FEMS Microbiology Ecology 53 (1988) 95-100
Published by Elsevier
95
FEC 00153
In situ rumen hydrogen concentrations in steers fed eight times
daily, measured using a mercury reduction detector
Walter J. Smolenski and Joseph A. Robinson
Microbiology and Nutrition Research, The Upjohn Company, Kalamazoo, MI, U.S.A.
Received 2 September 1987
Accepted 17 September 1987
Key words: Ruminal methanogenesis; H 2 probe; Post-feeding H 2 fluctuation; Fermentation monitoring
1. SUMMARY
Dissolved hydrogen was measured in the bovine
rumen using an in situ hydrogen probe coupled to
a mercury reduction detector. The probe can
quantitate dissolved hydrogen from low nM concentrations to saturation. In the rumen of steers
fed every 3 h, basal hydrogen concentrations averaged 1.38 #M :t: 0.26, and the basal level remained
stable throughout an 18-25 h period. In contrast,
a steer fed once a day had a basal hydrogen
concentration of 1.40/~M, but the level was not
stable between feedings. For the steers fed every 3
h, the reticulum displayed the most dramatic
fluctuations in the hydrogen concentration after
the feeding event. Hydrogen spikes (10-20 #M) in
the reticulum were detected 2 rain after feed ingestion, and lasted for 30 rain. In the center of the
rumen the feeding response was observed 30 min
after feeding and typically lasted 1 h. The magnitude of hydrogen spikes in the center of the rumen
was reduced in comparison to the reticulum. The
magnitude of the hydrogen spikes indicates that
Correspondence to: W.J. Smolenski, Microbiology and Nutrition Research, The Upjolm Company, Kalamazoo, MI 49001,
U.S.A.
feeding steers as frequently as eight times a day
does not establish a steady-state with respect to
hydrogen concentration. However, frequent feedings do minimize drift from the basal hydrogen
level. Assuming Michaelis-Menten kinetics our
data predict that methane production from hydrogen proceeds at 22% of its maximal velocity.
2. INTRODUCTION
The importance of molecular hydrogen (H2) in
methanogenic ecosystems and interspecies electron transfer has been described [1,2]; however,
measurement of H 2 concentrations in natural
habitats has been elusive. Many approaches to the
measurement of dissolved H 2 in anaerobic
ecosystems have been explored. Hungate was the
first to investigate H 2 metabolism in an anaerobic
habitat (bovine rumen) [3]. He developed an extraction procedure enabling quantitation of H 2 in
the /~M range. However, the method involved
several steps (including the boiling of a closed
glass vessel) and cannot be used for intensive
time-course studies due to the time required to
process a single sample. Hungate's method was
improved by others [4,5], but the techniques developed still required a long time to extract the liquid
0168-6496/88/$03.50 © 1988 Federation of European Microbiological Societies
96
phase and to quantitate this sparingly soluble gas.
Further, these improved methods are not easily
applied to measuring H 2 concentrations in lake
sediments, where the dissolved H 2 pool may be
100-fold lower than the ruminal H 2 pool [3,5-7].
Accurate measurement of in situ H 2 concentrations in sediments is now possible, due to the
development of the ultra-sensitive mercury vapor
detector [8]. This detector has been used in recently published studies to investigate the dynamics of H 2 utilization in sewage and sediments
[6,7]. It has not heretofore been applied to a
re-investigation of H 2 metabolism in the rumen.
Although the mercury vapor detector is not required for obtaining an estimate of the ruminal
H 2 pool at one particular time point, the sensitivity of the instrument allows accurate dynamic
measurement (2 min between time points) of
ruminal H 2 concentrations.
An ideal approach to in situ H 2 measurement
should not require the investigator to obtain a
sample from the ecosystem of interest. This approach is desirable, since the turnover time for H 2
is so short (0.08 s in the rumen [3]) that the
process of sampling can appreciably affect the
estimate of the H 2 pool. For this reason investigators have developed in situ H 2 probes. Lloyd and
co-workers have used membrane-inlet mass spectrometry to quantitate dissolved H 2 in the rumen
of sheep fed various diets [9]. While this approach
produces essentially a continuous record of the H 2
pool, membrane-inlet mass spectrometry suffers
from a number of constraints. The detection limit
for H 2 of membrane-inlet mass spectrometry is
about 0.5 /~M [9], a concentration which greatly
exceeds the in situ H 2 concentration in other
anaerobic habitats [7]. Further, water vapor and
methane both interfere with the quantitation of
H 2, since these two molecules contribute to the
signal at mass 2 (the major peak for H2). Other
researchers have used Clark-type oxygen electrodes to quantitate dissolved H 2 in culture media
[10-12], but successful application of this technology to direct measurement o f H 2 in anaerobic
habitats may be limited because the electrodes are
sensitive to other compounds commonly found in
reduced environments.
In the present manuscript we describe a H 2
probe that allows direct in situ measurement in
real time (2 min between time points). The probe
makes use of the mercury vapor detector and the
signal for H 2 is not interfered with by other
gaseous molecules dissolved in rumen fluid. The
probe was developed to gain a better understanding of the temporal dynamics of the H 2 pool in
various regions of the rumen, in response to different feeding regimens.
3. MATERIALS A N D M E T H O D S
3.1. Cattle and diet
Three rumen fistulated steers (Angus Hereford
cross, 310 kg) were fed a high forage maintenance
diet every 3 h for 8 months. One steer in the study
was fed the same diet once a day.
3.2. Hydrogen probe design
Hydrogen was measured in situ using a probe
constructed from 6 m Teflon tubing (1.5 mm o.d.,
0.5 mm i.d.) wrapped in a coil around a water-filled
Balch tube (Bellco Glass, Vineland, N J). The ends
of the Teflon coil were connected to 6 m (1.5 mm
o.d., 0.5 mm i.d.) of stainless steel tubing. Helium
(Chromatographic grade, Linde Gas Division, Union Carbide, Chicago, IL) served as the carrier gas
for both the probe and gas chromatograph (Trace
Analytical, Menlo Park, CA), with respective flow
rates of 10 and 40 ml/min. As helium passed
through the probe, dissolved H 2 diffused through
the Teflon into the helium stream. The helium
effluent from the probe, which contained trace
levels of H2, was routed to a 0.5-ml sample loop
on the injection valve (No. A4C6UWT, Valco
Inst. Co., Houston, TX) (Fig. 1). Hydrogen was
separated on a Molecular Sieve 5A column (0.9
m × 0.64 cm o.d. 8 0 / 1 0 0 mesh) at 125°C, and
detected using a Mercury Vapor Reduction Gas
Analyzer (Trace Analytical). A HP3393A integrator (Hewlett Packard, Avondale, PA) was used for
quantification and valve automation. A diagram
of the gas chromatograph and valving is shown in
Fig. 1. The hydrogen probe is similar in operation
to a previously described oxygen probe, which
employed porous Teflon tubing [13]. Probe
calibration was achieved by sparging tap water
97
STEEL
STAINLESS
TUBING
h
~131~-"~"~
J/
#
DORSAL
\
GAS
FLOWCONTROLLER
CHROMATOGRAPH
~
H_
FISTULA
/i t
'
I
'°'L'''"
I t - -
40
"L/MIN"
~
l
. . . . . . . . . . . . . . .
Lt C°LU'N:'S'At
.
1"
.
.
.
.
Tl II agoDETECTOR
REDUCTION [~
I-
L /~' ~ J
VENT LOAD F-__~AM_P_L-E_~/~9~
,ET,CDLUM
PRORE -
:
L ......
F i g . 1. S c h e m a t i c
indicate
representation
direction
of experimental
unit with
_LN_~C_T . . . . . . .
sagittal view of the rumen
of carrier gas flow. The flow path within the sampling
v a l v e is s h o w n
and
FI 2 s a m p l i n g
instrumentation.
in both the load and inject positions;
Arrows
see text
for details.
A
Retieulum
c 22: ',
~20 ',
018
i
"016I
`012
~10
8 Feedings/Day
i
i
i
i
i
i
i
i
i
i
i
q
i
i
i
I
i
i
I
I
t
I
I
I
I
I
I
I
I
I
I
t
I
I
I
I
I
I
I
4
I
I
I
I
I
I
I
i
o8:
6,
"_. 24!
,,
,a
It.
, ,
0
i ,
,I,
2
'LA
,,,,JJ:
i , ,
4
, ii,
, ,
6
'
~20i
'l,
o;B!
:
-1-141
',
"o12!
"i
>e10 i
i ,
8
Time
C
,I,
v,
.~- L-,t
"ml,
,'
i
, ,
"~ 2:
0i
'~r,W,-
i I
I
i , ,
, I
10 12 14 16 t8
i
0
i
i
i
i
i
i
i
1
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
t
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
t,
i
1
i
I
i
i ,
i
i
i
i
i
i
i
i
i
i
i
i
:
,L ~, k--,w~; - . ~ ,,,t
0'ii,
c 22~ :
i
i'
D0rsal/Ventral 8 Feedings/day
B
i
i
i
i
i
i
i
t
i
l
i
i
i
i
2
4
I
6
I , ,
8
i
, i
, ,I,
i
I
i
i
i
i
+
i
i
i
i
i
i
i
i
i
i
i , ,
, ii,
, ,
i
10 12 14 16 18
Time (hours)
(hours)
Dorsal/Ventral
I Feeding/Day
O"
0
5
10
15
Time
F i g . 2. D i s s o l v e d
The horizontal
received
H 2 fluctuations
solid line is drawn
eight feedings
center of the rumen.
observed
20
in the rumen
at the median
25
30
35
(hours)
as a result of feeding events (vertical dashed
concentration
p e r d a y . B, t h e d o r s a l / v e n t r a l
for H 2 (basal level). A, feeding response
region or center
of the rumen.
C, H 2 response
lines indicate
in the upper
in once-a-day
time of feeding).
reticulum
which
feedings in the
98
with k n o w n c o n c e n t r a t i o n s of H 2 a t 38.6 ° C . The
dissolved H 2 c o n c e n t r a t i o n was c a l c u l a t e d using
an O s t w a l d coefficient b a s e d u p o n the solubility
of H 2 in p u r e w a t e r at 3 8 . 6 ° C [14].
4. R E S U L T S
Changes in the H 2 concentration in the reticulum and dorsal/ventral region (center) within the
rumen are shown in Fig. 2A and B respectively.
Feeding eight times a day did not eliminate transient surges in the H 2 concentration upon feeding,
but the magnitude of the increases differed depending upon location. In the top of the reticulum, where feed enters the rumen, the basal
(median) H 2 concentration was 1.73 # M (Fig. 2A,
horizontal solid line). Upon feeding (vertical
dashed line) a rapid increase in dissolved H 2 was
observed. The feeding response in the reticulum
was seen only 2 min after feeding and reached its
maximum concentration 10 min after feeding
(10-20 #M). After 30 min the concentration returned to the basal level. In the lower reticulum
(data not shown) the feeding response was as
rapid as that seen in the upper reticulum, and the
duration was as long, but the magnitude of the H 2
spikes was reduced (6-12/~M). In contrast to the
upper reticulum, the basal H 2 concentration in the
lower reticulum was maintained at a reduced level
(1.00 #M, Table 1).
In the dorsal region a spike in dissolved H 2 at
feeding was not always observed. Also, when a
feeding response was seen, the magnitude of the
c h a n g e (4.79 # M ) was the lowest of a n y region
within the rumen. T h e b a s a l H 2 c o n c e n t r a t i o n in
the d o r s a l region was 1.41/~M ( T a b l e 1).
I n c o n t r a s t to the i m m e d i a t e feeding response
seen in the reticulum, we o b s e r v e d a time lag
b e t w e e n feeding a n d the increase in dissolved H 2
in the center o f the r u m e n (Fig. 2B, d o r s a l /
ventral). T h e time lag before the H 2 increase was
t y p i c a l l y 30 rain after feeding a n d the basal conc e n t r a t i o n was 1.43 /zM ( T a b l e 1). T h e basal H 2
c o n c e n t r a t i o n in the lower ventral region was 1.34
/~M ( T a b l e 1).
F o r c o m p a r a t i v e purposes, H 2 m e a s u r e m e n t s
were also m a d e in the r u m e n of a steer fed the
s a m e diet once a d a y (Fig. 2C). In the d o r s a l /
ventral region, H 2 c o n c e n t r a t i o n s were the lowest
b e f o r e feeding a n d increased after feeding. T h e
b a s a l H 2 c o n c e n t r a t i o n d i d not r e m a i n c o n s t a n t
over the run, b u t drifted steadily d o w n w a r d after
the initial rise u p o n feeding. T h e m e d i a n H 2 conc e n t r a t i o n over the entire run was 1.40 # M (Table
1).
5. D I S C U S S I O N
The time it takes to replace the H 2 pool in the
rumen (turnover time) has been estimated to be
0.08 s [3]. Because of this rapid hydrogen flux,
obtaining a representative sample becomes practically impossible. The physical action of sampling
releases sequestered organics to hydrogen-producing bacteria, increasing the rate of H 2 production.
This increase in production coupled with the rapid
Table 1
Hydrogen concentrations in various regions of the rumen
Probe location
Upper reticulum
Lower reticulum
Dorsal
Dorsal/ventral
Lower/ventral
Dorsal/ventral a
Hydrogen concentration (# M)
Sample number
(n)
Mean
Median
Maximum
Minimum
563
854
595
586
879
1249
2.39
1.25
1.55
2.23
1.73
1.67
1.73
1.00
1.41
1.43
1.34
1.40
18.45
7.50
4.79
20.13
11.32
8.23
0.858
0.521
0.610
0.796
0.645
0.356
a Steer was fed once a day. All other samplings were from steers fed every 3 h. Dorsal/ventral refers to the central region of the
rumen.
99
turnover time might yield biased estimates of the
H 2 pool. Accurate quantitation of dissolved H 2 is
best obtained using a non-disruptive environmental probe. The advantage of using the H 2 probe
described in this text over other techniques
[3-6,9-12,15,16] is the combination of high sensitivity and specificity, with rapid real time analysis.
Although fluctuations in the H 2 concentration
caused by feeding differed in magnitude within
the rumen ecosystem, the basal levels were comparable between locations (arithmetic average of
medians = 1.38 ~tM + 0.26). This concentration is
similar to dissolved H 2 values (1/~M) reported by
Hungate [3]. Using the average H 2 concentration
we observed in the rumen with reported halfsaturation constants (5 ~tM) for H 2 consumption
by methanogens [17], the Michaelis-Menten equation predicts that at basal H 2 levels methanogens
are producing methane from H 2 at 22% of their
maximal velocity. This estimate is strictly theoretical and ignores the juxtapositioning [6] between
hydrogen producers and methanogens.
Hydrogen production, as a result of feeding,
was more rapid and defined where feed enters the
rumen (Fig. 2A). In regions removed from where
feed first enters the reticulo-rumen, the response is
delayed, and the magnitude of H 2 surges is less
predictable than in the reticulum (Fig. 2B). Although soluble sugars were not measured in this
experiment it is our belief that the increase in H 2
production is due to an increase in soluble sugars
after feeding. Other authors have recorded an
increase in soluble sugars 30 min after feeding and
lasting up to 90 min [18]. This observation correlates with the increase and decrease of H 2 observed in the center of the rumen over this time
interval (Fig. 2B).
A comparison betwen the steers fed every 3 h
(Fig. 2A and B) with the one fed once a day (Fig.
2C) indicates that frequent feedings do reduce
drift from the basal H 2 concentration. For once-aday feedings the concentration rises above the
basal level after feeding and steadily drops below
this level until the next feeding. Our results indicate that feeding eight times a day is not frequent
enough to establish steady-state conditions with
respect to the H 2 concentration within the bovine
rumen ecosystem,
Although the dissolved hydrogen probe is superior to other hydrogen probes because of its
sensitivity, stability and selectivity, it does have
disadvantages. Among these is its obligate use in
systems that approach a steady-state with respect
to H 2. However, the response time of the probe
could be decreased by using thinner-walled Teflon
tubing, or an alternative material. At present the
probe gives a 90% response in 2 min. Another
disadvantage is its large size. Thus, the readings
represent a system averaging of concentrations
over the length of the probe, and not a point
source measurement.
Despite the few disadvantages, the probe has
m a n y possible applications for remote measurement of H 2 in environments where other approaches cannot be easily employed. The H 2 probe
could be used for monitoring dissolved H 2 concentrations in fermentors, sludge digesters,
anaerobic groundwater, or the water column and
sediments of lakes.
REFERENCES
[1] Mclnerney, M.J. and Bryant, M.P. (1981) Basic principles
of bioconversions in anaerobic digestion and methanogenesis. In: Biomass conversion Processes for Energy and
Fuels (Sorer, S,S. and Zaborsky, O.R., Eds.) pp. 277-296.
Plenum Press, New York.
[2] Wolin, M.J. (1980) The rumen fermentation: A model for
microbial interactions in anaerobic ecosystems. Adv. Microb. Ecol. 3, 49-77.
[3] Hungate, R.E. (1967) Hydrogen as an intermediate in the
rumen fermentation. Arch. Mikrobiol. 59, 158-164.
[4] Czerkawski,J.W. and Breckenridge, G. (1971) Determination of concentration of hydrogen and some other gases
dissolved in biological fluids. Lab. Pract. 20, 403-405,
413.
[5] Robinson, J.A., Strayer, R.F. and Tiedje, J.M. (1981)
Method for measuring dissolved hydrogen in anaerobic
ecosystems: Application to the rumen. Appl. Environ.
Microbiol. 41,545-548.
[6] Conrad, R., Phelps, T.J. and Zeikus, J.G. (1985) Gas
metabolism evidence in support of juxtaposition of hydrogen-producing and methanogenicbacteria in sewagesludge
and lake sediments. Appl. Environ. Microbiol. 50,
595-601.
[7] Conrad, R., Schink, B. and Phelps, T.J. (1986) Thermodynamics of H2-consuming and H2-producing metabolic
reactions in diverse methanogenic environments under in
situ conditions. FEMS Microbiol. Ecol. 38, 353-360.
[8] Seiler, W., Giehl, H. and Roggendorf, P. (1980) Detection
100
[9]
[10]
[11]
[12]
[13]
of carbon monoxide and hydrogen by conversion of
mercury oxide to mercury vapor. Atmos. Technol. 12,
40-45.
Hillman, K., Lloyd, D. and Williams, A.G. (1985) Use of
a portable quadrupole mass spectrometer for the measurement of dissolved gas concentrations in ovine rumen
liquor in situ. Curt. Microbiol. 12, 335-340.
Kristjansson, J.R., Schonheit, P. and Thauer, R.K. (1982)
Different K s values for hydrogen of methanogenic
bacteria and sulfate-reducing bacteria: An explanation for
the apparent inhibition of methanogenesis by sulfate. Arch.
Microbiol. 131,278-282.
Jones, L.W. and Bishop, N.I. (1976) Simultaneous measurement of oxygen and hydrogen exchange from bluegreen alga Anabaena. Plant Physiol. 57, 659-665.
Wang, R., Healey, F.P. and Myers, J. (1971) Amperometric measurement of hydrogen evolution in Clamydomonas.
Plant Physiol. 48, 108-110.
Dairaku, K. and Yamene, T. (1979) Use of the porous
teflon tubing method to measure gaseous or volatile sub-
[14]
[15]
[16]
[17]
[18]
stances dissolved in fermentation liquids. Biotech. Bioeng.
21, 1671-1676.
Wilhelm, E., Battino, R. and Wilcock, R.J. (1977) Low
pressure solubility of gases in liquid water. Chem. Rev. 77,
219-262.
Lloyd, D., Davies, K.J.P. and Boddy, L. (1986) Mass
spectrometry as an ecological tool for in situ measurement
of dissolved gases in sediment systems. FEMS Microbiol.
Ecol. 38, 11-17.
Whitmore, T.N. and Lloyd, D. (1986) Mass spectrometric
control of the thermophilic anaerobic digestion process
based on levels of dissolved hydrogen. Biotech. Lett. 8,
203-208.
Robinson, J.A. and Tiedje, J.M. (1984) Competition between sulfate-reducing and methanogenic bacteria for hydrogen under resting and growing conditions. Arch. Microbiol. 137, 26-32.
Counotte, G.H.M., Lankhorst, A. and Prins, R.A. (1983)
Role of DL-lactic acid as an intermediate in rumen
metabolism of dairy cows. J. Anim. Sci. 56, 1222-1235.