feeding
termites, most termites emit some CH4 (Chapter 1). Furthermore,
enrichments for Hz/COZ-utilizing bacteria from the guts of wood-feeding
termites often result in methanogenic bacteria overgrowing acetogens,
unless BES is added to the medium (Kane and Breznak, unpublished
results). However, an interesting feature of P. occidentis, the

termite used for this study, was that H2/CO acetogenesis occurred in

2
the gut of this termite species to the virtual exclusion of

methanogenesis (Chapter 1). Moreover, no Fazo-fluorescent cells were

observed in P. occidentis gut contents or in H2/CO2 enrichments from gut

contents, and no CH4 was produced in such enrichments (even in medium

without BES). However, cells similar in morphology to A. elongate were

71

observed in gut contents of P. occidentis and appeared to dominate the
Hz/COZ-utilizing enrichments.

It seems likely that A. elongata contributes to the nutrition of
P. occidentis termites by forming volatile fatty acids (eg. acetate,
propionate and butyrate) which are important carbon and energy sources
for the insect (Breznak and Odelson, 1983; Obrien and Breznak, 1984).
In R. flavipes termites, acetate derived from H2/CO2 acetogenesis by gut
bacteria could support up to 1/3 of the energy requirements of the
insect (Breznak and Switzer, 1986). Rates of H2/C02 acetogenesis by P.
occidentis gut homogenates (with or without exogenously supplied H2)
were, in fact, more than twice those of R. flavipes gut homogenates
(Chapter 1). However, culture methods may be ineffective in enumerating

H2/CO acetogenic bacteria such as S. termitida or A. elongata in

2
termite gut contents (Breznak et al., 1988). Consequently, we are
investigating the possibility of using 168 rRNA-directed hybridization
probes (Stahl et al., 1988), or analysis of membrane phospholipid fatty
acid "signitures" (Guckert and White, 1986) as means of assessing

populations of Gram negative endospore-forming H2/CO acetogens in vivo.

2
Description of Acetonema elongata, gen. nov. sp. nov.

Acetonema gen. nov. (A.ce.to.ne'ma. L.n. acetum vinegar; M.L.n.
nema thread; M.L.neut.n. Acetonema vinegar-forming thread)

Thin, straight rods with rounded ends. Motile, multi-
flagellate, but specific location of flagella insertion remains to be
confirmed. Gram negative by staining and by cell wall ultrastructure.
Lipopolysaccharide is present. Heat resistant endospores formed.

Catalase positive, oxidase negative.

72

Strict anaerobes. Chemoorganotrophs. Ferment H2 +CO2 to
acetate. Sugars and organic acids are preferred substrates, from which
butyrate is usually also a major product.

Do not respire anaerobically with nitrate or sulfate.

Cytochromes not detected. Mesophiles. DNA base composition:

51.5 mol% G + C (for the type species) Type species: Acetonema

elongata. Acetonema elongata sp. nov. (e.lon'ga.ta.

L.fem.part.adj. elongata
elongated, stretched out). Straight rods with round ends, 0.3-0.4 x 6-

60 pm. Cells single or in
short chains of 3 or 4. Long cells can be highly flexible.
Translational motility only observed for cells placed on agar-covered
slides, not in wet mounts; more than one flagellum present. Mature
endospores are 1 pm in diameter, spherical and terminal in location.
Endospores resist heating to 800 C for 10 min. Colonies of cells grown
on H2 + C02 are 1-2 mm in diameter, circular with uneven edges and
opaque with a slight brown color.

Strict anaerobe. Oxidase negative, catalase positive. Can obtain
energy for growth by acetogenesis from H2 + C02. Also ferments mannose,
glucose, fructose, rhamnose, ribose, mannitol, pyruvate and oxaloacetate
to butyrate and acetate, but ferments citrate, fumarate and propanol to
acetate and propionate. 1,2-propanediol is also a quantitatively
significant product from fermentation of rhamnose. Acetate was the sole
product detected from ethylene glycol and trimethoxybenzoate. H and

2

002 may be produced during growth on organic compounds.

73
pH optimum, 7.8 (range 6.4 to 8.6); temperature optimum, 330 C
(range 19 to 400 C). Trypticase or yeast extract required for good
growth. DNA base composition: 51.5 mol% G + C (strain APO-1; buoyant
density).

Source: Gut contents of the termite Pterotermes occidentis.

Type strain: Strain APO-1.

74
References

Breznak JA, Pankratz HS (1977) In situ morphology of the gut microbiota
of wood-eating termites [Reticulitermes flavipes (Kollar) and
Captotermes formosanus (Shiraki)]. Appl Environ Microbiol 33:406-
426

Breznak JA, Switzer JM (1986) Acetate synthesis from H plus CO2 by
termite gut microbes. Appl Environ Microbiol 52:623-630

Breznak JA, Switzer JM, Seitz H-J (1988) Sparomusa termitida sp. nov.,
an H /CO -utilizing acetogen isolated from termites. Arch Microbiol
150:282- 88

Cline E (1969) Spectrophotometric determination of hydrogen-sulfide in
natural waters. Limnol Oceanogr 14:454-458

Costilaw R (1981) Biophysical factors in growth. In: Gerhardt P (ed)
Manual of methods for general bacteriology, chapter 6. American
Society for Microbiology, Washington, DC

Dehning I, Stieb M, Schink B (1989) Sparomusa malonica sp. nov., a
homoacetogenic bacterium growing by decarboxylation of malonate or
succinate. Arch Microbiol 151:421-426

Doddema HJ, Vogels GD (1978) Improved identification of methanogenic
bacteria by fluorescence microscopy. Appl Environ Microbiol 36:752-
754

Eichler B, Schink B (1984) Oxidation of primary aliphatic alcohols by
Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe.
Arch Microbiol 140:147-152

Ghazvinizadeh H, Turtura CG, Zambonelli (1972) The fermentation of L-
rhamnose by clostridia. Ann Microbial Enzimol 22:155-158

Gottschalk G (1986) Bacterial metabolism. Springer-Verlag, New York
359pp

Green (1985) Gene 37:261-266

Hanson RS, Phillips JA (1981) Chemical composition. In: Gerhardt P (ed)
Manual of methods for general bacteriology, chapter 17. American
Society for Microbiology, Washington, DC

Hermann M, Papoff M-R, Sebald M (1987) Sparomusa paucivorans sp. nov., a
methylatrophic bacterium that forms acetic acid from hydrogen and
carbon dioxide. Int J Syst Bacteriol 37:93-101

Guckert JB, White DC (1986) Phospholipid, ester-linded fatty acid
analysis in microbial ecology. In: Megusar F and Center M (eds)
Perspectives in microbial ecology. Slovene Society for
Microbiology, Ljubljana, Yugoslavia

75
Hermann M, Papoff M-R, Sebald M (1987) Sparomusa paucivorans sp. nov., a
methylatrophic bacterium that forms acetic acid from hydrogen and
carbon dioxide. Int J Syst Bacteriol 37:93-101.

Honigberg BM (1970) Protozoa associated with termites and their role
digestion. In: Krishna K, Weesner FM (eds), Biology of termites,
chapter 1, vol.2. Academic Press, Inc, New York, pp1-36

Hungate RE (1969) A roll tube method for the cultivation of strict
anaerobes. In: Norris JR, Ribbons DW (eds), Methods in
microbiology, vol. 3B. Acedemic Press, Inc, New York, pp 117-132

Kluyver AJ, Schnellen C (1937) Uber die vergarung van rhamnose.
Enzymologia 4:7-12

Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin M, Pace NR (1985) Rapid
determination of 168 ribosomal RNA sequences for phylogenetic
analyses. Proc Natl Acad Sci USA 82:6955-6959

Lewis JC (1967) Determination of dipicolinic acid in bacterial spores by
ultriviolet spectrometry of the calcium chelate. Anal Biochem
19:327-337

Ljungdahl L0 (1986) The autotrophic pathway of acetate synthesis in
acetogenic bacteria. Ann Rev Microbiol 40:415-450

Mannarelli B (1988) Deoxyribonucleic acid relatedness among strains of
the species Butyrivibrio fibrosolvens. Int J Syst Bacteriol 38:340-
347

Moller B, Ossmer R, Howard B H, Gottschalk G, Hippe H. (1984) Sparomusa,
a new genus of gram-negative anaerobic bacteria including Sparomusa

sphaeroides spec. nov. and Sparomusa ovata spec. nov. Arch
Microbiol 139: 388—396

Montgomery L, Flesher B, Stahl D (1988) Transfer of Bacteroides
succinogenes (Hungate) to Fibrobacter gen. nov. as Fibrobacter
succinogenes comb. nov. and description of Fibrobacter
intestinalis sp. nov. Int J Syst Bacteriol 38:430-435

Odelson DA, Breznak JA (1983) Volatile fatty acid production by the
hindgut microbiota of xylophagous termites. Appl Environ Microbiol
45:1602-1613

Ollivier B, Cordruwisch R, Lombardo A, Garcia J-L (1985) Isolation and
characterization of Sparomusa acidovorans sp. nov., a
methylatrophic homoacetogenic bacterium. Arch Microbiol 142:307-310

Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR, Stahl DA (1986) Microbial
ecologoy and evolution: a ribosomal RNA approach. Annu Rev
Microbiol 40:337-365

Obrien RW, Breznak JA (1984) Enzymes of acetate and glucose metabolism
in termites. Insect Biochem 14:639-643

76
Oren A, Pohla H, Stadkebrandt E (1987) Transfer of Clostridium lortetii
to a new genus Sporohalobacter gen. nov. as Sporahalobacter

lortetii comb. nov., and description of Sporohalobacter
marismotui. Syst Appl Microbiol 9:239-246

Parker JH, Smith GA, Frederickson HL, Vestal JR, White DC (1982)
Sensitive assay, based on hydroxy fatty acids from
lipoplysaccharide Lipid A, for Gram negative bacteria in sediments.
Appl Environ Microbiol 44:1170-1177

Potrikus CJ, Breznak JA (1977) Nitrogen-fixing Enterobacter agglomerans
isolated from guts of wood-eating termites. Appl Environ Microbiol
33:192-399

Rogosa M (1971) Transfer of Peptostreptococcus elsdenii Gutierrez et al.
to a new genus, Megasphaera [M. elsdenii (Gutierrez et a1.)
comb.nov.]. Int J Syst Bacteriol 21:187-189

Smibert RM, Krieg NR (1981) General characterization. In: Gerhardt P
(ed) Manual of methods for general bacteriology. chapter 20.
American Society for Microbiology, Washington, DC

Stahl DA, Flesher B, Mansfield HR, Montgomery L (1988) Use of
phylogenetically based hybridization probes for studies of ruminal
microbial ecology. Appl Environ Microbiol 54:1079-1084

Stackebrandt E, Pohla H, Droppenstedt R, Hippe H, Woese CR (1985) 168
rRNA analysis of Sparomusa, Selenomonas, and Megasphaera: on the

phylogenetic origin of Gram-positive eubacteria. Arch Microbiol
143:270-276

Wiegel G, Quandt L (1982) Determination of the Gram type using the
reaction between polymyxin B and lipopolysaccharides of the outer
cell wall of whole bacteria. J Gen Microbiol 128: 2261-2270

CHAPTER 3
112/002 ACETOGENIC BACTERIA FROM TERMITE GUTS:
II. Clostridium mayombei, SP. NOV., ISOLATED FROM GUTS OF THE

AFRICAN SOIL-FEEDING TERMITE, Cubitermes speciousus

77

78

Abstract

Strain SFC-S, a previously undescribed H2/C02 acetogenic
bacterium was isolated from gut contents of the African, soil-feeding
termite Cubitermes speciosus. Cells of this strain were anaerobic, Gram
positive, catalase and oxidase negative, endospore-forming, motile rods
which measured 1 x 2-6 pm. Optimum conditions for growth on H2 + CO2
were at 330 C and pH 7.3, and under these conditions cells produced
acetate according to the equation 4H2 + 2002 ---> CH3COOH + 2H20.
Growth also occurred between 150 and 450 C and pH 5.5 to 9.3, and other
substrates supporting good growth included certain carbohydrates (eg.
glucose, xylose, starch), organic and amino acids and alcohols. The
major fermentation product was almost always acetate alone. Based on
comparative analysis of 168 rRNA nucleotide sequences, strain SFC-S was
closely related to various members of the genus Clostridium. However,
the morphological and physiological differences between strain SFC-S and
other homoacetogenic clostridia were significant. Consequently, it is
proposed that strain SFC-S constitute the type strain of a new species,
Clostridium mayombei. The properties of C. mayombei are compared to
those of Sparomusa termitida and Acetonema elongata (Hz/CO2 acetogens
previously isolated from the guts of wood-feeding termites) as part of a
continuing study to determine why acetogenic bacteria compete

effectively for H in the guts of wood-feeding, but not soil-feeding,

2

termites.

79
Introduction
The ability of Hz/CO2 acetogenic bacteria outcompete Hz/CO2

methanogenic bacteria for H is a widespread phenomenon in the hindgut

2
fermentations of wood- and grass-feeding termites (Chapter 1). This
process is important to termite nutrition, inasmuch as up to 1/3 of the
insects' respiratory requirement can be met by the oxidation of acetate
derived from bacterial H2/CO2 acetogenesis in the hindgut (Breznak and
Switzer, 1986). By contrast, this phenomenon does not appear to be as
important to termites which feed on the humic component of soil. Rates
of microbial H2/CO2 acetogenesis in the guts of soil-feeding termites
were considerably less, and rates of CH4 emission were substantially
higher, than those of their wood- and grass-feeding counterparts
(Chapter 1).

To learn more about termite gut acetogens in general, and to
begin to evaluate factors which might affect their competetiveness for
H2 in situ, H2/CO2 acetogenic bacteria were sought from the guts of
wood-feeding termites. Two Gram negative, endospore-forming isolates,
Sparomusa termitida (from Nasutitermes nigriceps) and Acetonema elongata
(from Pterotermes occidentis), were obtained and studied in detail
(Breznak et a1, 1988; Chapter 2). Although the competetiveness of S.
termitida for H2 could not be attributed to an unusually high affinity
or low threshold for H2 (Breznak et al., 1988; Cord-Ruwisch et al.,
1988), certain other aspects of their physiology which may bear on their
success in the termite gut (eg. the ability to grow mixotrophically),
are the subject of a continuing investigation (Breznak and Switzer,

1989). However, it also seemed useful to obtain, for comparative

purposes, H2/CO2 acetogens from the guts of termites in which this

80
activity was not a dominant electron sink reaction. Accordingly, we
attempted to isolate such bacteria from the hindguts of soil-feeding
termites.

In the present study, we describe the isolation and
characteristics of strain SFC-S, an Hz/CO2 acetogen from guts of the
African soil-feeding termite, Cubitermes speciosus. Unlike S. termitida
and A. elongata, strain SFC-S was Gram positive, and exhibited other
properties characteristic of members of the genus Clostridium. However,
strain SFC-S did not correspond closely to any H2/CO2 acetogenic
Clostridium that has been described previously, so it is propsed that
strain SFC-S constitute a new species, Clostridium mayombei.

[A preliminary report of these findings has been presented (M.
D. Kane and J. A. Breznak, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989,

1100, p.234)].

Materials and Methods
Termites

C. speciosus (Termitidae) specimens were collected from the
Mayombe tropical rain forest near Dimonika, People's Republic of Congo.
Termites were degutted on site and the guts were immediately immersed in

an anaerobic salts solution consisting of (g/l) KH P0 (0.2), NH Cl

2 4 4
(0.3), KCl (0.5), NaCl (1.0), MgClz.6H20 (0.4), NaHCO3 (2.5) and
CaC12.2H20 (0.15). Dissected guts were transported to Michigan over a

period of ten days, where they were used within 48h of receipt.

81
Isolation of strain SEC-5
Isolation of strain SFC-S was done by using media with (AC-K1)
and without (AC-K2) the addition of 2-bromoethane sulfonate (BES) as

described for the isolation of A. elongata (Chapter 2).

Growth studies

COz/bicarbonate-buffered medium (AC-K4) was used for growth
studies. It was identical to AC-Kl medium (Chapter 2), except that the
amount of trypticase was reduced to 1.0 g/l and rumen fluid was omitted.
Other aspects of growth studies have been described previously (Chapter

2)

Nutrition studies

Nutrition studies were performed by using AC-KS medium, which
was identical to AC-K4 medium, except that amounts of trypticase and
yeast extract were each increased to 2.0 and 1.0 g/l, respectively.

Nutrition studies were performed as described in Chapter 2.

Fermentation studies

A fermentation balance of H2/002 grown cells was done by using
AC-KS medium and an Hz/CO2 (80/20, vol/vol) atmosphere. The basal growth
medium for material balances of glucose, xylose and sodium succinate
fermentations was AC-KS modified by omitting NaHCO3, and including 3-(N-
morpholino) propanesulfonic acid buffer (sterilized separately, adjusted
to pH 7.4) at a final concentration of 10 mM. Incubation was under 100%

N2, and material balance calculations were corrected for the amount of

82
products formed from cells grown in basal medium containing no
additional substrate.
14 14
Measurements of H2-dependent reduction of CO2 to C-acetate
by cell suspensions, and determination of the distribution of 14C in

acetate by Schmidt degradation, were performed as described previously

(Breznak and Switzer, 1986 Breznak et al., 1988).

Chemical assays and other procedures
All chemical assays and other analyses (including comparative

16S rRNA sequence analysis) were performed as described in Chapter 2.

Results
Isolation of bacteria

After two weeks incubation, primary enrichments for H2/C02
acetogenic bacteria in AC-Kl medium (containing BES) exhibited
turbidity, negative pressure in the headspace, and acetate production up
to 30 mM (with periodic replenishment of H2/COZ). By contrast, H2/CO2
enrichments in AC-K2 medium (without BES) exhibited production of CH4

and contained Fazo-fluorescent cells. From the former enrichments
H2/002 acetogenic bacteria were isolated by using agar roll tubes.
Seven strains of Hz/CO2 acetogenic bacteria (strains SFC-l through SFC-
7) were isolated, all of which exhibited similar morphology and Gram

stain reaction. Strain SFC-5, which grew slightly faster than the other

strains, was chosen for further characterization.

83

Morphology

subsurface colonies of H2/CO2 grown cells were white to slightly
yellow, oval-shaped with smooth edges and about 2 mm in diameter. Cells
of strain SFC-S were straight, motile rods measuring 1.0 x 2-6 pm (Figs.
1a & 1b). In old cultures (2 7d), longer (10-15 pm), slightly curved
cells were also observed. Cells stained Gram positive, and electron
micrographs of thin sections revealed a typical Gram positive cell wall
morphology (Fig. 1b). In addition, cells formed central to subterminal,
oval endospores which sometimes casused cells to swell slightly (Fig.
1a). Viable cells could be recovered from sporulated cultures held at
800 C for 10 min. Moreover, dipicolinic acid (DPA) could be extracted
from sporulated cultures and exhibited a UV spectrum similar to that of

authentic DPA.

Growth and nutritional studies

Cells of strain SFC-5 were capable of growth only in 02-free
medium to which a reductant [eg.dithiothreitol (1.0 mM, final
concentration)] had been added. Growth required the addition of
trypticase or yeast extract (1.0-2.0 and 0.5-1.0 g/l, respectively), but
best results were obtained when both constituents were added to the
medium at ratio of 2:1 (trypticase:yeast extract). Addition of rumen
fluid (5%, vol./vol.) to the growth medium had no effect on growth.
Optimum growth in AC-K4 medium occurred at 330 C and pH 7.3. Cells also

grew within a temperature range of 15 to 450 C and a pH range of 5.5 to

9.3. No growth occured at 15 or 450 C, or at a pH of 5.2 or 9.6.

84

Figure la, b. Morphology of strain SFC-S. a. Phase contrast micrograph;
bar - 10.0 pm. b. Transmission electron micrograph of a thin section;

bar - 1.0 pm. Note oval endospores (arrows a, b).

85

 

86

When cells were grown at 300 C in AC-K5 medium with H2/C02 as
substrate, they exhibited a doubling time of 5h; achieved a final

0 of 0.47-0.50 (approx. 0.160-180 pg dry mass/m1); and

“”400 n.m.
produced 18 to 23 mM acetate (without replentishment of H2/C02) (Fig.
2). Cell yields and acetate production were considerably less when
cells were grown in the same medium with N2/CO2 in the headspace.

Strain SFC-S cells also grew on a variety of other substrates,
including sugars, organic and amino acids, and alcohols (Table l). The
major product formed from most substrates was acetate, although a trace
amount of isovalerate was also formed. The only exceptions were that
cells converted succinate to propionate + C02, and valine to
isobutyrate. Molar growth yields for selected substrates are given in

Table 2. Neither sulfate or nitrate were reduced by cells grown with

glucose as substrate.

Fermentation balances
Cells of strain SFC-S used H2/CO2 as a substrate for growth and

acetogenesis according to the equation: 4H2 + 2C02 ---> CH3COOH + 2H20

(Table 2). The ability of strain SFC-S to effect a complete synthesis

of acetate from H2 + CO2 was confirmed by incubating H2/CO2 grown cells

with H2 + 14CO2 as substrates. Under these conditions, cells formed

1.70 pm 1tic-acetate per h per mg protein. Degradation of 1l‘C-acetate by

the Schmidt reaction demonstrated that 40% of the 14C was associated

with the COOH-group and 43% with the CHB-group.

87

Figure 2. Utilization of H2 by strain SFC-S for growth and
acetogenesis. Cells were grown with shaking at 300 C in bottles
containing 245 m1 AC-K5 medium and 463 m1 gas phase at 1 atm. The gas
phase was either H2/C02 (80/20, vol./vol.; closed symbols, solid lines)

or N2/002 (open symbols, broken lines).

 

88

xx. IN .2 Im>Um_u>Om A: 03 32. >Om._.>._.m A>.DV

 

  

/9\
e
/A
...o’
-za’f.’
\4
O 20 40 60 80

1.0

TIME (H)
Figure 2.

  

 

 

A0 I\\.A
.
Ill/me OIIA/ A
I . I I!
_F______ .____4_AAT .111
O O
. 1..
1

o.
63538.3

 

0.01

89

Table l. Sustrates used for growth by strain SFC-S. Compounds were
supplied at a final concentration of S to 10 mM, except for H2/602

(80/20, vol./vol.).

 

Used for growth by strain SFC-S:

HZ/COZ’ fructose, glucose, maltose, xylose, starch, cellobiosea,
sorbitol, dulcitol, glycerol, formate, pyruvate, malate, succinate,
syringatea, alanine, glutamate, serine, valine, salicin, dextrin and

esculin.

Tested, but not used:

Mannose, rhamnose, ribose, melibiose, raffinose, arabinose,
galactose, lactose, sucrose, trehalose, L-fucose, lactate, citrate,
oxaloacetate, fumarate, D-gluconate, acetate, oxalate, gallate,
caffeate, 3-hydroxybenzoate, benzoate, 3,4,S-trimethoxybenzoate,
pyrogallol, methanol, ethanol, propanol, mannitol, ethylene glycol,
adonitol, erythritol, butanol, isobutanol, pectin, xanthine, betaine and

N,N-dimethylglycine.

 

a
Poor growth on this substrate.

90
Table 2. Molar growth yields and fermentation balances for strain SFC-S

grown with various substrates.

 

Substrate Growth yield Product (mmol/100 mmol substrate fermented)

(g dry cell matter

 

 

/mol substrate H2 002 Acetate Propionate % C
fermented) recovery

H2 (+ 50
mmol coz)a 1.9 - n.d.b 22.9 0.0 91.6
Glucose 43.1 13.4 6.7 251.5 0.0 85.0
Xylose 27.5 0.0 0.0 206.4 0.0 82.6
Sodium
succinate 0.9 0.0 58.2 0.0 122.8 106.7

 

gAssumed for calculation of material balance.

n.d., not determined.

Similar to most 112/002 acetogenic clostridia, strain SFC-S
produced acetate as the sole major product when fermenting organic
compounds such as glucose or xylose (Table 2). In addition, cells
fermented succinate according to the equation: HOOCCH CH COOH --->

2 2

CH3CH2000H + C02. This mildly exergonic reaction, first demonstrated
for the bacterium Propionogenium modestum (Schink and Pfennig, 1982),
has also been demonstrated for two 112/002 acetogens belonging to the

genus Sparomusa (Breznak et al., 1988; Dehning et al., 1989).

91

16S ribosomal RNA sequence analysis

Comparison of the nearly complete sequence of the 163 rRNA from
strain SFC-S with those of S. termitida, A. elongata and certain other
bacteria has been described in a companion paper (Chapter 2). The
phylogenetic tree resulting from such comparisons is given in Appendix
I. The analysis used to construct this tree included the unpublished
168 rRNA sequences from a variety of other clostridia, which were kindly
provided by C. R. Woese, U. Illinois. Strain SFC-5 showed a distinct
and close relationship with members of the genus Clostridium. The
relationship between strain SFC-S and its closest relative (Clostridium
lituseburense) was 0.053 evolutionary distance units (- 0.05 changes per
nucleotide position). The evolutionary distance between strain SFC-S
and six other clostridia was 2 0.117, and the evolutionary distance
between strain SFC-S and eubacteria from genera other than Clostridium

was 2 0.193.

Other characteristics
Strain SFC-S was catalase and oxidase negative. Cytochromes

were not detected in crude cell extracts.

Discussion
Taxonomy

Inasmuch as cells of strain SFC-S were strictly anaerobic, Gram
positive, endospore-forming rods that did not carry out dissimilatory
sulfate reduction, it seemed clear that this new isolate should be

classified in the genus Clostridium. This conclusion was supported by

92
analysis of the 168 rRNA sequence of strain SFC-S, which revealed a
close evolutionary relationship with various clostridia, especially 0.
lituseburense (Appendix I). However, the latter species is not
homoacetogenic (with glucose as substrate), and uses mannose, but not
xylose as a substrate for growth (Cato et al., 1986). In fact,
homoacetogenic carbohydrate fermentations such as those exhibited by
strain SFC-S are relatively uncommon among the more than 80 described
species of Clostridium (Cato et al., 1986). To date, only five species
and one unnamed isolate of this genus carry out homoacetogenic
fermentations of carbohydrates, and strain SFC-S differs from these
bacteria in several respects. For example, Clostridium thermaceticum
(Fontaine et al., 1941) and C. thermautotrophicum (Wiegel et al., 1981)
grow optimally at 55-600 C, whereas strain SFC-S did not grow above 450
C. Moreover, unlike strain SFC-S, these two thermophiles use lactate
and galactose, but not maltose as growth substrates. Cells of the other
four homoacetogenic clostridia are clearly distinguishable from those of
strain SFC-S with respect to their morphology. C. aceticum (Adamse 1980;
Braun et al., 1981), C. formicoaceticum (Andreesen et al., 1970) and
Clostridium strain CV-AAl (Adamse and Velzeboer, 1982) form round,
terminal endospores which markedly swell the sporangium, whereas those
of strain SFC-S are oval, usually subterminal, and swell the cells only
slightly or not at all. Although the endospores of C. magnum (Schink,
1984) are oval and often subterminal, the cells themselves have a
greater width to length ratio than those of strain SFC-S, especially
when the former produces endospores. Moreover, neither C. magnum or C.

fbrmicaceticum use Hz/CO as a substrate for growth and acetogenesis.

2
Other differences between strain SFC-S and the four previously described

93
mesophilic clostridia are listed in Table 3. Due to the morphological
and physiological differences described above, it is proposed that
strain SFC-S constitute a new species of Clostridium, named Clostridium

mayombei, a concise description of which is given below.

Ecological considerations

0. mayombei and the two other HZ/CO2 acetogenic bacteria
isolated from termite guts clearly do not constitute a single taxon.
Although S. termitida, A. elongata and C. mayombei are all motile and
form endospores, examination of other properties exhibited by these
isolates indicates that in fact, termite gut acetogens appear to be
quite diverse (Table 4). From an ecological standpoint, the increased
availability of H2/002 acetogenic bacteria from the guts of various
termites provides an opportunity to begin to systematically examine
factors which bear on the ability of such diverse bacteria to compete

effectively for H in the guts of wood-feeding, but not soil-feeding

2
termites. Experiments with S. termitida indicated that this isolate
does not have a higher affinity or lower threshold for H2 than that of
various HZ-utilizing methanogens (Breznak et al., 1988). Nevertheless,
other factors (such as the ability to grow mixotrophically) may be
important to the success of Hz-utilizing acetogens in the guts of wood-
feeding termites. The observation that S. termitida and A. elongata
(both Gram negative bacteria isolated from the guts of wood-feeding
termites) are relatively closely related to one another, but not to C.
mayombei (Chapter 2), suggests that H2-utilizing, Gram negative

acetogens in the guts of wood-feeding termites may have some properties

(not possessed by Gram positive acetogens such as C. mayombei) that

94

Table 3. Substrates useful for distinguishing strain SFC-S from other

 

 

 

 

mesophilic, homoacetogenic clostridia.a’b
Growth Isolate
on:
Strain C. aceticum C. formicoaceticum C. magnum Strain
SFC-S CV-AAl
H2 +002 + + - - +
Ethanol - + n.d. - n.d.
Glycerol + n.d. n.d. - n.d.
Methanol - - n.d. - +
Formate + + + - n.d.
Fumarate - + n.d. - n.d.
Succinate + - n.d. - n.d.
Glucose + - - + +
Maltose + - n.d. - n.d.
Ribose - + n.d. - n.d.
Sucrose - - - + n.d.
Xylose + - n.d. + n.d.
a+, growth; -, no growth; n.d., not determined.

bData compiled from Andreesen et al., 1970; Adamse 1980; Braun et al.,

1981; Adamse and Velzeboer, 1982 and this study.

95
Table 4. Characteristics useful for distinguishing HZ/CO2 acetogenic

bacteria isolated from termite guts.

 

 

 

Property Isolate
S. termitida A. elongata C. mayombei
Cell dimensions (pm) 0.5-0.8 x 2-8 0.3 x 6-60 1 x 2-6
Cell wall type Gram - Gram - Gram +
Endospore location terminal/subterminal terminal subterminal
pH optimum/range 7.2/6.2-8.l 7.8/6.4-8. 7.3/5.5-9.3
Temp. optimum/range (0C) 30/19-37 33/19-40 33/15-45
Catalase/oxidase +/- +/- -/-
Growth on:
H2 + CO2 + + +
Formate + - +
Methanol + - -
Lactate + - -
Ethanol + - -
Mannose - + -
Ribose - + -
Propanol - + -
Maltose - - +
Xylose - - +
Starch - - +

Glycerol - - +

 

96
confer on them a competitive advantage over H2/C02 methanogens in vivo.
“2 competition experiments between C. mayombei, S. termitida and A.
elongata may also help clarify these issues.

It is intriguing that a close relative of C. mayombei is C.
lituseburense (Appendix 1), a non-homoacetogenic bacterium which was
isolated from West African soil (Cato, et al., 1986). Possibly, some
bacteria that occur in the guts of soil-feeding termites could more
accurately be described as soil bacteria whose residence in the gut is
only temporary. This may well be the case for C. mayombei, which is
obviously not an effective competitor for H2 in the guts of C. speciosus
termites. 0n the other hand, Hz-independent fermentative activities of
C. mayombei may contribute to the nutrition of C.speciosus termites,
even if the bacterium's presence in the gut is of a transient nature.
In any case, the isolation and characterization of C. mayombei increases
our understanding of the diversity of Hz/CO2 acetogenic bacteria as well
as the intestinal microecology of one of the Earth's most abundant
groups of termites. It also provides an important new organism for

further studies on the ecological significance of H2 consumption by

acetogens and their competitiveness in natural habitats.

Description of Clostridium mayombei sp. nov.

Clostridium mayombei sp. nov. may.omb'e.i. M.L. adj. mayombei,
pertaining to the Mayombe tropical rainforest (People's Republic of
Congo), from which this bacterium was isolated by using the gut contents
of Cubitermes speciosus termites.

Straight to slightly curved rods 1 x 2-6 pm, with rounded ends.

97
Cells single or in pairs. Motile, but location and number of flagella
are uncertain. Gram positive. Heat resistant endospores formed that
are 1 pm in diameter, oval and subterminal to terminal in location.
Colonies grown on H + C0 are oval shaped with smooth edges, about 2 mm

2 2
in diameter and white to slightly yellow in color.

Strict anaerobe. Catalase and oxidase negative.
Chemoorganotroph. Ferments H2 + C02 to acetate. Also ferments
fructose, glucose, maltose, xylose, starch, cellobiose, sorbitol,

dulcitol, glycerol, formate, pyruvate, malate, syringate, alanine,
glutamate, serine, salicin, dextrin and esculin yielding acetate as the
sole major product. Succinate is fermented to propionate + C02, and
valine is fermented to isobutyrate.
Does not respire anaerobically with nitrate or sulfate.
Cytochromes not detected.
Temperature optimum, 330 C (range 15 to 450 C); pH optimum, 7.3
(range 5.5-9.3). Trypticase and yeast extract required for good
growth.
Source: Gut contents of the termite Cubitermes speciosus
collected from the Mayombe tropical rainforest, Peoples' Republic of

Congo.

Type strain: Strain SFC-S.

98
References

Adamse AD (1980) New isolation of Clostridium aceticum (Wieringa).
Antonie van Leewenhoek 46:523-531

Adamse AD, Velzeboer CTM (1982) Features of a Clostridium, strain CV-
AAl, an obligatory anaerobic bacterium producing acetic acid from
methanol. Antonie van Leeuwenhoek 48:305-313

Andreesen JR, Gottschalk G, Schlegel, MG (1970) Clostridium
formicoaceticum nov. spec. isolation, description and distinction
from C. aceticum and C. thermoaceticum. Arch Mikrobiol 72:154-170

Braun M, Mayer F, Gottschalk G (1981) Clostridium aceticum (Wieringa), a
microorganism producing acetic acid from molecular hydrogen and
carbon dioxide. Arch Microbiol 128:288-293

Breznak JA, Switzer JM (1986) Acetate synthesis from H2 plus CO2 by
termite gut microbes. Appl Environ Microbiol 52:623-630

Breznak JA, Switzer JM (1989) Abstr Ann Meet Amer Soc Microbiol, p.160.

Breznak JA, Switzer JM, Seitz H-J (1988) Sparomusa termitida sp. nov.,
an H /CO -uti1izing acetogen isolated from termites. Arch Microbiol
150:282- 88

Cato EP, George WL, Finegold SM (1986) Genus Clostridium. In: Sneath PHA
(ed) Bergey's manual of systematic bacteriology, Vol. 2. Williams &
Williams, Baltimore, p.114l-1200.

Cord-Ruwisch R, Seitz H-J, Conrad R (1988) The capacity of
hydrogenotrophic anaerobic bacteria to compete for traces of
hydrogen depends on the redox potential of the electron acceptor.
Arch Microbiol 149:350-357

Dehning I, Stieb M, Schink B (1989) Sparomusa malonica sp. nov., a
homoacetogenic bacterium growing by decarboxylation of malonate or
succinate. Arch Microbiol 151:421-426

Fontaine FE, Peterson WH, McCoy E, Johnson MJ, Ritter GJ (1941) A new
type of glucose fermentation by Clostridium thermoaceticum n.sp. J
Bacteriol 43:701—715

Schink B (1984) Clostridium magnum sp. nov., a non-autotrophic
homoacetogenic bacterium. Arch Microbiol 137:250-255

Schink B, Pfennig N (1982) Propionigenium modestum gen. nov. sp. nov. a
new strictly anaerobic, nonsporing bacterium growing on succinate.
Arch Microbiol 133:209-216

Wiegel J, Braun M, Gottschalk G (1981) Clostridium thermoautotrophicum

species novum, a thermophile producing acetate from molecular
hydrogen and carbon dioxide. Curr Microbiol 5:255-260

CHAPTER 4

EFFECT OF HOST DIET ON THE PRODUCTION OF ORGANIC ACIDS AND METHANE

BY COCKROACH GUT BACTERIA

99

100
ABSTRACT

The effect of high fiber diets on microbial processes in
cockroach guts was investigated by feeding Periplaneta americana
cockroaches milled cereal leaves (MCL), milled corn cob (MCC), or
commercial bran-type breakfast cereal (RAB) in place of the commonly
used laboratory diet of dog chow (PDC). The activities and numbers of
specific gut bacteria varied significantly with the insect's diet and
life stage. Acetate and lactate were the dominant organic acids present
in gut fluid of adult cockroaches and occurred at concentrations up to
17 mM and 8 mM, respectively. These acids were most abundant in the gut
fluid of PDC-fed insects and were generally highest in the foregut and
midgut regions. The foreguts of PDC-fed cockroaches contained an
abundant population of lactic acid bacteria which formed acetate and
lactate from endogenous hexoses. When adult cockroaches were fed PDC
amended with antibacterial drugs: 1) the concentrations of acetate,
lactate and total hexoses in gut fluid decreased significantly; ii) the
numbers of lactic acid bacteria in the foregut also decreased
significantly; and iii) the production of acetate and lactate by foregut
homogenates was suppressed. It was estimated that acetate and lactate
production by bacteria in the foregut of PDC-fed adult P. americana
could support up to 14% of the insect's respiratory requirement. When
insects were fed high fiber diets of KAB, MCL or MCC, bacterial
production of acetate and lactate in the foregut diminished. The main
electron sink processes accompanying the gut fermentation of P.

americana were microbial reduction of C02 to CH4, formate and acetate.

101

Methanogenesis occurred exclusively in the hindgut region and was
favored when insects were fed high fiber diets. Moreover, larvae
generally emitted more CH4 per gram fresh weight than adult cockroaches,
particularly when insects were fed the high fiber diets. These results

indicate that host diet has a significant effect on the intestinal

microecology of P. americana .

102
INTRODUCTION

Presence of the American cockroach, Periplaneta americana, in
human dwellings causes damage and distress world wide. Nevertheless,
because of its ubiquity, relatively large size and nonfastidious nature,
P. americana has served as an important model for studies of insect
physiology, biochemistry and behavior (23). This seemingly omnivorous
and opportunistic insect is commonly reared in the laboratory on a diet
of dog chow and water, yet the nutritional ecology of P. americana is
poorly understood (a situation that prevails for most cockroach
species).

Previous studies of P. americana have demonstrated the presence
of bacteria in the foregut and midgut regions (3). However, the most
dense and diverse bacterial flora occurs in the hindgut - a region
wherein extensive attachment of bacteria to the gut wall is observed
(13). Facultative and obligate anaerobic bacteria (including
methanogens) have been described from the midgut and hindgut (4,6,13).
Although several studies have suggested that the hindgut flora may
contribute to the nutrition and development of P. americana
(2,3,6,7,12), the biochemistry of such interactions has not been
examined in detail (13).

Results from our laboratory indicated that microbial reduction
of 002 to acetate (rather than to CH4) represented the main electron
sink reaction in the hindgut fermentation of wood-feeding termites
(Chapter 1). In fact, in Reticulitermes flavipes termites acetate
derived from CO2 reduction could subsequently support up to 33% of the
termite's respiratory requirement (8,20). By contrast, CO reduction to

2

methane was found to be the main eletron sink reaction in the hindgut

103

fermentation of P. americana (8,13). These and other observations (13)
indicated that gut microbial fermentation patterns in P. americana were
qualitatively and quantitatively different from those in termites.
Consequently, this study was initiated in order to examine further the
metabolic and nutritional relationships between P. americana and their
gut bacteria, as well as to increase our limited understanding of the
intestinal microecology of insects in general.

This report describes the production of organic acids and CH4 by
P. americana gut bacteria; the importance of the foregut as a site of
bacterial metabolism; and the effects of high and low fiber diets on

gut-associated bacterial processes.

MATERIALS AND METHODS
Animals. A colony of P. americana L. (Blattidae) was maintained
in an insect rearing cage (no. 145008; Bio Quip Products, Santa Monica,
CA) at 250 C. Insects were fed dog chow (Ralston Purina, St. Louis, MO)

and water ad libitum.

Feeding experiments. To evaluate the effect of diet on gut
microbe activity, individual cockroaches were held for up to two weeks
before assay in wide-mouth 30-m1 glass serum vials (no. 223553; Wheaton
Scientific, Millville, NJ) containing a wetted cotton swab and an
appropriate food pellet. Cotton swabs were remoistened daily. The
vials were stoppered with non-absorbant cotton. Individuals were fed
ca. ZOO-mg pellets of dog chow (PDC), ball-milled cereal leaves (MCL;
no. C-7l4l; Sigma Chemical, St. Louis, MO), ball-milled corn cobs (MCC;

a gift from R. Hespell, USDA, Peoria, IL) or 200-mg of All-Bran cereal

104
flakes (KAB; Kellogg, Battle Creek, MI) (Table 1). For treatment with
antibacterial drugs, individuals were fed dog chow pellets wetted with
0.5 m1 of a solution containing chloramphenicol, penicillin G and
tetracycline (400 pg/ml each). Food pellets and cotton swabs were

replaced periodically to discourage mold growth.

Table 1. Protein and fiber content of P. americana dietsa.

 

 

Diet % Crude protein (wt./wt.) % Crude fiber (wt./wt.)
PDC 21.0 4.5
MCL 2.5 15.0
KAB 14.1 31.7
MCC 2.7 33.4

 

a Data were supplied by the manufacturer/supplier, except for MCC (22).

1460 fixation assay and analysis of 1460 fixation products.

2 2
H2-dependent fixation of 14CO2 by cockroach gut microbiota was assayed

as described previously (8) except that cockroaches were dissected in an
anaerobic glove box (Coy Laboratory Products, Ann Arbor, MI). Entire

adult cockroach gut tracts were used for the assay. Soluble products of

14C02 fixation were separated by using high-performance liquid

chromatography (8).

105

Gas emission measurements. Emission of CH4 and H2 by live
cockroaches was measured by incubating individuals in stoppered 30-ml
vials at 300 C. Periodically, 0.2 ml samples of headspace gas were
removed for analysis of H2 and CH4 by gas chromatography (20). The
headspace gas thus removed was replaced with an equal volume of air.

In other experiments, guts from adult cockroaches were removed
by dissection in an anaerobic glove box and sectioned into foregut,
midgut and hindgut regions. Individual gut sections were pooled from
five animals and incubated at 300 C in stoppered 5-ml glass serum vials

containing 1 ml of a buffered salts solution (B88) (8) and an initial

(v/v). CH emission by gut segments was

atmosphere of 95%N2/5%H 4

2
measured by sampling the headspace gas at the end of the experiment.

H25 emission was measured by incubating live adults in stoppered
30-ml vials each containing a 1.0-ml stemmed glass well (no. K-882330-
0000; Kontes, Vineland, NJ) suspended about 3 cm above the insect.
Wells contained 0.75 ml of a 2% (wt/vol) solution of zinc acetate and a
fluted piece of filter paper. Following 5 h incubation, the entire
contents of each well was transferred to a vial containing 10 m1 H20.
The sulfide content of the solution was then determined by colorimetric
assay (11). The detection limit for H S was determined to be 40 nmol x

2
g (fresh weight insect).1 x h-l.

Sampling of cockroach gut fluid. Adult P. americana were held at

O

2 C until they became immobilized (about 5 minutes). The legs were
then removed and the alimentary canal was exposed by making a ventral

incision extending from the terminal sternum to the prothorax, with care

being taken not to tear any portion of the gut wall. Various regions of

106
the alimentary tract were then sampled by puncturing the gut wall with a
25 gauge syringe needle and immediately aspirating the gut contents into
a 20 p1 capillary pipet. Samples were then ejected into a small
polypropylene centrifuge tube and centrifuged at 11,310 x g for 20 min
at 4° C. 5.0 pl portions of supernatant fluid (hereafter referred to as

extracellular gut fluid) were used for subsequent analyses (below).

Organic acid production by foregut homogenates. Intact foreguts
were dissected out of five adult P. americana and pooled in a small
glass tissue homogenizer containing 4.5 m1 of Ringers solution, pH 7.8
(18). Foreguts were homogenized for 1 min, and 1 ml portions of the
homogenate were added to S-ml glass serum vials. The vials were sealed
with butyl rubber stoppers and incubated at 300 C. One vial in each
experiment was a heat-inactivated (1000 C, 10 min) control. Reactions
were~ terminated at appropriate times by placing the vials on ice.
Homogenates were centrifuged at 11,310 x g for 20 min at 40 C, and the
supernatant fluids (hereafter referred to as foregut homogenate fluid)

were collected for analyses (below).

Quantification and characterization of bacteria. Homogenized
foreguts from adult P. americana were serially diluted in sterile
Ringers solution, pH 7.8, and 0.1—0.5 ml of each dilution were spread on
plates of two different media. One medium was selective for
lactobacilli (22). The other medium contained (g/100ml): tryptone
(Difco Laboratories, Detroit, MI), 1.0, yeast extract (Difco), 0.5, beef

extract (Difco), 0.1, sucrose, 10.0, agar, 2.0, and NaN3 (sterilized

107
separately as a 1% solution), 0.005. Dilution and plating were done by
using an anaerobic glove box.

Spread plates were placed in a Gas Pak jar (Becton-Dickinson)
which contained an H2 + C02 generator envelope. The jar was incubated
at room temperature (22-250 C) in the anaerobic glove box until colonies
developed (3-4 days). After this time, colonies were enumerated and
representative ones selected for further characterization were streaked
for purification onto fresh plates of the same medium from which they
had been picked. Pure culture isolates were inoculated into homologous
broth medum for growth studies and for determination of organic acid
production (see below). In order to test for gas production, test tubes
(18 x 150 mm) containing inoculated liquid media were overlayed
sequentially with 1.0 ml 2% agar, 1.0 ml vaspar and 1.0 m1 2% agar.

Other characteristics of the bacteria were evaluated by routine

microbiological methods(17).

Analysis of organic acids and hexoses. For analysis of volatile
fatty acids (VFA's), 5 p1 samples of extracellular gut fluid were mixed

in small centrifuge tubes each containing 35 pl H 0, 5 pl of 85% H3P04,

2
and 5 pl of a 10.0 mM isobutyrate solution (internal standard). For
analysis of foregut homogenates, sample preparation was the same except
that 40.0 pl of sample was used and no H20 was added. Mixtures were
then centrifuged at 11,310 x g at 4° C for 10 min, and the supernatant
fluid from each was used for quantification of VFA's by gas-liquid
chromatography (8,20).

Lactic acid was assayed colorimetrically by the method of Barker

(1) which included a sample clarification step, but which was scaled

108
down to accommodate small sample sizes. For this procedure, 3 to 5 pl
gut fluid or 30 pl foregut homogenate were made up to lSOpl with water
prior to the clarification step. Lactic acid present in samples of gut
fluid was also assayed enzymatically by using L-lactic dehydrogenase (EC
1.1.1.27) as described by Everse (l4).
Lactic acid production by bacterial isolates grown in broth
cultures was assayed by gas-liquid chromatography (19).
Total hexoses present in foregut homogenates were measured by

the anthrone method (25) with glucose as a standard.

Electron Microscopy. Excised cockroach foreguts were ligated at
each end with cotton thread, then immersed in a 2.5% (vol/vol) solution
of glutaraldehyde for 2.5 h. Samples were subsequently dehydrated by
immersion in a graded ethanol series (70%, 80%, 90%, 95%, 99% and 100%).
The foreguts were then critical point dried under C02 and sliced midway
between the two ligated ends using a razor blade. Foregut halves were
mounted on studs (with the cut end facing up) and sputter coated with
gold for 3 min. Coated samples were viewed by using a JEOL model JSM-

35C scanning electron microscope.

RESULTS

Gas metabolism by cockroach gut microbiota. Cockroach whole-gut

-dependent 14CO2 fixation, and formate and

2
acetate accounted for virtually all of the 14C02 fixed (Table 2). The
-dependent fixation of ll‘CO

homogenates were capable of H

data in Table 2 represent strictly H

2 2

109

whereby tabulated values were corrected for 14002 fixation which

occurred (in controls) under an incubation atmosphere of 100% N2 and

which was supported by endogenously-produced H2 or some other reductant.

Consequently, since the endogenous rate of 14C02 fixation was often as

much as 40% of that occurring under an atmosphere of 100% H2, the values
in Table 2 are probably minimum rates. PDC-fed cockroaches exhibited

the highest rates of H -dependent formicogenesis and acetogenesis, but

2
MCC-fed cockroaches had the highest acetate/formate ratio.

Table 2. Effect of diet on Hz-dependent fixation of 14002 into organic

acids by adult P. americana whole-gut homogenates.a

 

l4

 

 

 

Diet nmol C02 fixed into product

x g (fresh weight insect).1 x h"1

Formate + Acetate

Total Formate Acetate (% of Total)
PDC 177.0 156.5 11.5 94.9
MCL 63.6 59.1 5.5 101.6
KAB 57.7 51.7 4.3 97.1
MCC 46.9 35.1 9.9 96.6
aTabulated values are corrected for 1400 fixation under an incubation

2
atmosphere of N2.

110
P. americana reared on a diet of dog chow and water emitted 29.6

i 24.2 nmol CH x g (fresh weight insect).1 x h.1 (grand mean; n - 130).

4

Although CH emission rates varied considerably between individuals of

4
similar weight, and from day to day for each individual, immature
cockroaches (larvae) almost always produced more CH4 per gram than did
large (adult) cockroaches (Fig. 1). By contrast, cockroaches fed high
fiber diets emitted CH4 at rates 4 to 6 times greater than that of dog

chow fed cockroaches and exhibited greater differences in CH“ emission
rates between immature and adult cockroaches. This was particularly
true for those cockroaches fed MCC and KAB (Fig. l).
Excised hindguts from dog chow fed adult cockroaches emitted
1

31.3 nmol CH4 x g (fresh weight insect).1 x h- , a rate which fully

accounted for the CH4 emissions displayed by intact adult insects.

Significant amounts of CH could not be detected from excised foregut or

4
midgut regions incubated under similar conditions, indicating that the
hindgut microbiota was the origin of gut methanogenic activity.

Adult P. americana fed PDC did not evolve significant amounts of
H2 or H28.

Organic acids in cockroach gut fluid. Acetate and lactate were
the predominant organic acids found in extracellular fluid from all
regions of the gut of P. americana. Trace amounts of propionate,
butyrate and iso-valerate were also detected. Concentrations of acetate
and lactate were highest in the guts of dog chow reared cockroaches, and
generally highest in the foregut and midgut regions (Table 3). However,

concentrations of these acids were markedly reduced in the foregut and

midgut, and to a lesser extent in the hindgut, when cockroaches were fed

111

Figure 1. Effect of diet on methane emission from P. americana. For
two weeks before sampling, cockroaches were fed either: MCC, circles;

KAB, asteriscs; MCL, triangles; or PDC (control), boxes.

112

 

 

 

 

 

 

0.4

0 0
:1: x T635 m x .053 m5}. zo.wm_s_m mz<zkm2

1.2

I ..0.
/ W1
r. L
A
.. _ 18.
o _ O
A
.. ..e.
* O
I 14.
_ 0
T I l
I * \ 12.
T .1! * ‘ — I.
ll I
.|111. o
_ _ _ _ _ t IIEO
2 1. o

WEIGHT (G)

Figure1.

113

 

 

 

 

 

 

Table 3. Acetate and lactate content of gut fluid of adult P.
a

americana.
Diet Acetate (mM)

Foregut Midgut Hindgut
PDC 17.37 i 2.13 14.09 i 1.47 .22 i 2.27
PDC + Antibioticsb 4.76 1 6.75 6.90 i 1.79 .06 i 5.02
MCL 1.67 i 0.11 11.68 i 5.08 .15 i 2.24
KAB 7.48 i 7.30 6.51 i 5.04 .95 i 1.51
MCC 4.74 i 4.93 8.93 + 2.50 .21 i 1.94
Starved 12.28 i 5.03 13.57 i 5.20 .94 i 1.69

Lactate (mM)

Foregut Midgut Hindgut
PDC 6.07 i 3.11 8.21 i 3.00 .71 i 2.52
PDC + Antibioticsb < 3.00 < 3.00 < 3.00
MCL 5.07 i 3.64 3.51 i 2.80 < 3.00
RAB < 3.00 < 3.00 < 3.00
MCC 6.18 _+_ 5.17 3.72 i 1.82 < 3.00
Starved < 3.00 < 3.00 < 3.00

 

8Values are the mean 1 standard deviation of 3 or more determinations.

bPenicillin G, chloramphenicol and tetracycline (@ 400 pg/g dog chow)

114
dog chow containing antibacterial drugs (Table 3). This observation
suggested that bacteria were primarily responsible for the presence of
acetate and lactate in P. americana guts.

Although acetate and lactate concentrations were generally lower
in cockroaches fed diets other than dog chow, such differences were not
always dramatic. For example, concentrations of lactate in the foregut,
and acetate in the midgut and hindgut, of animals fed cereal leaves were
not significantly different than those of dog chow fed animals (Table
3). Interestingly, animals starved for two weeks still maintained
relatively high levels of acetate in the foregut and midgut, but not in

the hindgut.

SEM of cockroach foreguts. SEM of foreguts from PDC-reared P.
americana revealed widespread, but generally sparse populations of
bacteria attached directly to the foregut wall (Fig. 2A). In contrast,
food particles in the foregut were extensively colonized by bacteria
(Fig. 2B). Bacteria found on such particles were mainly coccoid in
shape and formed a dense lattice of cells on the surface of the

particles (Fig. 2C,D).

Production of acetate and lactate by cockroach foregut
homogenates. Inasmuch as bacteria were suspected to be responsible for
the high concentrations of lactate and acetate in the foregut of PDC-
reared cockroaches, it was important to measure the production of these
acids by resident foregut microbiota. When foregut homogenates from
PDC-reared adult P. americana were incubated at 300 C, acetate and

lactate were produced and total hexoses (measured as glucose

115

Figure 2. Scanning electron micrographs of the luminal surface of the
foregut of P. americana (A) and of bacteria attached to the surface of
food particles in the foregut (B,C); D is higher magnification of the

enclosed area in B. (Bar in A, B and C - 10 pm; bar in D - 1 pm.)

116

, . .,.o

 

_15KU x1

117

equivalents) decreased (Fig. 3A). Production of acetate and lactate
alone in foregut homogenates from PDC fed animals could, in theory,
account for about 40% of the total hexose used. By contrast, foregut
homogenates from cockroaches fed PDC containing antibacterial drugs did
not produce significant amounts of acetate or lactate and contained
lower amounts of total hexoses (Fig. 3B). These results suggested that
gut bacteria were responsible for the presence of a nutritionally
significant hexose fraction in cockroach foreguts from which they
produced acetate and lactate in situ.

Foregut homogenates from cockroaches fed MCL also produced
acetate and a small amount of lactate, accompanied by a decrease in
total hexoses (Fig. 3C). Acetate and lactate production in foregut
homogenates from cockroaches fed MCL could theoretically account for
about 60% of the total hexose used. Foregut homogenates from
cockroaches that were starved, or fed KAB or MCC produced little or no
acetate or lactate and contained lower amounts of total hexoses than
foregut homogenates from PDC fed insects (Fig. 3, D-F). Zero time
values for acetate and lactate in the time course assay were
surprisingly similar between cockroaches fed different diets (Fig. 3, A-
F). This observation seemed inconsistent with the considerable
differences observed for steady state concentrations of these two acids
in foregut fluid (Table 3). However, this discrepancy was more apparent
than real. Owing to dilution in preparation of the foregut homogenate
(see Materials and Methods), the maximum concentrations of acetate and

lactate at the beginning of the time course experiment depicted by Fig.

118

Figure 3. Effect of diet on acetate and lactate production by foregut
homogenates of adult P. americana. For two weeks prior to assay,
cockroaches were fed either: A, PDC (control); B, PDC + antibiotics; C,
MCL; D, KAB; E, MCC; or F, no food. Acetate (circles), lactate (boxes)
and total hexose (measured in glucose equivalents) (triangles) are given

in pmol per gram fresh weight insect.

119

 

 

 

. . .305... .25» .68:

 

 

 

 

 

0. o o.
o o o
3 2 «I oo
1 d d d 4 4 u a 4 4.1% 5
0
=4
0
3)
H
E
om
. A—ALT
o
4 .
4|
8
PD P b P h b P h b
c 0 0 100
3 2 cl

... 5555 so .. o 5:53 .05:

. . .390: .25» .651

 

 

 

 

 

o. o o.
o o o
3 2 1- 0°
aqauJila_11< .
5
o
.--A‘.
O.
3)
H
E
om
‘Ah‘T
0.
1
berr_. ».»»..
3 2 1..

. a $5.55 :0 .. o . upchwua BE:

A C .mewa ..(hO... _OE:

 

 

 

 

 

o o. o.
o o o
3 2 cl 0°
4 a q _ a 4 a a 4 A u .
5
O
O I .
A 4
O.
.3)
H
E
om
.—.2T
.0.
1
F.
> F r p _ h p b p 5’
o. o. o. oo
3 2 1

.o 5.555 :0 .. o 3.553 .251

 

 

 

 

 

 

 

. c .30me 459. .33

 

 

 

 

o. o o.
o o o
3 2 1 0°
14¢«44dq444 .
5
I e a O.
\ 4
\ o.
31!
H
\ ...
0m
O .. 2T
.0.
1.
hpiribyr>s0lm|~ o
c. o. o. o
3 2 1

x. 5555 no .. 6 5.253 .65:

 

 

A . 5.8.3.: 439. .951

 

 

 

0. o. o.
0 0 O
3 2 1| 00
1.1.1.4111 4.14173
0
o a
A 4
o.
3 11
H
E
0 m
o.
1
. e r p a a p » LI..>' o
G O. 0 O
3 2 1.

A u _ 3255 no .. o a .3453 .05:

 

 

. . .30me 43b» .06:

 

 

 

 

o o. o.
o O O
3 2 “I: 00
«as «444 434 5
0
. .
O .44
O.
31]
W
m.
0|
O.
i
E
bhkhi—[bb
0. 0 mm! 00

3 a. 1
.- 5:55 :0 .. a $553 .951

 

 

 

 

Figure 3.

120
Table 4. Effect of diet on populations of lactic acid bacteria in adult

0 a
P. americana foreguts.

 

 

 

Diet Viable cells x 104 per g (fresh weight insect)-1
Streptococci Lactobacilli
PDC 23,200.0 72,800.0
PDC + Antibiotics 1.6 0.9
MCL 0.8 1,010.0
KAB 32.0 4.3
MCC 230.0 53.0
Starved 800.0 1,300.0

 

8Results are the values of individual analyses, except for insects fed

PDC, where results are the mean for n - 3.

3 were close to the detection limits of the assays used, somewhat
masking the significant differences _observed when steady state

concentrations of acetate and lactate in gut fluid were determined.

Lactic acid bacteria in cockroach foreguts. The presence and
production of lactic and acetic acids in P. americana foreguts suggested
that the resident bacteria observed by SEM (above) were saccharolytic
lactic acid bacteria. This was verified by quantitative culture
techniques employing selective, as well as rich non-selective, media.

Foreguts of PDC-reared cockroaches contained nearly 109 bacteria per

121

gram fresh weight insect. All isolates examined were Gram positive,
catalase negative, nonmotile rods and cocci that did not form
endospores. All produced lactic acid from growth on glucose, and none
required strict anaerobic conditions for growth. Four rod isolates and
four coccus isolates were chosen for further characterization, and
tenatively identified (by testing growth at various temperatures, and
for acetate and/or gas production from glucose) as members of the genera
Lactobacillus (both homo- and heterofermentative species), Streptococcus
and Enterococcus. Lactic acid bacteria were significantly less abundant
in the foreguts of cockroaches fed diets other than dog chow (Table 4).
Suprisingly, the number of lactic acid bacteria in the foreguts of
starved cockroaches was as higher than that in foreguts of cockroaches
fed diets other than dog chow, but only about 2% of that of PDC reared
cockroaches.

A water extract of PDC pellets from the cockroach rearing cage
contained about 107 viable bacteria per gram PDC. Since adult P.
americana consume about 18 mg PDC per day (4), they could ingest a
quantity of organisms equivalent to about 0.01% (105 bacteria) of their
resident population of lactic acid bacteria per day. This observation
indicates that the lactic acid bacteria observed in the foregut are

proliferating in that habitat and not merely transient organisms.

122

DISCUSSION

Results of this study showed that production of organic acids
and CH4 by gut bacteria of P. americana cockroaches were significantly
affected by the insect's developmental stage and diet. In addition, the
significance of the foregut as a site for lactate and acetate production
by lactic acid bacteria was also demonstrated. The general trend
accompanying a shift from PDC to a low protein-high fiber diet was: i) a
decrease in populations of lactic acid bacteria in the foregut; ii) a
decrease in total soluble hexose and a suppression of bacterial
production of lactate and acetate in the foregut; and iii) an increase
in bacterial methanogenic activity, and a modest decrease in H2-
dependent formico- and acetogenesis in the hindgut. The only exception
to this trend was that a diet of MCL did not substantially supress
acetate production by cockroach foregut bacteria.

The decrease in total hexose observed in foreguts of insects fed
MCL, RAB and MCC probably reflects the lower amounts of soluble,
anthrone-reactive material (soluble hexose monomers and polymers)
present in the diets themselves. However, foreguts of cockroaches fed
PDC + antibiotics also had lower amounts of soluble hexose compared to
PDC-fed controls. This observation suggests that foregut bacteria may
be capable of liberating anthrone-reactive material from certain
ingested foodstuff such as PDC.

At present we cannot explain why starved P. americana maintained
relatively high concentrations of acetate and lactate in the foregut and

midgut regions (Table 3) as well as a significant population of lactic

acid bacteria in the foregut. This is particularly confusing inasmuch

123
as lactic acid bacteria appear to colonize mainly food particles in the
foregut (Fig. 2). Although food particles can have a residence time in
the foregut of over 100 h (24), this does not explain the enigma of
lactic acid bacteria remaining in the foregut following 2 weeks of food
starvation.

A previous study demonstrated the presence of bacteria in the
foregut of P. americana (3), however subsequent research emphasized the
dense and diverse microflora present in the hindgut region (4,13) and
the relationship of foregut bacteria to the host was not examined. An
important finding in this study is that the foregut of adult cockroaches
fed PDC contained significant populations of lactic acid bacteria which
appear to heavily colonize certain food particles, and whose activities
were responsible for high concentrations of acetate and lactate in this
region. It seems likely that bacterial production of organic acids
contributes to the lower pH of the foregut (5.4 - 6.8) compared to the
rest of the alimentary canal (6.5 -7.6), as occurs in the crop of
chickens (15). However, preliminary experiments by Greenberg et a1.
(17) found that cockroaches fed antibiotic-treated banana for 5 d also
retained a low pH in the foregut region. Therefore, non—bacterial (i.e.
host) factors apparently also influence the pH of the foregut.

P. americana possess the enzymes necessary for complete
oxidation of acetate and lactate (26,27). [Although only low levels of
lactic dehydrogenase (LDH) are present in most cockroach tissue, such is
not the case for the midgut, which exhibits high LDH activity (9,10),
and is the primary sight of metabolite absorption in most insects (5)].
Based on acetate and lactate production rates (0.52 and 0.40 umol x g

[fresh weight insect].1 x h-1, respectively; calculated from Fig. 3A) it

124
can be estimated that the subsequent uptake and oxidation of these acids
by P. americana could support as much as 14% of the animal's respiratory
requirement.

An additional interesting aspect of the present study was the
discovery that immature (larval) cockroaches produced significantly more
CH4 on a per gram basis than did adult cockroaches, particularly when
cockroches were fed diets other than PDC. Bracke et a1 (6) observed
that loss of the cockroach hindgut anaerobic community by feeding
insects metronidazole adversely affected the development of larvae, but
did not seem to affect adult cockroaches. It seems likely that the
nutritional requirements of immature P. americana differ from adult
animals, and this is reflected by changes in activities or populations
of hindgut anaerobic bacteria, including methanogens.

Undoubtedly, considerable research remains before the complex
nutritional interactions between cockroaches and their intestinal
microflora are fully appreciated. Nevertheless, the present study
underscores the importance of using a carefully defined diet, and

considering the potential contributions of gut bacteria, in studies of

cockroach nutrition and physiology.

125
LITERATURE CITED

1. Barker, S. B. 1957. Preparation and colorimetric determination of
lactic acid, p.241-246. In S. P. Colowick and N. 0. Kaplan (ed.),
Methods in enzymology, vol. 3. Academic Press, Inc., New York.

2. Bignell, D. E. 1977a. An experimental study of cellulose and
hemicellulose degradation in the alimentary canal of the American
cockroach. Can. J. 2001. 55:579-589.

3. Bignell, D. E. 1977b. Some observations on the distribution of gut
flora in the American cockroach, Periplaneta americana. J. Invert.
Pathol. 29:338-343.

4. Bignell, D. E. 1981. Nutrition and digestion, p.57-86. In Bell, W.
J., and K. G. Adiyodi (ed.), The American cockroach. Chapman and Hall,
New York.

5. Bignell, D. E. 1984. The arthropod gut as an environment for
microorganisms, p.205-227. In J. M. Anderson, A. D. M. Rayner and D. W.
H. Dalton (ed), Invertabrate - microbial interactions. Cambridge
University Press. Cambridge, England.

6. Bracke, J. W., D. L. Cruden and A. J. Markovetz. 1978. Effect of
metronidazole on the intestinal microflora of the American cockroach,
Periplaneta americana. Antimicrob. Agents Chemother. 13:115-120.

7. Bracke, J. W., D. L. Cruden and A. J. Markovetz. 1979. Intestinal
microbial flora of the American cockroach, Periplaneta americana L.
Appl. Environ. Microbiol. 38:945-955.

8. Breznak, J. A. and J. M. Switzer. 1986. Acetate synthesis fron H

plus CO 2

by termite gut microbes. Appl. Environ. Microbiol. 52:623-630.

2
9. Cherfuka, W. 1958. Glucose metabolism in insects, p.115-l37. In L.
Levenbook (ed.), Proceedings oth the international congress of

biochemistry, vol. 12. Pergamon Press, New York.

10. Cherfuka, W. 1965. Intermediary metabolism of carbohydrates in
insects, p.581-667. In M. Rockstein (ed.), The physiology of insects,
vol. 2. Academic Press, Inc., New York.

11. Cline, E. 1969. Spectrophotometric determination of hydrogen-sulfide
in natural waters. Limnol. Oceanogr. 14:454-458.

12. Cruden, D. L. and A. J. Markovetz. 1979. Carboxymethyl cellulose
decomposition by intestinal bacteria of cockroaches. Appl. Environ.
Microbiol. 38:369-372.

13. Cruden, D. L. and A. J. Markovetz. 1987. Microbial ecology of the
cockroach gut. Ann. Rev. Microbiol. 41:617-643.

14. Everse, J. 1975. Enzymic determination of lactic acid, p.4l-43. In
W. A. Wood (ed.), Methods in enzymology, vol. 41. Academic Press, Inc.,
New York.

126
15. Fuller, R. and B. E. Brooker. 1974. Lactobacillic which attach to
the crop epithelium of the fowl. Amer. J. Clin. Nutr. 27:1305-1312.

16. Gerhardt,P., R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A.
Wood, N. R. Krieg and G. B. Phillips. 1981. Manual of Methods for
General Bacteriology. American Society for Microbiology. Washington,
D.C.

l7. Greenberg, B., J. Kowalski and J. Karpus. 1970. Micro-potentiometric
pH determinations of the gut of Periplaneta americana fed three
different diets. J. Econ. Entomol. 63:1795-1797.

l8. Griffiths, J. T. and O. E. Tauber. 1943. Effects of pH and various

concetrations of sodium, potassium, and calcium chloride on muscular
activity of the isolated crop of Periplaneta americana (Orthoptera). J.
Gen. Physiol. 26:541-558.

19. Holdeman, L. V., E. P. Cato and W. E. C. Moore. 1977. Anaerobe
Laboratory Manual, 4th ed. V. P. I. Anaerobe Laboratory. Blacksburg, VA.

20. Odelson, D. A. and J. A. Breznak. 1983. Volatile fatty acid
production by the hindgut microbiota of xylophagous termites. Appl.
Environ. Microbiol. 45:1602-1613.

21. Preston, R. L. 1987. Ingredient table helps balance rations.
Feedstuffs p. 18-23.

22. Rogosa, M., J. A. Mitchell and R. F. Wiseman. 1951. A selective
medium for the isolation and enumeration of oral and fecal lactobacilli.
J. Bacteriol. 62:132-133.

23. Roth, L. M. 1981. Introduction, p.1-l4. In Bell, W. J. and K. G.
Adiyodi (ed.), The American cockroach. Chapman and Hall, New York.

24. Snipes, B. T. and O. E. Tauber. 1937. Time required for passage
through the alimentary tract of the cockroach, Periplaneta americana
Linn. Ann. Entoml. Soc. Amer. 30:277-284.

25. Spiro, R. G. 1966. Analysis of sugars found in glycoproteins, p.3-
26. In E. F. Neufeld and V. Ginsburg (ed.), Methods in enzymology, vol.
8. Academic Press, Inc., New York.

26. Storey, K. B. and E. Bailey. 1978a. The intracellular distribution
of enzymes of carbohydrate degradation in the fat body of the adult male
cockroach. Insect. Biochem. 8:73-79.

27. Storey, K. B. and E. Bailey. 1978b. Intracellular distribution of
enzymes associated with lipogenesis and gluconeogenesis in fat body of
the adult cockroach, Periplaneta. Insect. Biochem. 8:125-131.

APPENDIX

127
ANALYSIS OF 168 rRNA SEQUNECES OF H2/002 ACETOGENIC

BACTERIA ISOLATED FROM TERMITE CUTS

Table 1. Nearly complete sequences of 16S rRNA from H2/CO2
acetogenic bacteria isolated from termite guts. SFC-S, Clostridium
mayombei strain SFC-S (Chapter 3); APO-1, Acetonema elongata strain APO-
1 (Chapter 2); JSN-2, Sparomusa termitida strain JSN-2 (Breznak et al.,
1988, Chapter 2). The sequence of Bacillus subtilus (B.subt; provided

by C. R. Woese, U. Illinois) is given as a reference.

128

“Luna
EuQL
LMZL

9.2.2

.2604
ESL
LMZL

9.22

.2..an
ESL
LMZL

mes?

«$0.9
ESL
.355

P?!

2..an
ESL
32L

FEE

.2606
ESL
LMZL

Pei:

who...“
ESL
LMZL

FEE

Sung
E5L
EZL

was?

9.an
EVQL
LMZL

FEE

 

11111111 cm>m
IIIIIII com>o
Inlnc>ccmmom
11ccc>comm>m

Z>OZO>COOZ
D>OZOZCQOZ
>>O>O>COO>
>>C>O>COO>

OOQQ>OOQOC
OOQ>>OOOQC
QOQ>>OOOOC
OOOO>OOOQC

>C§OOIO>>>IQ
QCCOOIO>>>IO
>OOOOIQ>>>IQ
COOOQIO>>>IO

>CC>ICO>>>IQ
CCC>IOC>>>IO
QCC>I>O>>>IQ
>>O§IC>>>>IO

CDQOC>QCCQ
C>QOC§QCCQ
C>OOCOOCCO
C>QOC>QCCQ

OCIQVQVIQQOIC
OCIO>Q>IOQ>IC
OCIQ>O>IQQ>IC
OCIQ>Q>IQOOIC

momovmmowm
nmmm>mon>m
nmma>mmn>m
nmmmwmon>m

Cybomononm
O>>OOOOOOO
O>>OOOOOOQ
Owbnmnnonm

>QCCCQ>COO COOOCO>®Q> CDDZOQOCQQ OOQOOICQOIOC
>QCCCQ>EOO CQQOCOvmmw OQ>>OOOCOQ OOOOQICQOICG
OQCCCDmDOO COQOCO>QQ> OQ>>ODOCOQ OQOOQICQOIOC
>QCCCQ>COO CQQOCO>QQ> OQ>>OQOCQQ OOOOQICQOIOC
>QCOQ>QOQ> IIIIIII CCOC OICCOQIO>Qm> IIIIII Q>OOG
CQCOQ>>OQQ IIIII Omwmcc >IOO>>IC>ZOC OOIIII>>QCQ
>OCOO>>OOQ IIIIII >QCZC >IOOV>IC5>EC ZIIIOCC>OCQ
>OCOQ>QOQQ >O>QQICOOQ> QIOCCGIOCOOO OI>CQCCVQOQ
Q>OCD>OOOQ CQQQC>>OOC GOOCO>C>O> O>CQQ>C>>O
O§QC>>OQOQ C>Q>OZ>COC GOOOCCQ>O> DQOQQ>O>>O
Q>QC>>OOOQ C>Q>O>>OOC QCOCOCC>®O CQQOQ>O>>O
O>OC>>O>OQ CQQQC>>OOC OOOCQC>>Q> OCQQQ?G>>O
QCZCOOC>>C >OfiOO>CQ>C I>C>>O>Q>ICCO I>O>CIZO§ICGC
O>OOOOCm>C >OOO>>CQCC IOCQQ>Q>OI§XC IQO>CIZOCICCO
COOCOOC>>C >000>>CQCQ IOC>>OCOQIOCC IQO>CI>>OIOQ>
OOOOOOC>>C >OOOO>CQOC ICQCCCQ>1I>OO IOO>CIOQCICO>
O IIIIIIIII C000 IIIIII IIIOOOOC>C Q>Q>COO>OO OOOQCOCQ>C
OCOOOOCOCI>CZCIZCZ>Q OC>OO>OCO> >Omwmmmmcn COOQCOCO>G
>Cmmnownllcocml>>0>> OC>COQOC>> Q>Q>CQOOCO CQOOCOCQvC
OCOQO IIIII CCOO IIIII Q OC>00>OCC> O>O>COO>OO OQOOOOQO>G
QC>I>QOC>>C QOOCC>OO§> QQOQ>OQ>CO >QC>OOZQ>O
QCQI>QOC>>O OOOCO>OZ>> GOOZ>OQ>CO >QC>QOOOOC
OCCI>OQC§>O QOfiCO>OOW> omnmwnowcn >OC>QOOOQC
OCOI>QOC>>O QGOCO>OO>> QQO>>00>CQ omcvmnomvn
Q>COQ®OO>O wccmmvbocm >QvO§OQOCO OZmVOCnOC>
O>>OOQOO>O >OCOQO>OCO >Q>O§OQQOO ZZO?OCOOC>
m>>OOOOO>O >OCOQQ>OCQ >Q>OvOQOOO ZZQ>OCOOC>
ODCOGOOO?O Docmmmwocm >O>O>OOOOO O>Q>OCOOC>
O>QCQQOO>> C>CCQO>O>> COOOOIO>>>IO OOCZ>COO>O
O>QCOOOO>> COCCOOQO>> COOQOIm>>>IQ OOCQ>OQQ>Q
O>QCQOQG>> COCCOOOO>> COO>010>>>IO COCQ>OOQ>Q
0>OC>OOO>> COCCOOOO>> COQ>OIO>>>IO COCO>OQO>®
CQ>OCO>CO> VOQOOICCOOIQ QCOOC>>>>O COCOCOOCO>
CQ>OCO>>O> >OOCCICCOOIG >CCOC>>>QO COCQCOCCCO
CQ>QCQ>>Q> >QQOOICCOQIO QCOOC>>>GO COCOCOQCCC
cowmco>co> >OOCCICCOOIO >COOC>>>OO COCQCCOCCW

a
a
8

mm
mm
o—
89

Law
5m
3—
So

5e
30
SN

N8
Q3
Q6
who

N3
N3
N8

wwm
w:
”Xe
wuo

3m
m3
woe

#8
as
to
ac

129

ESQ
ESL
.32.».

FEE

9.an
ESL
\QZL

FEE

ESQ
ESL
EZL

FEE

ESQ
ESL
32L

FEE

ESQ
ESL
\QZQ

FEE

. Ens

ESL
LQZL
FEE

ESQ
ESL
LQZL

FEE

who...“
ESL
32L

FEE

ESQ
ESL
32L

FEE

 

>00>§QIIII
QQO>OQ>>CO
QQQ>OQ>>OQ
QQO>>Q>>05

Q>QQ>>QOOO
Q>Q00m000>
Q>QO>>OOO>
O>Q>>>QOO>

mzzzmnmccw
mnzvmooccm
QO>>QOQCCQ
mnwbmnmccm

C>>QCOI>QQ>
C>>QCOICQCC
C>>QCOIQCOC
C>>QCOIC0>C

cmmo»m>o-cc
cmcxwvc01cc
cmcno>601cc
commo>>oucc

>>>ICOOQC>Q
>>>ICOOQCZQ
w>>ICQOQC§O
>>>ICGOQC>O

OCQC>>OCQV
OCQCC>OCZ>
OCQCQCOCQ?
OCQC>>OCQ>

monocsoc>o
vnonnmmcpm
vnnnommcwm
>nnocmmc>o

OOOOIOOMCOO
Q>OOIODCOOC
Q>OOIOOCCOC
OOOIOOOOCCP

conoomcnoc
czomocwnmc
vocwnnmccn

OQQOC>>IOC>
OOQOC>>IOC>
OQQOC>>IOC>
OQQOC>>IOC>

commences»
conno>>ca>
cnooo>wcc>
cnooowwcc>

QCQ>>>QQOC
QCQ>>>>CQO
QCOC>>QCQO
QCO>>>OOOO

mvmcmonmmv
mvmcmn>00>
QDQCQO>®Q>
OWOCQO>O>>

munccwomwm
>c>ccmom>m
wc>ccoom>m
vmwcmcmmwo

O>OCQ>QQO>
OOOCQ>OOOQ
O>Ocmvmmnm
OQOCQ>00>Q

cooeomonoc
concymnzmc
coocmonnmc
coo>oonnmc

mamaomnsoo
acoozmmsmc
mannoam>mc
mcmocmowmo

un>cw> uuuuuuu
nvvvcvnnvcovo
um>>c>un>gnc>
nowvcvnuooonm

nocmonemn»
nmcmoowmnv
nmcmnnwoow
omcoonvonv

OCQOOOQC?)
CCQQQOQCZ>
CCQQQOQC>§
CCQQQOOC>>

>OQQIOCZ>>IO

ommmnocamvlm

Q>>QIOCO>>IO
OOOQIOCOPbIO

Q>0022>OC0
Q>QQ>>>QOQ
Q>QQ>>>QCQ
Q>Q0vmfimcm

Q>>OmOO>OIC
Q>>O>OO>QIC
Q>>O>OO>QIC
Q>>O§OO>QIC

OGW>>OOQCQ
O>>>>OOO§Q
OQ>>>QOO>Q
OO>>>QOOCQ

v>>Om>CQ>Q
Z>>OQ>CQOQ
>>>OQ>COOQ
>>>OQ>CO>Q

C>>OOO>CC>
C>>OQO>>C>
C>>OOO>>C>
C>>OOO>CC>

uuuuuuu amen
cnuocwwcowo
owuocvscoso
acusnoccovn

GOOQOQOC>>
OOOQOQQC>>
Gnomnmmcwv
QOOOOOOC>>

>OQOCGOQC>
>$QQOQOQO>
>QQOOQCOCO
>0000C000>

anabmcvllvm
KOOQC>QQIQC
CCOQC>CQIQQ
OQQQQ>QQIQC

Q>§CCOOC>Q
m>>CCOOO>Q
O>>CCOOO>Q
Owbccnnwnm

fifiomwbmmno
Omnmbbmmnm
QGOQ>§QQOQ
OOOO>>QGOQ

mammmnmgo
QQC>0C0>>O
OOO>QOQ>>O
QOQ>QOO§>O

C?OC>OQCQC
C>OC>OOCOC
CDOC>QOCOC
CQOC>>OCOC

mac>ncoom0
vmcmooawmo
wmcmnonnmo
mmowocnnmo

QOCVOCCQva
OOC>OOQ>>IQ
OOC>OO>§>IQ
00C>nncw>ln

CDOQC>OOQQ
C>OQC>QQCQ
C>OQC>QGCQ
CPOQCDQQCQ

QQCQQCCCOG
QQOQQQCCC>
QQCOQOOGOQ
QQOQQCGCOC

OCOCCQ>>>O
O>>CQQ>>>O
Omnvmmwvvn
OVCCQQ>>DO

cmcvmnmlmcm
C0C>®OQIOCQ
CQC>QDQIQCQ
CQCVQOQIQCQ

oncccncmow
onzccoczow
>ncccocoow
wncocncooc

>QQ>GC§Q§C
OQQ>CC>Q>C
QQQ>CC>®>C
>00>CC$O>C

ZZQQIIIICCZ
>Qm>OQIC>CO
>Q>OQGIC>CO
Cwmmmmmlccc

OCQQOQ>OC>
OCOOOO>OC>
OCQOOQ>QC>
OCQOOQ>OC>

30
#3
new
moo

Q3
Que

Que
mom
Q8

Q8
Q3
Q5
8o

OS
00m
08
<8

ace
.Km
<3
.Luo

.Em
3Q
.30

Sou
m3
mac
muo

mam
mom
m8

130

nmocoumo>>nm >ocmw>>ncn v>>nmm>wccm wnomoowoon
ommoouon>>uo occmvwwnna >>>umm>>cco vnmmmmzoon
nmooouonvvuo macm>vvoco >>>umm>>ccm vnmmmmaonn
anacouonwbum wncm>>vnco vkumo>>cco vommommnon
nmmvoo>cmc mmccc>>ccn mwvoowvooo a>>mwwnncc
cmmvmcmcoc zoccczwcco mwzzz>>omo m>>m>>oncc
comnmcncmc zocccv>ccm awaonwvnon my>o>>nocc
cmm>mn>cmc mmcccvwccn mwvmnwvnmo awwmwwnncc

awnwcounccc aumwonmncmnu
w>owcouoooz wu>>umvcnoc>
ownvccumvmo ou>>u>mmcco>
mvnwcnuncnc ouvouwwcnocw

unczwcnnmowmmm mumuuuucc0uccnm
unmwmwuucyomom m uuuuuu «mmuccoo
ucmwm>uucmmwcn ououcuu>cnuccom
numwmwuncwmm>n mienoaunnouccnm

mun>n>wmcvo
mlnwnwbmomm
mlnzoavmomm
mlnwnvvmnmm

>IOOC>>QOCC
>IOO>QOQOCC
vIOO>QQOOCC
>IOO>QQCOCC

IQQQI>O>Q§>QC
IQQWI>IO>QQCN
IO>CI>IOQOQ>>
IOOQIOIObG>mC

COCCQQQCC>
CZCCQOODDD
coccmmmccm
cmccmmmcc>

>IICC>>QCICQQ
DSIDDDDDDDDDS
OOIOC>>>QIQOQ
vinccomeICmm

mmmmwcmwnm
5355:5355:
OOQQ>CQ>OQ
mammwcmwnm

OC>O>>CQOQ
OCZOzvcme
OC>Om>CmQO
OC>O>>COO>

owccw>moo>
nw>>>wnm>m
owm>>>c>mm
owowvvcocm

Q>O>OQCOOC QO>COQCCOC OOCO>QOCOQ COCOIQCO>I0>
O>O>OQCOOC OO>CQQO£NC ZOZZ>QOCZQ COCZIQCZPIQ>
>>O>QOCOOC QO>COQOCQC OQCO>O>C>Q COCOIOCO>I0>
O>O>QQCOOC QO>COQCCOC COCOFQOCOQ CGCOIQCO>IQ>
>QCOOOOO>> 00>OOOO>>O OOCCOOOCCC >OCCQOO>OO
3555355355 5335555535 5355553335 5333533355
>QCOOOOO>> OO>QOOO>mO OOCC>COOCC CQCCQOOWQO
>OCOOOOO>> OQ>OOOO>>O OOCCO>COCC >QCCOOO>QO
QO>OCOC>O> GOQ>OCOOO> QQIIO>C>>IOOC QIQVOQ>>OOC
3355335333 3355335553 55ll33335l535 5|535555355
Om>mOCOZ>O mmwmncmnflm O>ISQ>O>>ICGO OIOCQQmmOQO
QO>OCOC>>G QCQ>OCOOOO OCIIQ>O>>I>OO QIO>QQ>>QOC
CZm>§CO>CO >COOOOOCC> COOCC>OQQO C>O>O>OOCQ
DDD>QCO>CO >COOOOOCC9 CQCOOCQQQO CZOZZZOOC:
CO§>OCO>CO >CQOOOOCM> COCOOCQOQO C>O>O§OQCZ
CO>>§CO>CO wCQOOOOCC> CQ>OOCQOOO C>O>O>OOCQ
CQOC>IO>O>O OOO>QOO>>Q COIOCO>IOOOO Q>OOC>§COO
OCOCC>ZZO>Q QO>>00>>>Q OOIOOOvIOmOO >>OOOm>COO
OCC>Z>O>>>Q OQ>>OO>>QO OCIOOQ>I>OQC >>OO>>>>OQ
O>O>>IO>>>O OQO>OOO>>> OOIQOQ>IOOCC >>0005>C00
CCOCO>QCCO OQ>CCOC>OQ OCO>>>OCOO OOCxO§CO>>
>COCZZQCCO QO>COQO>QQ OCOOZZOCOQ OOCCZQCQ>>
OCOCOWOCCO 00>meobmm OCQO>NOCOO OOCOOOCO>>
CCOCO>QCCO OO>COOO>OC OCOO>>OCOO >OCOOOCQD>

moczm>mccz
acnmm>>cno
mcooom>com
mocmm>>com

_ocu
——oo

_oca
_—EQ
__uo
-Qo

__&§
—_cu
_NO~
—Noc

_gcA
_nnm
_NQ_
_Nuo

_Nbu
_mba

_uoo

_Ncw
~uba
_uQN
_wuo

131

EOQ ocvocgcoo ovovcommng cmocmomocz $8888 o 3%

>50E ncmmcmvcnm ODQFCOmQIOD CQOCZOQOC 5.3
kmznw OC>OC>>COQ 0300 58
Pat? OCDQCECOO OOQvCOLrOIOLr COOOOOQQCQ gcvnmccnn OOQQOOCGOC zoo
ESQ 55¢
EEVSL 53
3.2.N 58
b.9459 3.05.0300000 OOCO>O>OO> OQ>OwOCCCQ anmrnnomv meCOQQCQVO 750
ESQ 5:
EESL 53
32$ 58
PER: IOCgOOCI I ICCC II I I>QO>OOO>OO OQOIOOgOQC OQQLVOLVQLEQ >CCQQQQCO> Goo
ESQ 55E
>LuQL 53
3.2-N 5am
FEES _ vmcnmcgnv >QQC>QOOQC LEOOQEOQC QOQOOCOQ>C O>OOCOOCCC awe
Eu. 0 Q 55>
EuSL 53
.LE'LE 58

FEE 0c 33

132

Figure 1a, b. 168 rRNA phylogenetic tree of 112/CO2 acetogenic
bacteria from termite guts with various other eubacteria presented in
bush (a) and bar(b) formats. The scale bars are in percent nucleotide
substitutions per sequence position. SFC-S, Clostridium mayombei strain
SFC-S (Chapter 3); APO-1, Acetonema elongata strain APO-1 (Chapter 2);
JSN-2, Sparomusa termitida strain JSN-2 (Breznak et al., 1988, Chapter
2). The following sequences were kindly provided by C. R. Woese, U.
Illinois: Sporo, Sparomusa paucivorans; Megas, Megasphaera elsdenii;
bsubt, Bacillus subtilis; C.ami, Clostridium aminovalericum; C.lit,
Clostridium lituseburense; C.sti, Clostridium sticklandii; C.pur,
Clostridium purinilyticum; C.lim, Clostridium limosum; C.pas,
pasteurianum; C.per, Clostridium perfringens. The tree was constructed
using the least squares method [Fitch, W. M. and E. Margoliash (19C7),
Construction of phylogenetic trees: a method based on mutational
distances as estimated from cytochrome 0 sequences is of general

applicatability. Science 155:279-284].

 

133

0.32
OLE“

0:5

0.35

Du:

ESQ

0.5

O.» .

Est

525

 

 

 

>10-—

our—V—

znwmm

134

 

$3—

 

 

5:3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Emu—2 |li ulii lili 4
_-;_<
WZIL .l ..
32.x ilL
con—U
nan—.0
IL
5:0
can—.0
:aU
nay—m
EU

 

 

_EaU

 

 

 

 

 

 

 

 

 

 

 

"IWEIEEWA'ENMMTE‘EMWE‘W