Fermentation and anaerobic decomposition in a hot spring microbial mat
by Karen Leigh Anderson
A thesis submitted in partial fulfillment of requirements for the degree of Master of Science in
Microbiology
Montana State University
© Copyright by Karen Leigh Anderson (1984)
Abstract:
Fermentation was investigated in a low sulfate hot spring microbial mat (Octopus Spring) according to
current models on anaerobic decomposition. The mat was studied to determine what fermentation
products accumulated, where in the mat they accumulated, and what factors affected their
accumulation. Mat samples were incubated under dark anaerobic conditions to measure accumulation
of fermentation products. Acetate and propionate (ca. 3:1) were the major products to accumulate in a
55&deg,C mat. Other products accumulated to a much lesser extent. Incubation of mat samples of
varying thickness showed that fermentation occurred in the top 4mm of the mat. This has interesting
implications for fermentative organisms in the mat due to the diurnal changes in mat oxygen
concentrations. Fermentation measured in mat samples collected at various temperatures
(50&deg,-70°C) showed acetate and propionate to be the major accumulation products. According to
the interspecies hydrogen transfer model, the hydrogen concentration in a system affects the types of
fermentation products produced. At a 65° C site, with natural high hydrogen levels, and at a 55°C site,
with active methanogenesis, fermentation product accumulation was compared. There was a greater
ratio of reduced fermentation products to acetate, with the exception of propionate, at 65°C. Ethanol
accumulated at the 65°C site, as did lactate, though to a lesser extent. Artificial induction of an elevated
hydrogen environment with the addition of 2-bromoethanesulfonic acid to 55°C mat samples only
produced a substantial difference in the ratio of acetate to ethanol. Mat samples incubated in the light
had less acetate accumulation than corresponding samples incubated in the dark. This might be due to
inhibition of product formation by photosynthetically-derived oxygen or to photoincorporation of
fermentation products. A heterotrophic potential experiment showed that acetate, lactate, and ethanol
had the greatest potential for uptake by the microbial population at a 65°C site. These results correlate
with the lack of propionate accumulation at 65°C (propionate had the least potential for uptake at 65°C
of the compounds tested), and with the accumulation of ethanol. The results also point out that placing
importance on fermentation products by their accumulation data alone may be misleading. F E R M E N T A T I O N A N D 'A N A E R O B I C D E C O M P O S I T I O N
IN A HOT SPRING M I C R OB I A L MAT
by
Karen Leigh A n d e rs o n
A thesis s u b m i t t e d in partial f u l f illment
of r e q u i r e m e n t s for the degree
of
Master of S c i ence
in
Microbiology
M O N T A N A STATE U N I V ER S I T Y
Bozeman, M o n t a n a
June 1984
ii
APPROVAL
of a thesis s u b m it t e d by
Karen Leigh A nderson
This thesis has been read by each member of the thesis
c o m m it t e e and has been found to be s a t i s f a c t o r y regarding
content, English usage, format, citations, b i b l io g r a p h i c
style, and consistency, and is ready for s u b m i s s i o n to
the Col l eg e of G r a d ua t e Studies.
G raduate C o m m it t e e
Ckf-Chai r p e r s o n ,
G raduate Com m it t e e
A p p r ov e d for the Major Department
z
/
Dat
i
/ < yy
^
/ /
Head, Major Department
Appr ov e d for the College of G raduate Studies
Difi
<r.
^
Gra d ua t e ^Dean
iii
S T A T E M E N T OF P E R M I S S I O N TO USE
In p r e s e n t i n g this thesis in partial f u l f il l m e n t of
the r e q u i r e m e n t s for a mas t er ' s deg r ee at M o n t a n a State
University, I agree that the L i b r ar y shall make it available
to b o r r o w e r s under rules of the Library.
Brief quotations
from this thesis are a l l o w a b l e wit h ou t special permission,
p r o v id e d that a c c u ra t e a c k n o w l e d g m e n t of s o urce is made.
P e r m i s s i o n for e x t e n s i v e q u o t at i o n from or r eproduction
of this thesis may be g r a n te d by my major professor, or
in his/ he r absence, by the D i r e ct o r of L i b r ar i e s when,
in the o p i n io n of either, the p r o p os e d use of material
is for s c h o l a r l y purposes.
Any cop y in g or use of the
material in this thesis for financial gain shall not be
allowed wit h ou t my w r i t te n permission.
Signature
Date
d. Ar\cLzA^as&ir~
8 ^ 19 8 V
V
ACKNOWLEDGEMENTS
I e x p r es s ray a p p r e c i a t i o n to ray advisor, Dr. David
Ward, for s h a r in g his insight and e n t h u s i a s m in tac k li n g
res e ar c h p r o b l e m s in microbial ecology.
I also thank ray
c o m m i t t e e members, e s p e c i a l l y Dr. Temple, for their
critical c o m m en t s on this project.
T h e use of e q u i pm e n t
and f a c i li t i e s was made p o s s ib l e by many patient faculty
members, g r a d u a t e students, and staff members in the
m i c r o b i o l o g y department.
In Dr. War d 's laboratory,
a s s i s t a n c e by Bill R u t h e r f o r d in gas c h r o m a t o g r a p h y a?
well as th^ help from Tim Tayne and Mary B a t e so n in the
field were indispensible.
And finally, I thank Mark for
h i s humor and e n c ouragement.
vi
T ABLE OF CON T EN T S
Page
V I T A ............... .................. .
. .
iv
ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
LIST OF T A B L E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
viii
LIST OF FIGURES
. . . ■. . . . . . . . . . . . . . . . . . . . . . . .
x
ABSTRACT....................................
xi
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . .
R a t i o n a l e for Study of Hot Spring
M i c r o b i a I Mats .. . . . . . . . . . . . . . . . . . . .
M i c r o b i o l o g y of Hot Spring
Microbial Mats . . . . . . . . . . . . . .
Current Mod e ls of A n a e r o b i c
. Decompositi o n . . . . . . . . . . . . . . . . .
A n a e r o b i c D e c o m p o s i t i o n in Hot
Spring Microbial M a t s . . . . . . . . . . . . . . .
M A T E R I A L S AND M E T H O D S
. . . .. . . . . . . . . . . . . .
Study Area .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A c c u m u l a t i o n of F e r m e n t a t i o n
P roducts .. . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors A f f e c t i n g F e r m en t a t i o n
Product A c c u m u l a t i o n . . . . . . . . . . .
H e t e r o t r o p h i c Potential . . . . . . . . . . . . .
Analytical M e t h od s . . . . . . . . . . . . . . . . . . . . . .
RESU LT S
....... , ............... .
F e r m e n t a t i o n Pro d uc t A c c u m u l a t i o n . . . . . . . . . . . .
L o c a ti o n of Pro d uc t A c c u m u l a t i o n . . . . . . .
Factors A f f e c t i n g Product
Accumulation........
P o p u l a t i o n Potential for U p t a ke of
F e r m e n t a t i o n P roducts . . . . . . . . . . . . . .
DISCUSSION...........................
I
3
6
15
19
19
20
22
23
25
32
32
?2
37
46
50
vii
T ABLE OF C O N T EN T S (continued)
Page
LITERATURE C I T E D . . . . . . . . . . . . . . . . . . . . . . . . . . .
63
APPENDIX................. ..................
71
viH
LIST OF TABLES
Table.
page
1.
F e r m e n t a t i v e o r g a n i s m s i 90] ate<t, f roip Optp pw s
Spring and their known f e r m e n t a t i o n
s u b s t r a t e s and prod uc t s ... . . . . . . . . . . . . . . . . . 17
2.
Ratios of f e r m e n t a t i o n prod uc t s to gcet at e
p r o d uc e d after a 96 h dark ana e ro b i c
i n c u b a t i o n of 55° and 6 5 * C Octopus
Spri ng m a t . . . . . . . . . . . . . . . . . . . . .
3.
4.
41
Ratios of f e r m e n t a t i o n p r o d u c t s to acet at e
p r o d uc e d after a 95 h dark a n a e ro b i c
i n c u b a t i o n of 55* C Oct o pu s Spr i ng mat
in the p r e s en c e and a b s e nc e of 2-bromoe t h a n e s u l f o n i c acid (BES) . . . . . . . . . . . . . . . . .
45
Effect of light on a c e t at e a c c u m u l a t i o n in
O c t o pu s Spring and M u s h r o o m Spring 55* C m$t
sam p le s . . . . . . . .
, .......
46
5.
V max for uptake and o x i d at i o p of ^ C 02 of
l ^ c - f e r m e n t a t i on p r o d uc t s in the 1-3 mm
interval of Octo pu s S p ring 6 5 * C m a t . . . . . . . . 49
6.
Res u lt s of h e t e r o t r o p h i c potential
e x p e r i m e n t to d e t e r m i n e V max for uptake
and m e t a b o l i s m of f e r m e n t a t i o n products in
the 1-3 mm interval of O c t o pu s Spring
65* C mat .. . . . . . . . . . . . . . . . . . . . . .
71
ix
LIST OF FIGURES
Figure
Page
1.
A c c u m u l a t i o n of v o l a t i l e fatty acids
d u r i ng dark a n a e r o b i c i n c u b a t i p n of mat
samp le s from the shoulder, 4 7 ° - 4 9 e C, and
the s o u t h e r n e f f l u e n t channel, 5 0 ® - 5 2 ° C,
of O c t o pu s S p r i n g . . . . . . . . . . . . . . . . . . . . . . . . . . p3
2.
Depth p r o f il e of a c e t a t e and p r o p io n a t e
a c c u m u l a t i o n after a 54 h dark ana e ro b i c
i n c u b a t i o n of mat s a m p le s from Octopus
S p r i ng 5 5 ® C . . . . . . . . . . . . . . . . . . . . . . . . .
35
A c e t a t g and p r o p i o n a t e a c c u m u l a t i o n in
mat samp le s c o l l e c t e d at various t e m p e r a t u r e s
in Octopus S p ring . . . . . . . .
36
A c c u m u l a t i o n of h y d r o g e n and met h an e in
mat s a m p l e s icgllep^g^ at 55® and 6 5 ® C in
Oct p pu s Spri n g . . . . . . . . . . . . . . .
38
A c c u m u l a t i o n of a c e t a t e and p r o p i o n a t e
in mat samples c o l l e c t e d at 55* and 6 5 ® C in
Oct o pu s Spring .. . . . . . . . . .
3^
A c c u m u l a t i o n of other f e r m e n t a t i o n prod uc t s
in mat samples c o l l e c t e d at 55* and 6 5 ® C in
O c t o pu s Spring . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
3.
4.
5.
6.
7.
A c c u m u l a t i o n of h y d r o g e n and met h an e in the
p r e s e n c e and a b s e n c e of 2- b r o m o e t h a n e s u l fonic
acid (BES) in s a m p l e s from a 5 5 ® C Oct o pu s
Spr i ng s i t e . . . . . . . . . . . . , . . . . . . . . . . . . . . . 42
8.
A c c u m u l a t i o n of a c e t at e and pro p io n a t e
in the p r e s e n c e and a b s e nc e of
2 - b r o m o e t h a n e s u l fonic acid (BES) in sam p le s
from a 5 5 ® C Oct o pu s S p r i ng s i t e . . . . . . . . . . . . 43
9.
A c c u m u l a t i o n of other f e r m e n t a t i o n prod uc t s
in the p r e s e n c e and a b s e nc e of
2 - b r o m o e t h a n e s u l fonic agid (BES) in samp le s
from a 5 5 ® C O c t o pu s S p ring site . . . . . . . .
44
X
LIST OF FIG U RE S (continued)
Figure
10.
Page
M o d i f i e d L i n e w e a v e r - B u r k plot of the uptake
and m e t a b o l i s m of l ^ C - f e r m e n t a t i o n pro d uc t s
in the 1-3 mm interval of the 6 5 e C Octopus
Spr i ng mat . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
xi
ABSTRACT
F e r m e n t a t i o n was i n v e s t i g a t e d in a low s u l f at e hot
s p ring microbial mat (Octopus Spring) a c c o rd i n g to current
models on a n a e r o b i c decomposition. T^e mat %as stud ie d
to d e t e r m i n e what f e r m e n t a t i o n p r o d u c t s accumulated, where
in the mat they accumulated, and what factors aff e ct e d
their accumulation. Mat samples w e r e incubated under
dark a n a e r o b i c c o n d i t i o n s to m e a s u r e a c c u m u l a t i o n of
f e r m e n t a t i o n products. A c e t a t e and p r o p i o n a t e (ca. 3:1)
were the major p r o d uc t s to a c c u m u l a t e in a 5 5 * C mat. Other
pro d uc t s a c c u m u l a t e d to a much lesser extent. I n c ubation
of mat sam p le s of vary in g t h i c k n e s s showed that
f e r m e n t a t i o n o c c u r r e d in the top 4 mm of the mat. Tfjis
has i n t e r e s t i n g i m p l i c a t i o n s for f e r m e n t a t i v e o r g a ni s m s
in the mat due to the diurnal c h a n ge s in mat oxygen
c o n c entrations. F e r m e n t a t i o n m e a s u r e d in fnat sam p le s
c o l l ec t e d at various t e m p e r a t u r e s (5 0 9 - 7 0 * C ) showed acetate
and p r o p i o n a t e to be the major a c c u m u l a t i o n products.
A c c o r d i n g to the 1 n t ^ r s p e c i e s h y d r o g e n t ransfer njodel,
the h y d r o g e n c o n c e n t r a t i o n in a s y s t e m affects th% type?
of f e r m e n t a t i o n p r o d u c t s produced. At a 6 5 * C site, Wiijh
natural high h y d r o g e n levels, and at a 5 6 * C site, with
active methan o g e n e s i s , f e r m e n t a t i o n product a c c u m u l a t i o n
was compared. T h e r e was a g r e a te r r a t j o pf reduced
f e r m e n t a t i o n p r o d uc t s to acetate, with the e x c e p t i o n of
propionate, at 6 5 * C. Ethanol a c c u m u l a t e d at the 6 5 * C site,
as did lactate, tho u gh to a lesser extent. Artificial
ind u ct i o n of an e l e v a t e d h y d r og e n e n v i r o n m e n t with the
add i ti o n of 2- b r o m o e t h a n e s u l fonic acid to 5 5 * C mat samples
only p r o d uc e d a substantial d i f f e r e n c e in the ratio of
acet at e to e t h a n o l . Mat s a m p le s i n c u ba t e d in the light
had less a c e t at e a c c u m u l a t i o n than c o r r e s p o n d i n g samples
i n c u ba t e d in the dark. This might be due to inh i bi t i o n
of product f o r m a t i o n by p h o t o s y n t h e t i c a l l y - d e r i v e d oxygen
or to p h o t o i n c o r p o r a t i o n of f e r m e n t a t i o n products. A
h e t e r o t r o p h i c potential e x p e r i m e n t sho w ed that acetate,
lactate, and ethanol had the g r e a t e s t potential for uptake
by the microbial p o p u l a t i o n at a 65*C site. T h e s e results
c o r r e l a t e with the lack of p r o p i o n a t e a c c u m u l a t i o n at 65*C .
(p r o p i o n a t e had the least potential fpr uptake at 6 5 * C of
the c o m p o u n d s tested), and with the a c c u m u l a t i o n of ethanol.
The res u lt s also point out that p l a c in g i m p o r t a n c e on
f e r m e n t a t i o n p r o d uc t s by their a c c u m u l a t i o n data alone
may be misleading.
I
INTRODUCTION
This r e s e a r c h r e p r e s e n t s a c o n t i n u i n g effort to
c h a r a c t e r i z e the microbial c o m m u n i t i e s in microbial mats
found in a l k a l i n e s i l i c e o u s hot springs.
Many of these
s y s t em s are l o c a te d in Y e l l o w s t o n e National Park and have
been well d e s c r i b e d by Brock (9).
My o b j e c t i v e was to further study f e r m e n t a t i o n as
a part of a n a e r o b i c d e c o m p o s i t i o n in the Oct o pu s Spring
microbial mat.
This s y s t em is located in thd White Creek;
area pf the Lower Gey s er Basin in Y e l l o w s t o n e National
Park.
Its 9 1 ° C s o u r c e s u p p li e s a c o n t i n u o u s flow of
a l k a l i n e w ater (pH 8.3) to the microbial mats w h ich thrive
from 74° to 30°C (51).
R a t i o n a l e for Study of Hot Spring
Microbial Mats
Much a t t e n t i o n has been f o c u s e d on hot s p ring
microbial mats largely b e c a us e of interest in P r e c a m b r i an
stromatolites, m a t - l i k e s t r u c t u r e s a p p a r e n t l y formed by
ancient microbial life (49).
T h e s e fossils are comprised
of lam i na t e d s e d i m e n t a r y rocks that house m i c r o s c o p i c
s t r u c t u r e s often m o r p h o l o g i c a l l y si mi liar to fil a me n t o u s
m i c r o o r g a n i s m s (2).
Microbial mats of hot spri ng s contain
I
2
conical or c o l u m n a r s t r u c t u r e s (16, 50) similar to
s t r u c t u r e s found in s t r omatolites, and are also laminated
with depth.
Both the conical s t r u c t u r e s and the mats
con t ai n f i l a m e n t o u s organisms, such as the p h o t o s y n t h e t i c
bacterium, C h l o r o f l e x u s a u r a n t i a c u s .
It is hoped that
stu d ie s of modern ecosystems, such as the mats of hot
springs, will reveal i n f o r m a t i o n r e l e va n t to t hese ancient
s t r u c t u r e s and c o n t r i b u t e to our u n d e r s t a n d i n g of early
life on Earth.
Ecologically, the Oct o pu s S p r i ng microbial community
should be a s i mple s y s t e m for study.
High ^ e m p e r & t ^ r e g
res t ri c t the d i v e r s i t y of m i c r o o r g a n i s m s in an e c o s y s t e m
(9).
This is true for the m a t - f o r m i n g p h o t g t r p p h s in
Oct o pu s Spring, and is p r e s um e d valid for other
m i c r o o r g a n i s m s in the mats (52).
E u c a r y o t i c organisms,
i n c l ud i n g m e t a zo a n grazers, are absent above 5 0 * C (56).
The t h i c k n e s s of the Octo pu s mat and the chemical and
physical p a r a m e t e r s of the s o u r ce w a t e r have r emained
c o n s ta n t for many years (8, 9).
The major a d v a n t a g e in
s t u d y i n g the O c t o p u s mats is the abi l it y to inv e st i g a t e
a natural e c o s y s t e m that rem a in s s t a b le with time.
Finally, t h e r e is an i n c r e a s i n g interest in using
a n a e r o b i c m i c r o b e s for p r o c e s s i n g w a stes from m u n i c i p a l ­
ities, agriculture, and i ndustry to yield chemical and
fuel pro d uc t s (75).
T h e r m o p h i l i c b a c t e r i a are being used
for the industrial p r o d u c t i o n of f e r m e n t a t i o n p roducts
3
such as met h an e and ethanol (57, 75).
A better
u n d e r s t a n d i n g of a n a e r o b i c p r o c e s s e s in t h e r m o p h i l i c
e n v i r o n m e n t s could ben e fi t d e v e l o p m e n t s in industry.
M i c r o b i o l o g y of Hot S p r i ng Microbial Mats
B e c a u s e much is k nown about Octo pu s Spring, I will
c o n s id e r its m i c r o b i o l o g y in detail.
have been i d e n t i f i e d from these mats.
Few m i c r o o r g a n i s m s
The cyanobacterium,
S y n e c h o c o c c u s I i v i d u s . c o m p r i s e s the top green layer of
the mat and is r e s p o n s i b l e for the c o m m u n i t y ' s primary
p r o d uc t i o n (9, 40).
Chloroflexus aurantiacus. a fila­
mentous bacterium, makes up much of the orgnge undermat
And is r e s p o n s i b l e for much of the mat's i n t egrity (17).
Lit t le isj k n own about the a e r o bi c o r g a n i s m s in ^jhis system
The a e r o bi c bacterium, T h e r m u s a q u a t i cus (10, 52) was
i solated from O c t o pu s Spring.
M i c r o s c o p y i n dicated that
Iso c ys t i s p a l l i d a , a f i l a m e n t o u s c h e m o h e t e r o t r o p h i c
bacterium, is also an inh a bi t a n t of Oct o pu s S p r i ng (Ward,
personal communi c a t i o n , 19).
W o r k e r s have pri m ar i l y
foc u se d on a n a e r o b i c p r o c e s s e s in this system b e c a us e
it was pre s um e d that the aerobic zone, p roduced by the
top layer p h o t o t r o p h s (17), was thin rel a ti v e to the
t h i c kn e s s of the mat.
This, t o g e t h e r with other reasons
to be d i s c u s s e d below, and the int e re s t in the e c o n o m i c
potential of t h e r m o p h i l i c a n a e ro b e s e x p l a i n s why the
4
maj o ri t y of o r g a n i s m s cul t ur e d were a n a e ro b i c and
fermentative.
A s u l fate-reducer, T h e r m o d e s u l f o b a c t e r i urn
comm un e (77) and a m e t h a n o g e n i c bacterium, M e t h a n o b a c t e r i urn
t h e r m o a u t o t r o p h i cum (76) have also been isolated.
The
f e r m e n t a t i v e b a c t e r i a will be c o n s i d e r e d in grea te r detail
below.
How these o r g a n i s m s interact has been the subject
of pre v io u s research.
Doemel and Brock (17) s u ggested
that the mat was d i v i d e d into two major zones:
an upper,
a e r o bi c zone in which a d e q u a t e light is a v a i l a b l e for
photosynthesis, and a lower, dark a n a e r o b i c zone where
d e c o m p o s i t i o n predominates.
A number of studies confirm
a p h o t o s y n t h e t i cal I y - a c ti v e upper mat.
Light does not
p e n e t r a t e below 2 mm from the mat's surface due to shading
by S v n e c h o c o c c u s and the primary photic zone is restricted
to the upper 0.5 to I mm (17).
The highest c o n c e n t r a t i o n
of chlorophyll a, r e p r e s e n t i n g S y n e c h o c o c c u s . is found
w i thin the top 0.5 mm and is absent below I mm.
The
highest levels of b a c t e r i o c h l o r o p h y l I s a and c, presumably
from Chl orof I e x u s . c o r r e s p o n d ijo the 0.5 to 3 mm interval
(5).
During full sunlight, o x y g e n i c p h o t o s y n t h e s i s occurs
in the 0.5 to 1,1 mm of a 5 5 * C Octo pu s mat.
The c o n c e n t r a ­
tion of oxygen in the top 3 mm is about 6 times that of
the o v e r l y i n g water and the high es t level of oxygen peaked
in the upper I mm of the mat.
T h e s e o b s e r v a t i o n s support
active p h o t o s y n t h e s i s by S y n e c h o c o c c u s .
Below 3 mm, oxic
5
c o n d i t i o n s are lower than the levels of oxygen in the
o v e r l y i n g water, and anoxic c o n d i t i o n s prevail near 7mm
and below (40).
Interest in the s t e a d y - s t a t e nature of this system
led to stud ie s on microbial d e c o m p o s i t i o n in the mats
for a number of reasons.
These mats are above the upper-
t e m p e r a t u r e limit of m e t a zo a n graz er s (56, 61) and no
fungal d e c o m p o s e r s have been iso l at e d (9)
Consequently,
the mats depend on a p r o c a r y o t i c food chain for the
m i n e r a l i z a t i o n of o r g a n i c matter.
Doemel and Brock (17)
i n v e s t i g a t e d the p o s s i b l e steady s tate nature of the mat,
and d i s c o v e r e d that the growth rate e q u a ll e d the
d e c o m p o s i t i o n rate, with c o m p le t e d e c o m p o s i t i o n occurring
in one year.
The rates of g r owth and d e c o m p o s i t i o n tested
at sites bet w ee n 70* and 4 2 * C were opti mu m b e t w ee n 55* and
52* C.
B e c a us e high t e m p e r a t u r e limits the s o l u b i l i t y of
oxygen, d e c o m p o s i t i o n was thought to occur thr o ug h
a n a e r o b i c processes.
R e v s be c h and Ward (40) found a
diurnal c h ange in oxygen c o n c e n t r a t i o n s in this system.
T h e . ma t s are oxic d u ring the day, as d e s c r i b e d above,
but at night they are a n oxic with the e x c e p t i o n of the
top 0.5 mm.
The v a r i a b i l i t y of o x ygen levels in this
s y stem raises the q u e s ti o n of w h e t h e r aerobic or anaerobic
decomposition, or both, is important.
As indicated, little
6
is known about a e r o bi c o r g a n i s m s in the mats, with much
more, i n f o r m a t i o n known about a n a e r o b i c isolates and
processes. Studies on a n a e r o b i c d e c o m p o s i t i o n in Octopus
Spring have been f o r m ul a t e d a c c o r d i n g to current models
of this process.
B e f o re d e t a i l i n g these studies, it is
imp o rt a n t to review p r o p os e d models for a n a e r o b i c
decomposition.
Current M o dels of A n a e r o b i c D e c o m p o s i t i o n
P r e s e n t l y i n v e s t i g a t o r s a d v o c a t e a t h r e e - s t a g e scheme
to d e s c r i b e the fate of o r g a ni c mol e cu l e s in ana e ro b i c
systems (11, 33, 34).
This s c h e me has been d e v e lo p e d
from o b s e r v a t i o n s of f e r m e n t a t i o n in a number of anaerobic
environments, i n c l ud i n g sewage slu d ge and other waste
digesters, s e d i me n t s of lakes, rivers, and m a rine systems,
flooded soils, and s e d i m e n t s from the tundra, swamps,
and bogs (11).
Other sys t em s stud ie d include the rumen
of h e r b i v o r e s and the c a ecum of certain n o n - r u m i n a n t s
as well as the g a s t r o i n t e s t i n al tract of humans and animals
(33).
The stages of d e c o m p o s i t i o n are divided by the type
of m i c r o o r g a n i s m i nvolved in the process.
Fermentative
b a c t er i a are r e s p o n s i b l e for d e g r a d i n g the carbohydrates,
proteins, and lipids to fatty acids, alcohols, carbon
dioxide, hydrogen, ammonia, and sulfide.
The s e cond group,
o b l i ga t e p r o t o n - r e d u c i n g a c e t o g e n i c bacteria, degrade
7
the f e r m e n t a t i o n pro d uc t s p r o p i o n a t e and longer chained
fatty acids, alcohols, and pos s ib l y some org a ni c acids,
such as b e n z o a t e (18).
The a c e t o g e n i c bact er i a convert
these s u b s t r a t e s to acetate, hydrogen, and in the case
of odd n umbered carbon energy sources, carbon dioxide.
Finally, in low s u l f a t e environments, m e t h a n o g e n i c bacteria
are r e s p o n s i b l e for the terminal d e c o m p o s i t i o n process
of meth an e production.
Other terminal d e c o m p o s i t i o n
pro c es s e s will be c o n s i d e r e d below.
M e t h a n o g e n i c bacteria
g e n e ra t e m e t h an e from acetate, carbon dioxide and hydrogen,
or other s u b s t r a t e s such as form at e and methanol.
It is imp o rt a n t to con s id e r not only the individual
p r o c e s s i n g of o r g a ni c c o m p ou n d s by these groups, but also
the i n t e r a c t i o n among them.
As noted above, f e r m en t a t i v e
b a c t e r i a prod uc e c o m p o u n d s w hich are used by the other
two groups of organisms.
B e f o r e the d i scovery of obligate
p r o t o n - r e d u c i n g a c e t o g e n i c bacteria, it was tho u gh t that
m e t h a n o g e n i c b a c t e r i a deg r ad e d f e r m en t a t i o n pro d uc t s to
produce methane.
This idea was dispelled, however, when
Bryant d i s c o v e r e d that an abun da n t sewage methanogen,
Methanobaci 11 us o m e l i a n s k i i . which d egraded ethanol to
methane, was a c t u al l y a c o c u l t u r e of two o r g a n i s m s (13).
The nonmethanogen, the s o - c a l l e d S organism, d egraded
ethanol to ace t at e and h y d r og e n (39), and the methanogen,
d e s i g n a t e d M.o.H. and later named M e t h an o b a c t e r i urn
8
b r y a n t i i , used the h y d r o g e n p r o d u c e d in the f e r m e n t a t i o n
for m e t h an e p r o d u c t i o n (72).
Dur i ng c h a r a c t e r i z a t i o n of the S organism, researchers
noted that the o r g a n i s m grew p o orly on alcohols with little
h y d r og e n production.
When it was grown with a methanogen,
its growth i n c r e a s e d dramatically, neither ethanol nor
hyd r og e n was produced, and m e t h an e a c c u m u l a t e d (39).
It was ass u me d that h y d r og e n p r o d u c t i o n by the S o rganism
i n creased in the p r e s e n c e of the methanogen.
The increased
p r o d uc t i o n of h y d r o g e n was r e f l e c t e d in the amount of
meth an e produced:
I mole of methane.
4 moles of h y d r o g e n are used to produce
The o b s e r v a t i o n of increased
m e t h a n o g e n e s i s in the p r e s en c e of a f e r m e n t a t i v e organism
was also o b s e r v e d by Schei finger, et a l . (44) in the growth
of S e l e n o m o n a s r u m i n a n t i u m with m e t h a n e - p r o d u c i n g bacteria.
Noting the shift in f e r m e n t a t i o n products, as well as
the i n c r ea s e d p r o d u c t i o n of h y d r o g e n in coculture,
r e s e a r c h e r s began i n v e s t i g a t i n g other culture systems.
Stud ie s on the i n t e r a c t i o n of bacterial iso l at e s
from the rumen showed dif f er e n t pat t er n s of f e r m en t a t i o n
product a c c u m u l a t i o n in pure c u l t u r e versus c o c u l t u r e
(71).
The c e l luly ti c R u m i n o c o c c u s albus p r o d uc e d ethanol,
acetate, formate, hydrogen, and carbon dioxide from
c e l lubiose when grown alone.
V i brio s u c c i n o g e n e s coupled
the o x i d a t i o n of h y d r o g e n or f o r m a t e with r e d u c t i o n to
s u c c i n a t e in pure culture.
When these two o r g a n i s m s were
9
grown t o g e th e r ethanol was not p r o d uc e d but s u c c i n a t e
a c c u m u l a t e d and a rise in a c e t at e c o n c e n t r a t i o n nearly
e q u a ll e d the amount of ethanol p r o d uc e d by the m o n o cu l t u r e
(22).
This example, as well as many others (14, 15, 27,
28, 55, 66, 67, 70) s u g g e s t e d that a h y d r o g e n - u s i n g
o r g a n i s m or a h y d r o g e n o t r o p h (68) caused a shift in
e l e c t r o n s away from more reduced f e r m e n t a t i o n products,
such as ethanol, to yield more o x i d i z e d products, such
as acetate.
It was also d i s c o v e r e d that the a c c u m u l a t i o n of
hyd r og e n i n h i b i t e d the growth of some organisms.
The
S o r g a ni s m grew poorly alone when grown on ethanol but
fer m en t e d ethanol to acet at e and carbon dio x id e in the
p r e s en c e of a methanogen.
D e s u l fovibrio spe c ie s fermented
ethanol or lac t at e to the same p r o d uc t s in the p resence
of a m e t h a n o g e n but grew poorly by the m se l v e s (12, 72).
P r o p i o n a t e - and b u t y r a t e - d e g r a d i n g b a c t er i a iso l at e d from
sewage slu d ge and a q u a ti c s e d i m e n t s will not grow unless
they are c u l t u r e d with a h y d r o g e n o t r o p h (7,35). These
org a ni s m s p r o v i d e the first e v i d e n c e of n o n m e t h a n o g e n i c
b a c t er i a that a n a e r o b i c a l l y d e g r a d e fatty acids without
light, sulfate, nit r at e or s i m i l a r e l e c tr o n acc e pt o r s
(35).
The b u t y r a t e - d e g r a d i n g o r g a n i s m also m e t a b o l i z e d
c a p r o a t e and capryI ate to a c e t at e and hydrogen and valerate
and h e p t a n o a t e to acetate, propionate, and h y d r o g e n (36).
T h ese b a c t e r i a have been termed o b l i ga t e p r o t o n - r e d u c i n g
10
a c e t o g e n i c bacteria, b e c a us e they must produce h ydrogen
to grow, but req u ir e an e n v i r o n m e n t in which hydr og e n
is removed (33, 34).
The d e g r a d a t i o n r e actions for the
p r o p io n a t e - and b u t y ra t e - a c e t o g e n s , for example, become
t h e r m o d y n a m i c a l l y f a v o ra b l e when the h ydrogen stress is
relieved.
This is e v i d en t when c o m p a r i n g the free energies
of pro p os e d r e a c t i o n s inv o lv e d in the c a t a b o l i s m of
p r o p i o n a t e and b u t y r a t e a I one:
A G 01(kcal/rxn)
p r o p i o n a t e + S H g O - ^ a c e t a t e + HCOg" + H+ + 3Hg0
18.2
b u t y ra t e + Z H g O - ^ Z a c e t a t e + H + + 2Hg
11.5
to the r e d u c t i o n in free e n e r g i e s to -24.4 and -9.4 kcal
per reaction, respectively, when grown in s y n t r o p h i c
a s s o c i a t i o n with h y d r o g e n - u s i n g m e t h a n o g e n s (11).
The i m p o r t a n c e of hyd r og e n r e g u l a t i o n in an e c o system
is e x p l a i n e d in a concept known as i n t e r s p e c i e s h ydrogen
t ransfer (33, 34).
In glycolysis, the r e g e n e r a t i o n of
M A D + by f e r m e n t a t i v e b a c t er i a is a c c o m p l i s h e d by shifting
e l e c tr o n s from NADH towards the production, of various
reduced products, such as ethanol, lactate, formate,
propionate, or h y d r o g e n (74).
The c o n c e n t r a t i o n of
hydr og e n in the s y stem d e t e r m i n e s whether redu ce d
f e r m en t a t i o n prod uc t s or h y d r o g e n will be formed.
This
can be seen in the f o l l ow i n g t h e r m o d y n a m i c reaction:
MADH + H + -^Hg + W A D +
A G 0 ' = +4.3 k c a l /r x n
11
Note that this r e a c ti o n is endergonic, and thus oxidation
of NADH to p r o d u c e hyd r og e n will not be fea s ib l e until
the products, namely h y d r og e n and N A D + are r e m o ve d from
the s y stem (34).
H y d r o g e n removal by h y d r o genotrophs,
such as methanogens, will force the r eaction to the right
and allow f e r m e n t a t i v e o r g a n i s m s to produce more oxidized
prod uc t s (33).
Not only does i n t e r s p e c i e s h y d r og e n
t ransfer result in a d i f f e r e n t p r o p o r t i o n of redu ce d
f e r m e n t a t i o n p r o d uc t s than if the f e r m e n t a t i v e o r g a ni s m
is grown in pure culture, but an inc r ea s e in s u b s t r a t e
use was shown in several c o c u l t u r e e x p e r i m e n t s in which
a h y d r o g e n o t r o p h was e m p l o y e d (74).
More ATP is
s y n t h e s i z e d by the n o n m e t h a n o g e n because p y r u va t e can
be oxi d iz e d to a c e t at e and c a rbon dioxide via acetyl -CoA
with the g e n e r a t i o n of I mole of A T P / m o l e of a c e t at e formed
(28, 74) and g r e a te r growth is seen for the f e rmentor
(15, 67 ,74).
From the above observations, i n t e rs p e c i e s hydrogen
t ransfer can be divi de d into two categories.
The first
involves n o n o b l i g a t o r y i n t e r a c t i o n s between m e t h a n o g e n i c
b acteria or other h y d r o g e n o t r o p h i c b acteria such as sulfate
reducing bacteria, and f e r m e n t a t i v e bacteria in which
h ydrogen use b e n e fi t s both organisms.
If the h y d r o g e n o ­
trophi c o r g a n i s m is removed from the c o culture c o n t ai n i n g
a f e r m e n t a t i v e bacterium, the f e r m en t o r finds a l t e r n a t i v e
routes to d i s p o s e of its e l e c t r o n s - - n a m e l y in s h i f t i n g
12
f e r m e n t a t i o n p r o d u c t i o n towards more reduced pro d uc t s
(71).
The sec o nd cat e go r y inv o lv e s an i n t e r a c t i o n in
which the removal of hyd r og e n is essential to the
f u n c t i o n i n g of o b l i g a t e p r o t o n - r e d u c i n g a c e t o g e n i c bacteria
(74).
M e t h a n o g e n s p e r f o r m two principal f u nctions in mixed
cult ur e fermentations.
T h r o ug h u t i l iz a t i o n of h ydrogen
for methano g e n e s i s , they keep e l e c t r o n flow from f e r m e n t a ­
tive b a c t e r i a towards proton reduction.
Thus a shift
in reduced to o x i d iz e d f e r m e n t a t i o n products t akes place.
A c e t o g e n s are also s u p p o r t e d by this proton transfer.
And, finally, some m e t h a n o g e n i c bac t er i a p r o d uc e methane
from acet at e g e n e r a t e d by f e r m e n t a t i o n and i n t e r s p e c i e s
hydr og e n t r a n sf e r rea c ti o n s (33).
Work er s rec e nt l y i n v e s t i g a t e d a bacterial mixture
cap a bl e of d e g r a d i n g s u c r os e to met h an e and car b on dioxide
(23).
The o r g a n i s m s i ncluded a f e r m e n t o r , two acetogens,
and two m e t h a n o g e h s w h o s e pure c u l t u r e c h a r a c t e r i s t i c s
were known.
By c u l t u r i n g the f e r m en t o r with various
c o m b i n a t i o n s of the other organisms, workers d e m o n s t r a t e d
the i m p o r t a n c e of i n t e r s p e c i e s h y d r o g e n tra n sf e r and
a c e t o g e n i c a c tion on the the types of f e r m e n t a t i o n products
formed.
I n t e r s p e c i e s h y d r og e n t r a n s f e r has also been studied
in natural systems.
The r uminant system, for example,
has been well c h a r a c t e r i z e d (21, 69, 71).
A c t i ve
13
m e t h a n o g e n e s i s in this e n v i r o n m e n t mai n ta i n s a low hydrogen
c o n c e n t r a t i o n (IO "4 a t m o sp h e r e s ) and the dom i na n t f e r m e n t a ­
tion p r o d uc t s are acetate, propionate, and butyrate.
Ethanol and other red u ce d f e r m e n t a t i o n p roducts are
c o n s id e r e d absent in the rumen due to i n t e r s p e c i e s hydrogen
t ransfer of e l e c t r o n s from h y d r o g e n to methane p r o duction
(69).
O b l i g a t e p r o t o n - r e d u c i n g ace t og e n s have not been
isolated frpm the rumen.
P r o d uc t s such as p r o p i o n a t e
and b u t y r a t e are a b s o rb e d by the rumen walls for use by
the r u m i n a n t (69) and a c e t o g e n i c b a c t er i a p r o b ab l y would
not occur to any s i g n i f i c a n t e x t e nt in the rumen unless
there was a r e d u c t i o n in the t u r n o v e r of rumen contents
(37).
A n a e r o b i c d e c o m p o s i t i o n has also been s t u d ie d in
aquatic sediments.
F e r m e n t a t i o n leading to m e t h an e
p r o d uc t i o n as the the terminal d e c o m p o s i t i o n p r o c es s was
noted in several syst em s (29, 54, 62, 64, 65) and acetate
is the pri m ar y s u b s t r a t e for m e t h a n o g e n e s i s in aquatic
s e d i me n t s (11).
S t u d ie s on microbial p o p u l a t i o n s in
sed i me n t s from a e u t r o p h i c lake i n dicated the imp o rt a n c e
of i n t e r s p e c i e s h y d r o g e n t r a n s f e r (25).
S e d i m e n t s labeled
with [ U - I4C] g l u c os e and i n c u ba t e d with 100% h y d r og e n
p roduced less a c e t at e and more lac t at e than c o r r e s p o n d i n g
samples i n c u b a t e d with 100% nitrogen. Evi d en c e for the
I
p r e s en c e of a c e t o g e n i c b a c t er i a in an aquatic s y s t e m was
14
noted In a f r e s h w a t e r river bed (4) and in a e u t r o p h i c
lake s e d i me n t (29).
In the river sediment, b u t y ra t e
t urnover was i n h i bi t e d by the a d d i t i o n of hydrogen.
In
the lake sediment, hyd r og e n c o m p l e t e l y inh i bi t e d the
m e t a b o l i s m of propionate, iso-butyrate, iso-valerate,
and val e ra t e added to the sediment, whereas gre a te r than
90$ of added vol a ti l e fatty acids were m e t a b o l i z e d in
controls.
I n h i b i t i o n of m e t h a n o g e n e s i s in t h e s e sediments
also r e s u lt e d in an i m m e di a t e a c c u m u l a t i o n of h y d r og e n
and fatty acids.
The i m p o r t a n c e of m e t h a n e - p r o d u c i n g b a c t e r i a in a
d e c o m p o s i t i o n s c h e me is fully r e a l i z e d when r e v i e w i n g
the e f f e c t s of a high hyd r og e n e n v i r o n m e n t on an anaerobic
community.
T h e s e are the most important o r g a n i s m s capable
of. catabol i zi ng a c e t at e and h y d r o g e n to gaseous products
in the a b s e nc e of light energy or e x o g en o u s e l e c t r o n
acc e pt o r s such as oxygen, sulfate, and nitrate.
Without
the m e t h a n o g e n i c bacteria, e f f e c t i v e m i n e r a l i z a t i o n would
stop b e c a u s e n o n g a s e o u s red u ce d products, of f e r m e n t a t i o n
would a c c u m u l a t e (11).
S u l f a t e - r e d u c i n g bac t er i a can o u t c om p e t e methanogen.s
for a v a i l a b l e h y d r og e n and a c e t a t e in high s u l f a t e
e n v i r o n m e n t s (11, 54) and s h ould be c o n sidered when
s t u d yi n g a n a e r o b i c d e c o mposition.
Mar i ne e n v i r o n m e n t s
(3, 26, 45, 47) and a hot s p r i ng microbial mat (53) are
e x a m pl e s of high s u l f a t e e n v i r o n m e n t s with a c t i ve sulfate
15
reduction. A d d i t i o n of s u l f at e to low sulfate e n v i r o n m e n t s
was also shown to inhibit m e t h a n o g e n e s i s with a c o n c omitant
s t i m u l a t i o n of s u l f at e r e d u c t i o n (63).
The major acetate-
users in a s a l t m a r s h and in m a r i ne sed i me n t s w e r e sulfater educing b a c t e r i a (3, 47).
The o x i d a t i o n of s h o r t - c h a i n
fatty acids was noted a s s o c i a t e d with sulfate r e duction
(26).
The a d d i t i o n of sod i um molybdate, an inh i bi t o r
of s u l f at e reduction, sto p pe d p r o p i o n a t e and b u t y ra t e
d e g r a d a t i o n (3, 47) as well as the m i n e r a l i z a t i o n of
propionate, lact at e and free a mino acids (46).
T h ese o b s e r v a t i o n s s u g g e s t e d that s u l f a t e - r e d u c i n g
b a c t er i a are imp o rt a n t in a n a e r o b i c decomposition.
A
two s tage pro c es s has been p r o p o s e d for the fate of organic
com p ou n d s in high s u l f a t e e n vironments.
Fermentative
bact er i a p e r f o r m the first s t age of d e g r ad a t i o n and
s u l f a t e - r e d u c i n g b a c t er i a o x i d i z e reduced f e r m e n t a t i o n
products, thus f u l f i l l i n g the roles of a c e t pg e n s and
m e t h a n o g e n s in low s u l f a t e s y s t em s (54).
A n a e r o b i c D e c o m p o s i t i o n in Hot
S p ring Microbial Mats
I n v e s t i g a t i o n s of a n a e r o b i c d e c o m p o s i t i o n in hot
spring microbial mats suggest that both the 2 and 3 stage
models exist.
Ward and 01 son (53) showed that sulfate
red u ct i o n d o m i n a t e d m e t h a n o g e n e s i s in Bath Lake, a high
sul f at e hot spring.
A c e t at e and propionate, as well as
16
other v o l a t i l e fatty acids, a c c u m u l a t e d as s u l f a t e was
dep l et e d in s a m p le s inc u ba t e d under dark a n a e r o b i c
conditions.
T h e s e o b s e r v a t i o n s suggest a 2 stage model
for a n a e r o b i c d e c o m p o s i t i o n in Bath Lake.
P r e v i o u s work
on the Octo pu s Spr i ng microbial mat sug g es t s a 3 stage
model for a n a e r o b i c d e c o mposition.
In this low sulfate
environment, m e t h a n o g e n e s i s is a c tive (51) and h ydrogen
and carbon dioxide, not acetate, are important methane
p r e c ur s o r s (43).
R a d i o l a b e l e d a c e t a t e added to the mats
was i n c o r p o r a t e d by long f i l a m e n t o u s o r g anisms r e s e mbling
the p h o t o t r o p h i c b a c t e r i u m from mat communities, C h l o r o f lexus a u r a n t i a c u s (43).
T ayne (48) i n v e s t i g a t e d the
fate of a c e t a t e t o g e t h e r with other f e r m e n t a t i o n products
and found that acetate, propionate, butyrate, lactate,
and ethanol were p h o t o i n c o r p o r a t e d by a strain of
C h l o r o f l e x u s i solated from the mat.
C a t a b o l i s m of these
com p ou n d s in the dark, e s p e c i a l l y under dark a n a e r o b i c
conditions, was not significant, with the e x c e p t i o n of
lactate, w h i c h was c a t a b o l i zed under all i n c u b a t i o n
conditions. T ayne also found e v i d e n c e for b u t y r a t e
acetogenesis, a l t h ou g h this was not c o n s idered an
important proc es s in the mats (48).
R e c y cl i n g of
f e r m e n t a t i o n pro d uc t s to C h l o r o f l e x u s was p r o p o s e d as
an a l t e r n a t i v e to f e r m e n t a t i o n pro d uc t c a t a b o l i s m often
noted in other natural systems.
17
L i ttle is known about f e r m e n t a t i o n in Octopus Spring.
Doemel and Brock (17) showed that the c o n c e n t r a t i o n of
protein d e c r ea s e d with depth in the mat, i m p l icating
f e r m e n t a t i o n processes.
As m e n t i o n e d above, more
f e r m e n t a t i v e bact er i a have been isolated from Octopus
Spring than any other m e t a bo l i c group (52).
T able I lists
these s a c c h a r l y t i c org a ni s m s and their c h a r a c t e r i s t i c
f e r m e n t a t i o n s u b s t r a t e s and products.
Table I.
F e r m e n t a t i o n org a ni s m s isolated from Octopus
Spring and their known f e r m en t a t i o n sub s tr a t e s
and products.
ORGANISM
Th e r m q a n a e r q b i u m
KNOWN SUBSTRATES
(76,78)
FERMENTATION PRODUCTS
SUGARS. CARBOHYDRATES
ETHANOL, LACTATE, ACETATE.
T H FR M nB A rT FR flinFS Af-FTOFTHYt IQ lIS (6)
SUGARS, CARBOHYDRATES
ETHANOL, ACETATE, H y CO^
T h f r m o a n a f r q b a q t f r f t h a n o i i q i i s (59)
SUGARS, PYRUVATE
ETHANOL, CO,, ACETATE, LACTATE
SUGARS, CARBOHYDRATES,
PYRUVATE
ETHANOL, LACTATE, ACETATE.
SUGARS
ACETATE
Cl
brockii
n S T R in illM THFRMQAllTQTRQPHt QtIM ( 58)
CV H2
H2
2
cY H2
My role in the c o n t i n u i n g i n v e s t i g a t i o n of anaerobic
d e c o m p o s i t i o n in the Octopus S p ring microbial mats has
been to i n v e s t i g a t e fermentation.
The f o llowing aspects
of f e r m e n t a t i o n were addressed:
1)
2)
3)
What f e r m e n t a t i o n pro d uc t s a c c u mulate?
Where do these p roducts a c c u mu l a t e in the mat?
What factors could affect the a c c u m u l a t i o n of
these pro d uc t s ?
The studies d e s i gn e d to answer these questions attempted
to further i n v e s t i g a t e a unique sys t em in which a
18
p h o t o h e t e r o t r o p h appears to play a role in the fate of
f e r m e n t a t i o n products.
19
MAT E RI A L AND M E T H O D S
.
Study Area
E x p e r i m e n t s were c a r r ie d out at Oct o pu s Spring located
in the Lower Geyser Basin of Y e l l o w s t o n e National Park
(see 17 for s p e c i f i c location).
This area was chosen
to study d e c o m p o s i t i o n as it has been the subject of
pre v io u s i n v e s t i g a t i o n s on a n a e r o b i c p r ocesses (17, SI,
52, 53) and the microbial s y stem has been well studied
by others (5, 9).
Much of the work was p e r f or m e d at a
site south of the main source.
This s houlder area was
s e p a r a t e d from the s o urce by pt sinter barrier which allowed
a g e ntle flow of water over the microbial mat.
Samples
were more h o m o g e n e o u s and t e m p e r a t u r e f l u c t u a t i o n s were
less ( 5 5 * C i 2 * C ) in the shou ld e r than at other areas of
the Spring.
Samples w e r e . a l s o c o l l ec t e d from sites at
50*, 60®, 65®, and 7 0 ® C in the s outhern e f f l u e n t channel.
Anot he r study area, M u s h r o o m Spring, also located
in the Lower Geyser Basin (9) has a microbial mat similar
in s t r u c t u r e and c o m p o s i t i o n to that found at Octopus
Spring.
The inf l ue n c e of light on f e r m e n t a t i o n product
a c c u m u l a t i o n was stu d ie d at a 5 5 ® C M u s h r o o m Spr i ng site.
20
A c c u m u l a t i o n of F e r m e n t a t i o n Pro d uc t s
Unless o t h e r w i s e noted, all e x p e r i m e n t s des i gn e d
to m e a s ur e t h e a c c u m u l a t i o n of f e r m e n t a t i o n prod uc t s
prod uc e d under dark a n a e r o b i c c o n d i t i o n s were p e r f or m e d
as follows.
Vertical core s a m p le s removed from the
microbial mat with a #4 cork borer (I cm x 50.3 m m 2 ) were
placed in I dram glass vials (14.5 x 45 mm, Kimble).
A n a e r o b i c c o n d i t i o n s were e s t a b l i s h e d by c o n t i n u o u s l y
f lushing a s t r e am of nit r gg e n gas over the c o n t a i n e d
samples.
Vials w e r e sealed with butyl rubber sto p pe r s
(00, Thomas) and then w r a p pe d at the g l a s s - r u b b e r interface
with black electrical tape to s e c u re the s t o p p e r s during
incubation.
Dark c o n d i t i o n s w e r e sim u la t e d by w r a p pi n g
the vials with black electrical, tape and several layers
of a l u m in u m foil.
Usu a ll y I ml of source water, which
had been b u b b le d with either h e l i u m or nitrogen, was added
to each core sample.
Vials were i n c ubated at in situ
t e m p e r a t u r e s d u ring the c o l l e c t i o n procedure.
For
t r a nsport to l a b o ra t o r y incubators, vials were t r a n sf e r r e d
to pla s ti c t h e r mo s bottles c o n t a i n i n g water at the in
situ t e m p e r a t u r e and the bottles were placed in s t yrofoam
coolers c o n t a i n i n g water 5 - 1 0 * C warmer.
Transportation
time to the l a b o ra t o r y was 2-3 hours, during w h i c h time
21
the t e m p e r a t u r e in the i n c u ba t o r s fell S - S 6C.
Vials were
inc u ba t e d in d a r k e n e d i n c u b a t o r s that m a i n t a i n e d a set
t e m p e r a t u r e to w i thin + S 6C.
Dur i ng dark a n a e r o b i c i n c u b a t i o n s in the lab,
s u b s a m p l e s were rem o ve d from the gas h e a d s p a c e and measured
dir e ct l y by gas c h r o matography.
Liq u id was s u b s am p l e d
and frozen (- 206C ) for later a n a l ys i s of f e r m e n t a t i o n
products.
Depth Pro f il e
From a S S 6 C site I cm cores were s e c t i o n e d with a
razor blade into the fol l ow i n g vertical intervals: the
top I inm, 0-2 mm, 0-4 mm, 0-6 mm, 0^6 mm, and OirlO mm.
T hese were trea te d as s p e c i f i e d abpve for $ t u d i ££ on
f e r m e n t a t i o n pro d uc t accumulation.
Temperature Distribution
One c e n t i m e t e r cores were sam p le d from the shoulder
at S O 6 and S S 6 C and from the s o u t he r n effl ue n t channel at
60°, 6 5 6 , and 7 0 6 C.
Vials c o n t a i n i n g samples from each
t e m p e r a t u r e were inj e ct e d with 2 ml of anoxic sou r ce water
and t r a n s p o r t e d to the l a b o ra t o r y as d e scribed above.
Upon r e t u r n i n g to M o n t a n a State University, it was noted
that the t e m p e r a t u r e of each t r a n s p o r t i n g thermos had
e q u i l i b r a t e d to 6 5 ® C .
The incubations, however, were
c o n t in u e d in lab o ra t o r y i n c u ba t o r s at S O 6 , S S 6 , 60°, 65°,
22
and 7 0 * C and s u b s a m p l e s were remo ve d at time intervals
for f e r m e n t a t i o n pro d uc t analysis.
Factors A f f e c t i n g F e r m e n t a t i o n
Pro d uc t A c c u m u l a t i o n
I n h i b i t i o n of M e t h a n o g e n e s i s
Tayne (48) sho w ed that m e t h an e p r o d uc t i o n in cores
i n c u ba t i n g under dark a n a e r o b i c c o n d it i o n s could be
i n hibited by the a d d i t i o n of 2- b r o m o e t h a n e s u l fonic acid.
A c o n c o m i t a n t rise in h y d r og e n a c c u m u l a t i o n was noted
with the i n h i bi t i o n of methanogenesis.
A #6 cork borer (I cm x 78.5 m m 2 ) was used in these
e x p e r i m e n t s as larger liquid volumes were needed or both
v o l a ti l e and n o n v o l a t i l e fatty acid analysis.
Cores
sampled at 5 5 ° C and in the s o u t he r n channel at 6 5 * C were
i n c u ba t e d using 2 dram glass vials (19 x 48 mm, Kimble),
butyl rubber s t o p pe r s (01, T h o m a s ) , and 3 ml of anoxic
sou r ce water.
Sam p le s were m o n i t o r e d for m e t h a n o g e n e s i s
When m e t h a n e levels reac he d a p p r o x i m a t e l y 1.0 umqle/vial
(48), 0.2 ml of a 0.5 M stock s o l u ti o n of 2 - b r o m o e t h a n e ­
sul f oni c acid (Sigma Chemical Company), adju st e d to pH
6.4 to match the pH of i n c u b a t i n g cores, was i njected
into half of the vials to obtain a final c o n c e n t r a t i o n
of 0.05 M ; the other half of the samp le s served as
controls.
Both gas and liquid s u b s a m p l e s were removed
at int e rv a l s until the c o m p l e t i o n of the experiment.
23
Light
Cores r e m o ve d from a 5 5 * C Octopus Spring site were
i n cubated with 1.5 ml of anoxic s o u r c e water.
Vials were
flushed with e i ther n itrogen or h e l i um and inc u ba t e d glass
}
end up in situ under light or dark c o n d i t i o n s for 5 and
10 hours.
At the end of each incubation, samp le s were
i njected with 0.15 ml of a 37% f o r m a l d e h y d e s olution
(formalin) and s h aken to stop all biological activity.
Al I formal In k i lled samp le s were assayed for fer m en t a t i o n
products.
Light i n t e ns i t y was m o n i to r e d with a Li-Cor
light meter (model L I r l 8 5 ) .
This e x p e r i m e n t was rep e at e d twice in M u s h r g o m Sprigg
at a 5 5 * C site.
After 5 1/2 and 7 hours of inc u ba t i o n
for each experiment, respectively, I ml syr i ng e s were
used to tran sf e r liquid s u b s am p l e s from vials to 1.5 ml
pla s ti c c e n t r i f u g e tubes (T h o m a s ) .
These subsamples,
d e s i g n a t e d for later f e r m e n t a t i o n product analysis, were
kept cool on ice in a s t y r o f o a m incubator d u ring transport
to the lab.
H e t e r o t r o p h i c Potential
P h o t o h e t e r o t r o p h y was i n v e s t i g a t e d in a b ioassay
of a d a p t a t i o n by the microbial mat's pop u la t i o n to take
24
up s p e c i f i c compounds.
The method of Hobbie and Wright
(73) was used to d e t e r m i n e a 6 5 * C p o p u l a t i o n ' s potential
for a s s i m i l a t i n g r a d i o l a b e l e d f e r m e n t a t i o n products.
T/F values (where T is the i n c u b a t i o n time in hours and
F is the dpm of lab e le d cells and COg divided by the total
dpm added per vial ) were r e g r es s e d on A (the c o n c e n t r a t i o n
of added s u b s t r a t e in y M ) .
The reciprocal of the slopes
of the r e g r e s s i o n lines gave the V max values for each
c o m p o u n d tested.
The V max values r e p r e s e n t e d the microbial
p o p u l a t i o n ' s potential to take up the c o m p ou n d s tested.
The method for m e a s u r i n g h e t e r o t r o p h i c potential
for a 6 5 * C microbial p o p u l a t i o n closely f ollowed that of
T ayne (48).
T h i r t e e n I cm cores g a t h er e d with a. #4 cork
borer were s e c t i o n e d to obtain, the top 1-3 mm interval.
This s e c t io n was used as it gave a max i mu m for uptake
and m e t a b o l i s m of f e r m e n t a t i o n pro d uc t s c o m p ar e d to other
int e rv a l s tested in a 5 0 ® C O c t o pu s Spring mat (48).
The
s u b s a m p l e s were h o m o g e n i z e d in a 40 ml hand t i s s u e grinder
(Wheaton) and d i l u te d in 150 ml of Octopus S p r i n g source
water.
Two m i l l i l i t e r s of the h o m o g e n a t e was added to
I dram glass vials.
T h e s e were s e a l ed with butyl rubber
sto p pe r s and the c l o s u r e s were secu re d with b lack
electrical tape.
V ials placed glass side up in a wire
rack in the flowing water were p r e i n c u b a t e d for 30 minutes
under full sunlight.
T e n - f o l d c o n c e n t r a t e d stock solutions
25
of I - 1^C labeled acetate, propionate, butyrate, lactate,
and ethanol were p r e w a r m e d in the Spring.
After
preincubation, each vial was inj e ct e d with a r a d i o labeled
f e r m e n t a t i o n product and ret u rn e d to the imm e rs e d wire
rack.
The final c o n c e n t r a t i o n s o b t a in e d of labeled
c o m p o u n d s were a p p r o x i m a t e l y 0.125, 0.250, 0.5, and
1.0 jjCi/vial, with the e x c e p t i o n of ethanol and acetate,
which were three t imes higher (see Analytical Methods).
Vials were inj e ct e d with 0.1, ml of for m al i n after incubat i on for 30 m i nutes.
For each r a d i o l a b e l e d c o m p o u n d tested, tri p li c a t e
samples at each of the four c o n c e n t r a t i o n s were analyzed
for
in the vial headspace, and for the p resence
of the radiolabel in the cell fra c ti o n and fil t ra t e from
the homogenate.
Analytical M e t h od s
Gas A n a l y s i s
H y d r og e n and m e t h a n e were m e a s u r e d by remo vi n g
0.2 ml s u b s a m p l e s from vial h e a d s p a c e s with a I ml Glaspak
s y r i ng e (Becton, D i c k inson) m o d i f i e d with a M i n i n e r t valve
(Supelco) to make it gas-tight.
S u b s a m p l e s were injected
into a gas c h r o m a t o g r a p h (Carle, model 8500) e q u i pp e d
with a thermal c o n d u c t i v i t y d e t e ct o r and s t a i n l e s s steel
column (2.3 meters by 3.18 mm 0.D) packed with Poropak N
26
(80 mesh).
The oven t e m p e r a t u r e was set at 42® C.
Nitrogen
was used as a car r ie r gas with a flow rate of 21 ml/min.
The column had been s t a n d a r d i z e d with known c o n c e n t r a t i o n s
of h y d r og e n and methane, and the area unit r e s p o n s e s of
samples i njected were c o r r e c t e d t o . v m o l e s / i n j e c t i o n by
an i n t e g r a t i n g c o m p ut e r (Spectra Physics model 4100).
Total h y d r o g e n and m e t h an e per vial were c a l c u l a t e d by
c o r r e c t i n g for h e a d s p a c e volume.
F e r m e n t a t i o n Product A n a l ys i s
Liquid s u b s a m p l e s (0.2 ml) remo ve d with a I ml syringe
flushed with nit r og e n were p l aced in 1.5 ml p l a s ti c
c e n t r i f u g e tubes and frozen (-20® C ) until analysis.
P r e p a r a t i o n of these samples for volatile fatty acid and
alcohol ana l ys i s (Rutherford, personal co m m un i c a t i o n )
was as follows.
80
jjI
T h awed s u b s a m p l e s were a c i d i f i e d with
of a 40% (w/v) a l u m i n u m s u l f a t e s olution and 4
jj!
of a 40 mM h e x a n o i c acid s o l u t i o n was added as an internal
standard.
V o r t e x e d samples were f iltered t h r o u g h 0.45 pm
m e m b ra n e filters (13 mm, M i l l i pore type HA) c o n t ai n e d
in Swihnex. filter holders.
Two m i c r o l i t e r s of the filtrate
was inj e ct e d into a t e m p e r a t u r e - p r o g r a m m a b l e gas c h r o m a t o ­
graph (Vari an model 3700) fitted with a glass column
(6 feet x 0.25 inches O.D., 2 mm I . D. ) p a cked with GP
15% S P - 1 2 2 0 / 1 % H 3P O 4 on C h r o m o s o r b W, AW (Supelco, mesh
27
size 100/120).
The i njector and flame i o n i za t i o n detector
were set at 1 7 0 * C and 2 5 0 * C respectively.
Dur i ng an
a n a l ys i s the oven t e m p e r a t u r e was p r o g r a m m e d to hold 1 0 5 * C
for 2 minutes, i n c r ea s e at a rate of 4 0 * C / m i n to 1 4 5 * C,
and hold this final t e m p e r a t u r e for 3 minutes.
Flow rates
for the dete ct o r gases were 300 ml/min for air and
30 ml/min for hyd r og e n and the carrier gas, helium, was
set at 30 ml/min.
Two s t a n da r d solutions, a v olatile
fatty acid (VFA) rumen s t a n da r d and an alcohol standard
mixture,
(both from Supelco), were used to c a l i b r a t e area
unit r e s p o n s e s (yM) m o n i t o r e d by the i n t e g r a t i n g computer.
The s t a n d a r d s o l u ti o n s could not be mixed to cal i br a t e
the c h r o matograph, as sim i la r r e t e nt i o n times for acetate
from the VFA s o l u ti o n and pentanol from the alcohol
s o l u ti o n made it i m p o s s i b l e to ac c u ra t e l y c a l i b r a t e the
two compounds.
Each s o l u ti o n was, therefore, treated
i n d i v i d u a l l y with a l u m i n u m s u l f at e and h e x a n o i c acid,
as d e s c r i b e d above, for calibration.
A p r o g ra m was
e s t a b l i s h e d on the i n t e g r a t i n g c omputer which included
data from both standa r d i z a t i o ns .
Twenty m i c r o l i t e r s of an internal standard, 10 mM
g l u t ar i c acid, was added to s u b s a m p l e s (0.2 ml) which
were then m e t h y l a t e d , a c c o r d i n g to (20) for n o n v olatile
fatty acid (nVFA) analysis. C h r o m a t o g r a p h s e t t i n g s were the
same as above, e x cept the oven t e m p e r a t u r e was initially
set at 8 5 ® C for 2 minutes, then p r o g ra m m e d to increase
J.
28
at a rate of 1 0 * C / m i n to 1 4 5 * C, which was m a i n ta i n e d for
5 minutes.
An nVFA s t a n da r d (Supelco) was used to
c a l i b r a t e this col u mn using an internal sta n da r d method.
After each run, it was nec e ss a r y to man u al l y reset the
oven t e m p e r a t u r e to 1 8 5 * C to flush out a c o n t a m i n a t i n g
peak hav i ng a later r e t e n t i o n time than the non v ol a t i l e
fatty acids.
i
This higher t e m p e r a t u r e was mai n ta i n e d for
5 minutes before the column was c o oled for another
i njection.
b e c a us e several liquid s u b s a m p l e s were s e q u e n t i a l l y
removed from a vial d u ring an a c c u m u l a t i o n study, it was
n e c essary to adjust each c h r o m a t o g r a p h i c m e a s u r e m e n t with
a c o r r ec t i o n factor to account for the prev io u s amount
of product removed.
The mM c o n c e n t r a t i o n m e a s ur e d was
a d j u st e d to pmoles/vial by m u l t i p l y i n g it by the liquid
volume in the vial at the time of subsampling.
This same
mM c o n c e n t r a t i o n was also m u l t i p l i e d by the volume of
s u b s a m p l e rem o ve d (0.2ml + 0.05ml for syr i ng e dea d sp a c e
= 0.25 ml) to d e t e r m i n e the amount removed for analysis.
Each sub s eq u e n t time point was c o r r ec t e d first to a per
tube amount and then for the amount removed in previous
analysis.
After data were; corrected, means, s t a n da r d deviations
and sta n da r d err o rs were c a l c u l a t e d for r e p l i c a t e samples.
29
P r e p a r a t i o n of R a d i o l a b e l e d Com p ou n d s
The 14C - I a b e l e d f e r m e n t a t i o n c o m p o u n d s used in the
65°C h e t e r o t r o p h i c potential e x p e r i m e n t were; [ 2 - 14C] acetic
acid (New Eng l an d Nuclear, NEN).,' 1.8 mCi/mmol; [ I - 14C]
p r o p i o n i c acid ( N E N )1 58.4 mCi/mmol; [ I - 1 4 C j b u t y r i c acid
( A m e r s h a m ) , 56 mCi/mmol; D L - [ 1 - 14C ] 1 actic acid ( A m e r s h a m ) ,
54 mCi/mmol, and [ I - 1 4 Cjethanol (NEN), 7.6 mCi/mmol.
These s o l u ti o n s were d i l u te d with anoxic fil t er e d Octopus
Spring water to obtain final c o n c e n t r a t i o n s of 0.125,
0.25, 0.5, and 1.0 yCi/vial and autoclaved.
Total
r a d i o a c t i v e counts d e t e r m i n e d for these s o l u t i o n s showed
that the counts for ethanol and a c e t at e were 2 and 3 times,
higher, respectively.
This can only be a c c o un t e d for
by dilu ti o n error in the original p r e p a r a t i o n of these
solutions.
1 4 COg Anal ys i s
S u b s a m p l e s from vial h e a d s p a c e s were i njected into
the thermal c o n d u c t i v i t y gas c h r o m a t o g r a p h (described
above) con n ec t e d by a teflon line to a gas proportional
counter (Packard, model 894) for 1 4 COg analysis.
The
gas c h r o m a t o g r a p h had a flow rate of helium of 21 ml/min.
30
The gas proportional counter had two c o m b u s t i o n furnaces
in series o p e r a t e d at 7 5 0 ° C.
H e l i um make-up gas was added
after c o m b u s t i o n to i ncrease the flow rate to 70 ml/min.
Propane, the quench gas, was added at 10% of the total
flow rate.
A M i n i g r a t o r (Spectra Physics) rec o rd e d the
gas proportional c o u n te r output.
R a d i o l a b e l e d Cells and Filtrates
After
analysis, vials w e r e vortexed, and a
1:10 dil u ti o n of the h o m o g e n a t e (0.2 ml sample, 1.8 ml
of d i s t i l l e d w a t e r ) was f iltered thr o ug h a Milli pore filter
(0.45 pm x 25 ym, type HA).
With a vacuum app l ie d to the
filter apparatus, the f iltrate was c o l l ec t e d in a small
glass vial s u s p e n d e d ben e at h the funnel.
Air dried filters
were e x p o s e d o v e r n i g h t to h y d r o c h l o r i c acid (12 N) fumes
to remove carbonates.
The filters were placed in 10 ml
of Aquasol ( M E N ) , and the fil t ra t e s (500 y l ) plus distilled
water ( 500 jj I) were p i p e tt e d into 2 ml of A q u a s o l .
Both
were cou n te d by a liquid s c i n t i l l a t i o n counter (Packard,
460 c) using the s a m p l e c hannels ratio method to correct
for quenching.
C o u n te r windows were set at (A) 0-156
KeVolts and (B) 4 - 1 5 6 K e V o l ts.
The data were r eported
as dpm per s a mple vial (average of t r i p l i c a t e vials) after
c o r r e c t i o n for s u b s a m p l e volume and dilution. Total dpm
31
r e c o v e r e d as cells and f iltrate was 83.99 I 1.27 standard
error for a s a m p le size of 60 vials.
Statistical A n a l y s i s
Linear r e g r e s s i o n was used in the h e t e r o t r o p h i c
potential study to d e t e r m i n e the s lope and co r r el a t i o n
coefficient, r, for each line plotted.
A two sample
S t u d en t ' s t test was used to c o m p a r e means c a l c ul a t e d
for the light studies.
Lund (31).
S t a t i s t i c s p rograms were from
32
R E S U LT S
Fermentation Product Accumulation
The a c c u m u l a t i o n of f e r m e n t a t i o n products in the
Octopus S p ring mat at 5 5 * C i n c u ba t e d under dark a n aerobic
c o n d i t i o n s is shown in Figure I.
The p r e d o m i n a n t
f e r m e n t a t i o n pro d uc t s found w e r e the volatile fatty acids
(VFA) acet at e and propionate.
Acetate, the major product,
a c c u m u l a t e d in a ratio of 3:1 r e l a t i v e to propionate.
Iso-butyrate, n-butyrate, iso-valerate, and n - v a le r a t e
also a c c u m u l a t e d over time.
T h e s e products, however,
reached much lower c o n c e n t r a t i o n s than did a c e t a t e and
p r o p i o n a t e (Figure I).
The trend of f e r m e n t a t i o n product
a c c u m u l a t i o n was r e p e at e d in a number of similar
e x p e r i m e n t s (data not shown).
T h e r e was no e v i d e n c e that
n o n v o l a t i l e fatty acids or a l c o h o l s a c c u m u l a t e d at 5 5 * C
during an i n c u b a t i o n period of 120 hours (data not shown).
L o c a ti o n of Pro d uc t A c c u m u l a t i o n
Depth P r o f iI e
The vertical p osition in which f e r m e n t a t i o n occurs
in the mat was i n v e s t i g a t e d by s t u d yi n g core s ections
V- >
cut to vary in t h i c kn e s s from the top to the full length
33
Acetate
Propionate
mMOLES/VIAL
O thers
A c e ta te
Propionate
40
6
INCUBATION TIME (h )
Figure I.
A c c u m u l a t i o n of v o l a ti l e fatty acids during
dark a n a e r o b i c i n c u ba t i o n of mat samples from
the shoulder, 4 7 6-49°C, and the s outhern
eff l ue n t channel, 50'-52*C, of Octopus Spring.
"Others" refers to iso-butyrate, n-butyrate,
iso-valerate, and n-valerate. Bars are standard
error (n=3).
34
of the core.
A c e t a t e and p r o p i o n a t e were again found
to be the p r e d o m i n a n t f e r m e n t a t i o n products (Figure 2).
A l t h o u g h these fatty acids a c c u m u l a t e d in the top green
layer, their a c c u m u l a t i o n was more rapid if the thickness
of the mat was i n c r ea s e d to 2 or 4 mm.
Further increases
in t h i c k n e s s did not result in more rapid a c e t a t e and
p r o p i o n a t e accumulation.
Temperature Distribution
The cy a n ob a c t e r i a ! mats at Octopus Spring extend
from sites located from 4 0 * C to 7 0 * C.
P r e v io u s studies
along this thermal gra d ie n t r e v e a l e d d i fferent patterns
of h y d r og e n a c c u m u l a t i o n and m e t h a n o g e n e s i s (43, SI).
It was, therefore, important to i n v e st i g a t e f e r m en t a t i o n
in microbial p o p u l a t i o n s found over this t e m p e r a t u r e
range.
A c e t a t e and p r o p i o n a t e were the major fer m en t a t i o n
products to a c c u m u l a t e at all t e m p e r a t u r e s (Figure 3).
A c e t at e a c c u m u l a t e d to a higher c o n c e n t r a t i o n at the higher
temperatures, wher ea s p r o p i o n a t e a c c u m u l a t e d to a higher
level at the lower temperatures.
Prof ou n d d i f f e r e n c e s
in the rates of a c c u m u l a t i o n of major f e r m e n t a t i o n products
were not observed.
35
pM O LES/VIA L
O
TOP
GREEN LAYER
2
T
3
I
PROPIONATE
4
r—
5
6
I------------ p
ACETATE
D E P TH
INTERVAL
0-2 m m -
0-4 m m -
0-6 m m -
0-8 m m _
0-10 m m L
Figure 2.
Depth pro f il e of a c e t a t e and p r o p io n a t e
a c c u m u l a t i o n after a 54 h dark ana e ro b i c
inc u ba t i o n of mat s a m p le s from Octopus
Spring 5 5 * C. Bars are sta n da r d error (n=3).
36
- PROPIONATE
- ACETATE
Y 50°
40
HOURS
Figure 3.
A c e t a t e and p r o p i o n a t e a c c u mu l a t i o n in mat
samples c o l lected at various t e m p e r a t u r e s
in Octo pu s Spring. Bars are s t a n da r d error
(n=4).
37
Factors A f f e c t i n g P r o d uc t A c c u m u l a t i o n
C o m p a r i s o n s of High and Low H y d r o g e n Sites
A c c o r d i n g to the i n t e r s p e c i e s hyd r og e n t r a n sf e r m o d e l ,
the partial p r e s s u r e of h y d r o g e n in a system aff e ct s the
types of f e r m e n t a t i o n pro d uc t s produced.
Ward (51) found
more h y d r o g e n at a 6 5 ® C site in Octopus Spring than at
a 55® C site.
Core samples taken from both a 65® C and a
5 5 ® C site were i n c u ba t e d under dark ana e ro b i c conditions.
H y d r og e n a c c u m u l a t e d at 6 5 ® C w h e r e a s methane a c c u mu l a t e d
at 5 5 ® C (Figure 4).
A c e t at e and p r o p i o n a t e were the major
f e r m e n t a t i o n prod uc t s (Figure 5).
Acet at e a c c u m u l a t e d
to higher level at 6 5 ® C wher ea s p r o p i o n a t e was higher at
5 5 ® C.
A c o m p a r i s o n of other f e r m e n t a t i o n p r o d uc t s at the
two t e m p e r a t u r e s sho w ed that the level of other V F A 1s
was higher at 6 5 ® C (Figure 6).
Ethanol also accumulated.
Lact at e a c c u m u l a t i o n was o b s e r v e d at 6 5 ® C, a l t h ou g h the
rate of a c c u m u l a t i o n was less in com p ar i s o n to acetate
and p r o p i o n a t e (data not shown).
Since the d e g r ee of f e r m e n t a t i o n may vary between
these two sites, the ratio of each fe r m en t a t i o n product
to the major product, acetate, should more a c c u rately
reflect the i m p o r t a n c e of any product.
Iso-butyrate,
38
65'
H.
55' H2
HOURS
F i g u r e 4.
A c c u m u l a t i o n of h y d r o g e n a nd m e t h a n e in mat
s a m p l e s c o l l e c t e d at 55° a n d 6 5 ' C in O c t o p u s
Spring.
B a r s a re s t a n d a r d e r r o r (55®, n=9 f i r s t
t w o p o i n t s , r e m a i n d e r n=4; 65®, n=2).
39
PROPIONATE
6
3
^
_________
_______ ___________ ___________- »
65°
_________— <
65°
O
ACETATE
2I
mMOLES/VIAL
_J
<
> 18
\
(Z)
Il I
y
O
5a.
y/
—- '" " 'I
55°
is
12
/
9
/
/
/
6
3
O
....
F i g u r e 5>.
I
20
I
40
I
HOURS
60
I
80
I
IOO
A c c u m u l a t i o n of a c e t a t e a n d p r o p i o n a t e in mat
s a m p l e s c o l l e c t e d at 55° a nd 6 5 ° C in O c t o p u s
noP Eed"9 in n E u r e rl S t a n d a r d e r r ° ri 5 ^ p 1 e
s1ze
40
mMOLES/VIAL
. ETHANOL
1-VALERATE
N-BUTYRATE
I-BUTYRATE
-• 55 °
HOURS
F i g u r e 6.
A c c u m u l a t i o n of o t h e r f e r m e n t a t i o n p r o d u c t s
in mat s a m p l e s c o l l e c t e d at 55° a nd 6 5 * C in
Oct o p u s Spring.
B a r s a r e s t a n d a r d e r ro r .
S a m p l e s i z e n o t e d in F i g u r e 4.
41
iso-valerate, and ethanol a c c u m u l a t i o n rel a ti v e to acetate
was greater at 6 5 * C than at 5 5 * C (Table 2).
T able 2.
CONDITION
Ratios of f e r m e n t a t i o n products to acetate
p r o d u c e d after a 96 h dark a n a e ro b i c incubation
of 55* and 6 5 * C Octo pu s Spring, m a t .
ACETATE
PROPIONATE ' I-BUTYRATE
N-BUTYRATE
I-VALERATE
N-VALERATE
ETHANOL .
55”
0.310
0.017
0.029
0.025
0.028
0,004
65”
0.074 -
0.039
0.039
0.069
0.025
0.076
0.24
2.29
1.34
2.76
0.89
RATIO AT
RATIO AT
65”
55”
19. PO
Artificial I n c r ea s e in H ydrogen
To further test the effect of an ele v at e d hydrogen
level on f e r m e n t a t i o n product accumulation, 2- b r o mo e t h a n e sul f on i c acid (BES) was added to a r t i fi c i a l l y induce a
high hydrogen, environment.
The data in Figure 7 showed
that control cores e x h i b i t e d m e t h a n o g e n e s i s with little
hyd r og e n accumulation, w h e r ea s those incubated with BES
showed an i n h i b i t i o n of m e t h a n o g e n e s i s and an increase
in. hydrogen.
VFA anal ys i s (Figure 8.) reve al e d little
d i f f e r e n c e in acetate, a c c u m u l a t i o n patterns between
samples c o n t a i n i n g BES and controls.
Propionate
(Figure 8) as well as iso-butyrate, n-butyrate,
iso-valerate, n - v a l erate, and ethanol were higher in the
samples c o n t a i n i n g BES (Figure 9).
Table 3 shows that
42
CONTROL
BES
BES
ADDED
---- §
HOURS
Figure 7.
A c c u m u l a t i o n of h y d r og e n and methane in the
p r e s e n c e and absence of 2- b r o m o e t h a n e s u l fonic
acid (BES) in samples from a 5 5 * C Octopus Spring
site. Bars are sta n da r d error. Sample size
for 5 5 * C noted in Figure 4; n=5 for BES samples.
43
.
PROPIONATE
ADDED
+ BES
CONTROL
mMOLES/VIAL
ACETATE
+ BES
BES
ADDED
CONTROL
HOURS
Figure 8.
A c c u m u l a t i o n of ace t at e and p r o p i o n a t e in the
p r e s en c e and absence of 2- b r o m o e t h a n e s u l fonic
acid (BES) in samples from a 55® C Octopus Spring
site. Bars are s t a n da r d error. Sample size
noted in Figure 7.
44
0.5
BES
ADDED
ETHANOL
+ BES
CONTROL
mMOLES/VIAL
O
I -BUTYRATE
0.5
O
I
40
+ BES
60
IOO
HOURS
F i g u r e 9.
A c c u m u l a t i o n of o t h e r f e r m e n t a t i o n p r o d u c t s
in t h e p r e s e n c e a n d a b s e n c e of 2 - b r o m o e t h a n e s u l f o n i c a c i d (BES) in s a m p l e s f r o m a 5 5 * C
O c t o p u s S p r i n g site.
B a r s a r e s t a n d a r d e rror.
S a m p l e s i z e n o t e d in F i g u r e 7.
45
n - v a l e r a t e a nd e t h a n o l a c c u m u l a t e d to a g r e a t e r level
r e l a t i v e to a c e t a t e in t h e B E S s a m p l e s v e r s u s t h e
controls.
L a c t a t e d i d not a c c u m u l a t e in B E S s a m p l e s
( d a t a not s h o w n ) .
In a s u b s e q u e n t B E S e x p e r i m e n t t h e
i n c u b a t i o n t i m e w a s t r i p l e d a n d t h e h y d r o g e n level h a d
d o u b l e d by t h e e n d of t h e e x p e r i m e n t .
The only
r e p r o d u c i b l e e f f e c t of h y d r o g e n w a s an i n c r e a s e in e t h a n o l
a c c u m u l a t i o n in t h e B E S s a m p l e s r e l a t i v e to t h e c o n t r o l s
( d a t a not s h o w n ) .
T a b l e 3.
R a t i o s of f e r m e n t a t i o n p r o d u c t s to a c e t a t e
p r o d u c e d a f t e r a 95 h d a r k a n a e r o b i c i n c u b a t i o n
of 5 5 * C O c t o p u s S p r i n g m at in t h e p r e s e n c e a nd
a b s e n c e of 2 - b r o m o e t h a n e s u l f o n i c ' a c i d ( B E S ) .
PROPIONATE
I-BUTYRATE
N-BUTYRATE
I-VALERATE
N-VALERATE
ETHANOL
55°
0.310
0.017
0.029
0.025
0.028
0.004
+BES
0.377
0.020
0.031
0.033
0.046
0.013
1.22
1.18
1.07
1.32
1.64
3.25
CONDITION
RATIO AT
RATIO
ACETATE
+BES
AT
E f f e c t s of L i g h t
C o r e s f r o m t h e 55* C O c t o p u s S p r i n g mat w e r e i n c u b a t e d
in s i t u u n d e r b o t h d a r k a nd s u n l i g h t c o n d i t i o n s for
10 h o u r s a n d w e r e s u b s a m p l e d f or f e r m e n t a t i o n p r o d u c t s
at t h e e n d of t h e i n c u b a t i o n p e r i o d .
Acetate accumulated
to a h i g h e r level d u r i n g d a r k i n c u b a t i o n t h a n in s u n l i g h t
( T a b l e 4).
T h e level in t h e d a r k w a s h i g h e r t h a n t h a t
46
in f ormalin c o n t r o l s s u g g e s t i n g that f e r m e n t a t i o n product
a c c u m u l a t i o n is s e n s i t i v e to light or other factors
c o n t ro l l e d by light.
For m al i n control data sho w ed that
at the time of s a m p l e collection, the level of acetate
in the mat was hig h er than after a 10 hour i n c ubation
in the light; this i n dicated that ace t at e c o n s um p t i o n
occurred.
Two other e x p e r i m e n t s p e r f or m e d on samples
from a 5 5 * C microbial mat at M u s h r o o m Spring s h owed a
similar trend of grea te r ace t at e a c c u m u l a t i o n in the dark,
versus the light (Table 4).
Table 4.
Effect of light on a c e t a t e a c c u m u l a t i o n in
Oct o pu s Spr i ng and M u s h r o o m Spring 55* C mat
samples. * i n dicates s i g n if i c a n t d i f f e r e n c e s
( p < 0 . 05) of dark sam p le s com p ar e d to light
samples.
SAMPLING
SITE
■ CONDITION
Oc t o p u s S p r i n g
M u s h r o o m Sp r i n g
EXPERIMENT # I
M u s h r o o m Sp r i n g
EXPERIMENT if
2
•
COLLECTION
TIME .
(VMOLES/VIALl SD)
ACETATE
Fo r m a l i n C o n t r o l
L i g h t (10 H r . i n c u b a t i o n )
D a r k (10 H r . i n c u b a t i o n )
0835
0835
0835
0.95 ± 0.57
0,04 ± 0.021.74 ± 0.05"
L i g h t (6 H r , i n c u b a t i o n )
D a r k (6 H r . i n c u b a t i o n )
1200
1200
0.17 ;t 0.07 .
0,45 ± 0.18*
L i g h t (6 H r . i n c u b a t i o n )
D a r k (6 H r , i n c u b a t i o n )
1200
1200
0.17 ± 0 . 1 4
0.78 ± 0.09*
•
P o p u l a t i o n Potential for Uptake
of F e r m e n t a t i o n P roducts
, .
Since the a c c u m u l a t i o n of ethanol and other products
was higher at 6 5 * C than at 5 5 * C, activity m e a s u r e m e n t s of
the higher t e m p e r a t u r e p o p u l a t i o n were made to det e rm i n e
47
if the mat m i c r o o r g a n i s m s were ada p te d to take up more
reduced f e r m e n t a t i o n products.
1 4 -C labeled acetate,
propionate, butyrate, lactate, and ethanol were tested
in the 1-3 mm interval of the m a t .
This seg m en t was
d e m o n s t r a t e d by T a y n e (48) to have a maximum for uptake
and m e t a b o l i s m for several r a d i o l a b e l e d c o m p o u n d s tested
at a 5 5 * C site..
Data for the 6 5 * C h e t e r o t r o p h i c potential
e x p e r i m e n t are r e p o rt e d on a m o d i fi e d L i n e w e a v e r - B u r k
plot in Figure 10.
on this plot.
(Note that all data are not reported
See T a b l e 6 in the A p p e n d i x for a listing
of all the data.)
The inverse s lope (Vm a x ) of each
c o m p ou n d was d e t e r m i n e d from linear r e g r es s i o n analysis
(Table 6).
The microbial c o m m un i t y at 6 5 * C had a greater
potential for a c e t at e and lact at e m e t a b o l i s m than for
the other c o m p o u n d s tested.
The potential for ethanol
m e t a b o l i s m was gre a te r than for fatty acids other than
acet at e and lactate.
48
Propionate
Ethanol
Butyrate
Acetate
actate
IO
20
30
ADDED SUBSTRATE (pM)
Figure 10.
M o d i f i e d L i n e w e a v e r - B u r k plot of the uptake
and m e t a b o l i s m of ^ C - f e r m e n t a t i o n products
in the 1-3 mm interval of the 6 5 ® C Octopus
S p ring m a t . TZF is the incubation time
divided by the fra c ti o n of label m e t a bo l i z e d
(bCi m e t a b o l ized/uCi added). Note data all
points are not shown. See Table 6 for a
c o m p l e t e listing of data.
49
T able 5.
V max for u p take and o x i d a t i o n to ^ C 02 of
l ^ c - f e r m e n t a t i o n p r o d uc t s in the 1-3 mm interval
pf O c t o p u s S p ring 6 5 * C mat. Units of V max are
pmoles of s u b s t r a t e i n c o r p o r a t e d / ! /h. r is the.
c o r r e l a t i o n c o e f f i c i e n t for a s t r a i g h t line
d e r i ve d from a linear r e g r e s s i o n of all data
points for each compound.
COMPOUND
vMAX
r
Ac
eta te
6.97
0.99
Pr
o p io n a t e
0.17
0.90
Bu
tyrate
0.52
0.93
l
1.37
0.99
c ta te
4.55
0.69
Et h a n o
La
50
DISCUSSION
S t u d ie s on a n a e r o b i c p r o c e s s e s in the O c t o pu s Spring
microbial mat showed act i ve m e t h a n o g e n e s i s and little
acetogenesis.
As little was known about f e r m e n t a t i o n
in this system, the goal of this r e s e ar c h was to further
study d e c o m p o s i t i o n of the Octopus Spring microbial mat
by i n v e s t i g a t i n g what f e r m e n t a t i o n p roducts a c c u mu l a t e d
in the mat, where they a c c u m u l a t e d within the mat, and
what factors a f f e ct e d their accumulation.
A c e t a t e and p r o p i o n a t e w e r e the p r e d o m i n a n t
f e r m e n t a t i o n p r o d uc t s to a c c u m u l a t e at a SfS0 C site.
a c c u m u l a t e d in a ratio of about 3:1.
Jhey
This a g rees well
with the i m p o r t a n c e of a c e t a t e as a d e c o m p o s i t i o n product
in many other a n a e r o b i c e n v i r o n m e n t s (54).
A c e t a t e was
r e p o rt e d the major f e r m e n t a t i o n product in lake sediments
(29).
In Lake W i n t e r g r e e n sediments, the r e l a t i o n s h i p
of the two major f e r m e n t a t i o n p roducts was sim i la r witjh
a ratio of 4.7:1 for acetate to p r o p i o n a t e (25),
Both
ace t at e and p r o p i o n a t e were the dominant f e r m e n t a t i o n
pro d uc t s found in 4 0 * C and 60*C cattle w aste dig e st e r s (32)
as well as in a high s u l f a t e salt marsh (3) and marine
s e d i me n t s (47). The low level of i m p o r t a n c e of other
v o l a t i l e fatty acids in O c t o pu s S p ring and s e d i me n t s was
also c o m p a r a b l e (3, 29, 32).
51
It is i m p o rt a n t to keep in mind that the methods
i n c o r p o r a t e d in my stu d ie s i n v o l v e d m e a s ur i n g the
a c c u m u l a t i o n of f e r m e n t a t i o n p r o d u c t s and not their
tur n ov e r by the natural populations.
A number of workers,
(3, 29, 64) i n v e s t i g a t e d the k i n e t i c s of f s r m e p ^ M p p
product d e g r a d a t i o n and their s u b s e q u e n t c o n t r i b u t i o n
to m e t h a n o g e n e s i s or s u l f at e r e d u c t i o n in natural systems.
The i m p o r t a n c e of a product in a s y s t e m may be u n d e r e s t i ­
mated by simply vie w in g a c c u m u l a t i o n data alone.
For
example, lac t at e does not a c c u m u l a t e in 5 5 ° C mat samples,
and yet T a y n e (48) sho w ed that this compound is metabolized
by the microbial p o p u l a t i o n s pre s en t under dark and light,
aerobic and a n a e r o b i c i n c u b a t i o n conditions.
Lactate,
then is pro b ab l y an imp o rt a n t f e r m e n t a t i o n s u b s t r a t e to
the microbial mat community.
To further u n d e r s t a n d a n a e r o b i c d e c o m p o s i t i o n in
the Octo pu s S p ring mat, the loc a ti o n of f e r m e n t a t i o n was
investigated.
Doemel and Brock (17) s p e c ul a t e d that
d e c o m p o s i t i o n p r o c e s s e s o c c u r r e d in the lower portion
of the mat, below the photic zone.
R evsbech and Ward
(40) d e m o n s t r a t e d that the mat was s u p e r o x i c in the upper
3 mm d u r i ng the d a y l ig h t hours with anoxic c o n d it i o n s
e x i s t i n g d u ring darkness.
S ince f e r m en t a t i o n is an
a n a e r o b i c process, one would e x p e c t f e r m e n t a t i o n to occur
either in the layers w h i c h are alw a ys anoxic (below
7 mm) or only at night, closer to the mat's surface.
52
The location of a n a e r o b i c d e c o m p o s i t i o n in the
Octo pu s Spring mat was stud ie d by i n c u ba t i n g sub s ec t i o n s
of I cm cores under dark a n a e ro b i c conditions.
That the
level of f e r m e n t a t i o n prod uc t s did not inc r ea s e when core
s e c t io n s greater than 4 mm were inc u ba t e d ind i ca t e s that
the majo ri t y of f e r m e n t a t i o n occurs within the upper
2-4 mm of the mat.
This finding relates well to the
vertical d i s t r i b u t i o n of other a n a e r o b i c p r o c es s e s in
the hot springs.
Ward (51) showed that rate of ^ethane
i
p r o d u c t i o n peaked I mm below the s u r f ac e and d e creased
with depth in a 45* and a 5 5 * C Octopus Spring.
At a 6 5 ® C
site, h y d r og e n production, and not methanogenesis, was
higher in the upper mat.
In the high sul f at e hot spring,
Bath Lake, sulf at e r e d u c t i o n was higher in the top 5 mm
interval than in the d e eper layers of the mat (53).
In studies on d e c o m p o s i t i o n in a n a e ro b i c sediments,
it has been r e p e a t e d ! y r e p o rt e d that highest rates of
a n a e r o b i c d e c o m p o s i t i o n are near the sed i me n t surface
(54).
D e p e n d i n g on the p a r t ic u l a r sys t em of study, either
m e t h a n o g e n e s i s (29, 64) or s u l f a t e - r e d u c t i o n (3, 30)
d o m i n a t e s in the upper sed i me n t interval, and decreases
with depth.
T u r n ov e r stud ie s of acetate in low and high
s u l f at e systems also reflect grea te s t rates of turnover
at the sedi me n t s u r f a c e (3, 29).
That f e r m e n t a t i o n o c c u rr e d in the upper 4 mm of the
mat has i n t e r e s t i n g i m p l i c a t i o n s with respect to daily
53
f l u c t u a t i o n s in o x y g en concentration.
The m e c h a n i s m of
survival for d e c o m p o s i t i o n organisms, which in the top
4 mm of the mats are a l t e r n a t e l y e x p o se d to s u p e r o x i c
and anoxic conditions, is unknown.
Some may be facultative
a n a e ro b e s able to s u r v i v e the diurnal changes in oxygen
levels.
Al I of the f e r m e n t a t i v e isolates from Octopus
Spr i ng are a n a e ro b e s (52); at least four are c o n s id e r e d
o b l i g a t e anaerobes.
However, one isolate T h e r m o a n a e r o -
bacter e t h a n o l i c u s . was not k i lled d u ring a one hour
i n c u b a t i o n in a e r o bi c media, but grew only under anaerobic
c o n d i t i o n s (59).
A n o t h e r isolate, C l o s t r i d i u m t h e r m o -
h v d r o s u l f u r f cum, was also shown to sur v iv e for several
days in aerated m e d i u m at 6 0 * C w i t h o u t growth (£7).
It is p o s s i b l e that in the natural mat e n v i ro n m e n t
some a n a e r o b i c o r g a n i s m s may be more t olerant to oxygen
than m i c r o o r g a n i s m s e x p o s e d to con s ta n t a n o x i c conditions.
R e v s b e c h and Ward (40) iso l at e d a m e t h a n o g e n from the
upper layers of the mat which was rather i n s e n s i t i v e to
oxygen.
This, is unusual since methanogens, as a group,
are c o n s i d e r e d strict a n a e ro b e s (33).
Oxygen c o n c e n t r a ­
tions, however, are not the only important par a me t e r when
c o n s i d e r i n g a n a e r o b i c environments.
The Eh, the
o x i d a t i o n - r e d u c t i o n potential, is also a major factor
which d e t e r m i n e s w h e t h e r or not certain o r g a n i s m s will
grow.
The c y n a n o b a c t e r i urn, O s c i l l a t o r i a t e r e b r i f o r m i s .
grows as a p h o t o h e t e r o t r o p h and a dark h e t e r o t r o p h only
54
when the redox potential is less than -100 mV.
An
o x y g e n - f r e e e n v i r o n m e n t is not s u f f ic i e n t for growth (42).
M e t h g n o g e n s r e q u ir e a low potential of less than
-300 mV (33).
It would be useful to d e t e r m i n e Eh profiles
in the Octopus S p ring mats, and comp ar e these with diurnal
f l u c t u a t i o n s of oxygen.
M i c r o n i c h e s in the top of the mat could allow these
org a ni s m s to s u r v i v e the e x t r e m e diurnal oxic f l u c t uations
The g e l a t i n o u s nature of the mat may hinder oxygen
dif f us i o n just as e x t r a c e l l u l a r p o l y s a c c h a r i d e s appear
to limit d i f f us i o n of n u trients and other mol e cu l e s to
bac t er i a e n c a se d in biofilms.
Current techniques, however
are not s e n s i t i v e e n ough for m e a s u r i n g oxygen gradients
s u r r o u n d i n g a s i n g l e bacterial cell.
As an a l t e r n a t i v e to oxygen tolerance, o r g anisms
may adjust their p l a c em e n t in the mat with cha n ge s in
oxygen concentration.
There are several e x a m p l e s of how
motile org a ni s m s p o s i t i o n t h e m s e l v e s in mats in relation
to chemical gradients.
O s c i l l a t o r i a t e r e b r i f o r m i s remain^
on the surf ac e of the microbial mat during most of the .
day and at night mig r at e s 1-2 mm down into the mat where
c o n d it i o n s are a n a e r o b i c (41).
(24) showed that B e g g i atoa
s p p
Jor g en s e n and Revsbech
.
formed layers, of cells
at the int e rf a c e of oxygen and h y d r og e n s u l f id e in a mat
from a marine sediment.
N e ! son and Jan n as c h (38)
d e m o n s t r a t e d in l a b o ra t o r y agar cultures th$t the growth
55
of a m a r i ne B e g g i atoa isolate d e p e n d e d on its p r e f er e n c e
for redu ce d oxygen c o n c e n t r a t i o n s and a limited sulfide
c o n c e n t r a t i o n in c o m b i n a t i o n with gli d in g motility.
In
the O c t o pu s S p ring ecosystem, Doeme] and Brock (17)
o b s e r v e d C h l o r o f I exus to glide to the mat's s u r f a c e d y r j n g
darkness.
C h l o r o f I exus can only grow as an a e r o b e in
the darkness.
If the mat o r g a n i s m s do adjust their p l a c em e n t within
the mat due to chan ge s in oxygen gradients, it should
be noted that four of the five f e r m e n t a t i v e isolates have
fla g el l a (58, 59, 60, 78) that could support such motility.
Some methanogens, such as a, str a in of M e t h a n o s a r c i na
barker! i s o l at e d from an e n r i c h m e n t of sewage s l u d ge anc)
a M e t h a n o t h r i x s p e c i e s i solated from a 5 8 ° C digester, have
gas vac u ol e s (I, 79).
A l t h o u g h M e t h a n o b a c t e r i u m thermoauto
t r o p h i cum, i s o l at e d from Octopus, was not o b s e r v e d to
have gas vacuoles, it is p o s s i b l e that other met h an o g e n s
not i s o l at e d could adapt to diurnal f l u c t u a t i o n s by using
such structures.
Light i n f l u e n c e d the a c c u m u l a t i o n of fe r m en t a t i o n
products.
Sam p le s i n c u ba t e d in the light had less acetate
than c o r r e s p o n d i n g sam p le s i n c p b a t e d in the dark;
Formalin
c o n t ro l s taken at zero time (early morning) and at 5 hours
into the e x p e r i m e n t rev e al e d h i gher c o n c e n t r a t i o n s of
ace t at e than in s a m p le s inc u ba t e d in the light for
56
10 hours.
( F ormalin c o n t ro l s for in situ a c e t at e m e a s u r e ­
ments at 10 hours were not t a k e n . )
This s u g g e s t e d that
l i g h t - d e p e n d e n t m e t a b o l i s m of a c e t at e occurred.
Tayne
(48) showed that f e r m e n t a t i o n prod uc t s were taken up under
light aero bi c c o n d i t i o n s by a f i l a me n t o u s bacterium, sho^n
to be C h l o r o f l e x u s a u r a n t i a c u s by a comb in e d i m m u n o f l u o r e s ­
c e n c e - a u t o r a d i o g r a p h i c procedure.
As few of these
f e r m e n t a t i o n p r o d uc t s were m e t a b o l i z e d in the dark, it
is likely that t hese c o m p o u n d s are taken up by this
p h o t o h e t e r o t r o p h d u r i n g the day;
L i g h t - d r i v e n p h o t o s y n t h e s i s not only aff e ct s oxygen
c o n c e n t r a t i o n s but pH c o n d i t i o n s as well.
Using
m i c r o e l e c t r o d e s in a 5 5 * C mat, R e v s be c h and Ward (40) found
the lowest pH value to be present b e fore s u n r i s e in t|ie
I to 6 mm depth interval.
The p h o t o s y n t h e t i c uptake of
b i c a r b o n a t e during the day as well as fatty acid production
at night are tho u gh t to i n f l u e n c e the pH cha n ge from basic
dur i ng the day t o w a rd s more a c idic at night (40).
o b s e r v a t i o n s of:
The
i
a h i gher a c e t at e level in the morning
mat, the a n a e ro b i c and acidic c o n d it i o n s of the night
mat, and my continual o b s e r v a t i o n s of f e r m e n t a t i o n product
a c c u m u l a t i o n in core samp le s i n c u ba t e d under dark anaerobic
c o n d i t i o n support act i ve f e r m e n t a t i o n in the Octo pu s 5 5 * C
mat at night.
F e r m e n t a t i o n o c c u r r e d in sam p le s c o l l ec t e d along
the thermal gra d ie n t from 70* to 5 0 * C.
The d ominant
X
57
f e r m e n t a t i o n p r o d u c t s w e r e a l w a ys ace t at e and propionate.
Ward (51) sho w ed that m e t h a n e p r o d u c t i o n in situ occurred
from 68° to 3 0 * C with very l i ttle m e t h a n o g e n e s i s observed
in the 68* to 6 3 * C range.
H y d r o g e n was noted to
a c c u m u l a t e in the 6 5 * C mats.
Carbon dioxide ar|$l hydrogep,
and not acetate, were found to be the i m portant methane
p r e c u r s o r s over a 60* to 4 5 * C r ange in the mats (43).
T h ese o b s e r v a t i o n s sup p or t the model p roposed for anaerobic
d e c o m p o s i t i o n in w h ich both f e r m e n t a t i v e and met h an e
p r o d u c i n g b a c t e r i a play i m p o rt a n t roles. In Octo pu s Spring,
h y d r o g e n and carbon d i o x i d e p r o d u c e d during f e r m en t a t i o n
are used by the m e t h a n o g e n s and C h l oro f le x u s is implicated
in the p h o t o i n c o r p o r a t i o n of acetate.
Zeikus, et a l . (76) s p e c u l a t e d that f e r m e n t a t i v e
a n a e r o b i c b a c t e r i a that p r o d uc e h ydrogen may function
in nature at t e m p e r a t u r e s gre a te r than 80* C but that
m e t h a n o g e n s may not.
E x p e r i m e n t s r evealed that hyplrog^n
f o r m at i o n but not m e t h a n o g e n e s i s was d etected at 8 0 * C
during a n a e r o b i c d e c o m p o s i t i o n of the Octopus Spr i ng m a t .
The 65* C mats p r o v i d e d a natural system in w h ich to
i n v e s t i g a t e the e f f e ct s of h y d r o g e n on f e r m e n t a t i o n
p r o d u c t i o n as rela te d to the i n t e rs p e c i e s h y d r o g e n transfer
theory. S a m p le s from a 6 5 * C c o m m u n i t y showed d i fferent
patt er n s of f e r m e n t a t i o n product a c c u m u l a t i o n than did
those from a 5 5 * C environment.
A n a l ys i s of fe r m en t a t i o n
p r o d uc t s in this hig h er h y d r o g e n e n v i r o n m e n t ( 6 5 ® C ) showed
58
a g r e a t e r ratio of redu ce d p r o d uc t s to acetate, as
s p e c i f i e d by the the i n t e r s p e c i e s hyd r og e n t r a n sf e r model.
The f i n d in g of a high ratio for ethanol a c c u m u l a t i o n
c o r r e l a t e s well with four of the f e r m e n t a t i v e isolates
which p r o d u c e e t h a n o l .
T h e r m o p h i l i c i solates from hot
spri ng s may prove useful for industrial p r o d u c t i o n of
chemicals, such as ethanol (57).
The lower p r o p i o n a t e a c c u m u l a t i o n at 6 5 * C, however,
was not expected.
In many o t h e r systems studied, such
as the rumen or a n a e r o b i c digester, when m e t h a n o g e n e s i s
ceased, a higher h y d r o g e n level caused p r o p i o n a t e to
a c c u m u l a t e (34).
The i n c r e a s e in h y d r og e n c o n c en t r a t i o n
r e s u l t i n g from the i n h i b i t i o n of m e t h a n o g e n e s i s either
i nhibits p r o p i o n a t e m e t a b o l i s m by a c e t o g e p i c bact er i a
or forces fer m en t o r s to p r o d uc e more reduced f e r m e ntation
p roducts due to the b r e a k d o w n in i n t e r s p e c i e s hydrogen
transfer. It was not p r o b a b l e that ace t og e n s b r o k e down
the p r o p i o n a t e at 6 5 * C, due to the high i n h i bi t o r y
c o n c e n t r a t i o n of h y d r o g e n at this temperature.
Tayne
(48) s h o w ed little e v i d e n c e for p r o p io n a t e a c e t o g e n e s i s
even at lower h y d r o g e n c o n c entrations.
A n o t he r appr oa c h in s t u d y i n g the effect of hydrogen
was to inhibit m e t h an e p r o d u c t i o n in the 5 5 ' C mat.
M e a s u r e m e n t s of s a m p le s i n c u b a t e d in the p r e s e n p ? apd
a b s e nc e of an i n h i bi t o r of methanogenesis, 2- b r o mo e t h a n e s u l f o n i c acid ( B E S )1 s h o w ed substantial d i f f e r e n c e s in
59
the ratio of f e r m e n t a t i o n pro d uc t s to ace t at e only in
ethanol accumulation.
It sho u ld be noted that the hydrogen
level dur i ng this e x p e r i m e n t did not reach that observed
after i n c u ba t i n g 6 5 * C s a m p le s and pos s ib l y was not high
enough to induce p r o d u c t i o n of r e d u ce d products.
T$yne
(48) found that b u t y r a t e a c e t o g e n e s i s was s e n s i t i v e in
a BES experiment.
A c e t o g e n e s i s, h o w e ve r is much more
s e n s i t i v e to h y d r o g e n c o n c e n t r a t i o n than is the production
of r e d u ce d f e r m e n t a t i o n products.
T h e r e are l i m i t a t i o n s in only viewing f e r m e ntation
product a c c u m u l a t i o n and a s s u m i n g that the r e s u lt i n g
p r o d uc t s are imp o rt a n t to a microbial community.
As noted
before, some c o m p o u n d s p r o d uc e d in the s y stem nf|ay be
metabolized, and therefore, will not accumulate.
To
further u n d e rs t a n d the i m p o r t a n c e of c o m p ou n d s in the
hot springs, p h o t o h e t e r o t r o p h y was used as a b ioassay
of what f e r m e n t a t i o n p r o d u c t s the microbial p o p ulation
were ada p te d to use.
T hese h e t e r o t r o p h i c potential results
r e p r es e n t a potential of the microbial p o p u l a t i o n to take
up and m e t a b o l i z e q u a n t i t i e s of s u b s t r a t e tho u gh t to
s a t u r a t e the organisms' p e r m e a s e systems.
Acetate, lactate, and ethanol had a grea te r potential
for uptake by the c o m m u n i t y at 6 5 * C while p r o p i o n a t e and
a c e t at e had the g r e a t e s t potential for i n c o r p o r a t i o n at
a 5 5 * C site.
L a c t a t e a c c u m u l a t e d in the 6 5 * C system, if
only slightly, but did not a c c u m u l a t e in the 5 5 * C site.
60
T Q yne (48) s h owed that lac t at e was m e t a b o l i z e d at 5 5 * C
under dark, a n a e r o b i c c o n d i t i o n s as well as under light
a e r o bi c and a n a e r o b i c and dark a e r o bi c conditions.
It
was not d e t e r m i n e d if lact at e was m e t a b o l i z e d at 6 5 * C.
P r o p i o n a t e a c c u m u l a t i o n at 5 5 * C was higher than at
6 5 * C.
Again, e x p e r i m e n t s were not p e r f o r m e d to d e termine
whether p r o p i o n a t e was m e t a b o l i z e d at 6 5 * C, but Tayne (48)
found that it was only p a r t i a l l y m e t a b o l i z e d under dark
a n a e r o b i c c o n d i t i o n s at 5 5 * C.
The V max value for p r o p ionate
uptake at 5 5 * C was the high es t for all c o m p o u n d s tested;
at 6 5 * C the V max for p r o p i o n a t e was the lowest.
The
r e l a t i v e d e c r e a s e in p r o p i o n a t e a c c u m u l a t i o n at 6 5 * C may
reflect the p o p u l a t i o n ' s i n a b il i t y to pro d uc e a? much
p r o p i o n a t e as was seen at 5 5 * C.
As men t io n e d above,
a c e t o g e n e s i s of this c o m p o u n d was highly unlikely, as
a high h y d r o g e n level inh i bi t s the a ctivity of these
organisms.
W h a t e v e r the reason, the d e c r ea s e in p r o p ionate
levels at the h i gher t e m p e r a t u r e c o i n ci d e d with a
p o p u l a t i o n which is not ada p te d to taking it up.
The Oct o pu s S p r i n g microbial mat is an i n t e re s t i n g
e c o s y s t e m for study of natural microbial interaction^.
We are limited, at this point, in u n d e r s t a n d i n g community
r e l a t i o n s h i p s by not k n o w in g the i d e n ti t i e s and pure
c u l t ur e a c t i v i t i e s of other b a c t e r i a in this system.
By s t u d y i n g gross processes, however, we have I earned
61
much about i m p o rt a n t factors w h ich i n f l ue n c e the microbial
population.
F e r m e n t a t i o n is an a c tive proc es s in O c t o pu s Spring.
A c e t a t e and p r o p i o n a t e are the major f e r m e n t a t i o n products
that a c c u m u l a t e under dark a n a e r o b i c conditions.
Z
.
' t
'Fermentation pro d uc t s a c c u m u l a t e p r e d o m i n a t e l y in the
upper 4 mm of the m a t .
This loc a ti o n of this process
has r a i s e d . s o m e i n t e r e s t i n g q u e s t i o n s c o n c e r n i n g
i n t e ra c t i o n s of the c o m m u n i t y ' s population.
For example,
how do f e r m e n t a t i v e o r g a n i s m s adapt to the diurnal changes
in o x ygen c o n c e n t r a t i o n ?
The o b s e r v a t i o n that mat s a m p l e
i n c u ba t e d in the light had less a c c u m u l a t i o n of f e r m e n t a ­
tion p r o d uc t s than c o r r e s p o n d i n g dark samples integrates
well with Tayn e' s f i n d in g that C h l o r o f l e x u s P h o t o i ncoroorated f e r m e n t a t i o n p r o d uc t s (48).
As Tayne poi n te d out,
this s y s t e m r e s e m b l e s the rumen in that the further
b r e a k d o w n of f e r m e n t a t i o n p roducts is not an important
process.
Instead, t hese pro d uc t s are cycled for use by
C h l o r o f I e x u s ; in the rumen, they are cycled for direct
use by the animal.
Also in both the rumen and in the
Octopus S p ring mats, a c e t a t e is not an imp o rt a n t methane
precursor.
That i n t e r s p e c i e s h y d r og e n tran sf e r is a c t i v e in
the 65°C e n v i r o n m e n t is s u p p o r t e d by the shift in the
a c c u m u l a t i o n of more reduced f e r m e n t a t i o n products, with
the e x c e p t i o n of propionate, at this higher t e m p e r a t u r e
62
comp ar e d to 55*0.
The i m p o rt a n c e of these red u ce d products
may be r e f l ec t e d in the het.erotrophic potential results.
The p o p u l a t i o n at 6 5 * C was more adapted to t a k i ng up lactate
and ethanol than was the com m un i t y at 5 5 * C.
Thus, at a
6 5 * C mat with less m e t h a n o g e n e s i s and more p r o d uc t i o n of
h y d r og e n than the 5 5 * C system, the i m p o rt a n c e of reduced
f e r m e n t a t i o n pro d uc t s agrees with the theory of
i n t e r s p e c i e s h y d r og e n tran sf e r and a d a p t i v e n e s s of the
bacterial c o m m u n i t y for these compounds.
F e r m e n t a t i o n in Octopus Spr i ng thus not only appears
to s erve as a means of a n a e r o b i c d e c o m p o s i t i o n in a low
s u l f at e e n v i r o n m e n t in which m e t h a n o g e n e s i s and little
a c e t o g e n e s i s occur, but it also appears to be important
in s u p p l y i n g the p h o t oh e t e r o t r o ph , C h l o r o f l e x u s . with
nutrients.
63
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71
APPENDIX
Table 6.
R e s u l t s of h e t e r o t r o p h i c potential e x p e r i m e n t
to d e t e r m i n e Vsax for u p t a ke and m e t a b o l i s m
of f e r m e n t a t i o n p r o d uc t s in the 1-3 mm interval
of O c t o p u s S p r i n g 6 5 * C mat.
l^c-fermentation
product
acetate
propionate
A
(vrnoles)
T/F
(hours)
77.78
14.28
15.15
15.62
171.21
33. 33
35.71
35.71
3 5 4.55
62.50
71.43
71.43
825.76
125.00
125.00
125.00
0.87
7,14
8.20
8.33
1.80
16.67
17.24
17.24
3.56
31.25
33.33
33.33
8.13
31.25
62.50
62.50
. .
72
Table 6.
Continued
l^ c - fe r m e n t a t i on
product
butyrate
lactate
ethanol
A
(VBioles)
T/F
(hours)
1.07
7.14
7. 81
8. 09
2.17
13. 51
13.98
14.29
4.20
13. 51
14.71
14.71
8,64
21.74
21.74
27. 78
1.06
5.26
5.38
6.17
I. 94
5.75
5.95
6.02
4. 34
5. 62
5.81
6. 85
8. 90
5.74
7. 94
8.47
10.94
6.85
7.69
7. 69
27.75
19.52
18.52
20.00
73
Table 6.
Continued.
l ^ C - f e r m e n t a t i on
product
ethanol
A
(ymoles)
T/F
(hours)
56.70
38.46
38. 46
41.67
122.49
83.33
83. 33
100.00
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