-2-
Microbial activity.
ABSTRACT
An attempt was made to measure microbial activity
in beach sand in relation to the black sulfide-containing
layer. Ihree biogeochemical conversions were investigated.
Changes in microbial activity as a function of sand depth
were observed and found to be primarily correlated with
differences in sand color. Conversion rates as determined
in the laboratory were found not to be good indices of
in situ activity. However, since all results were com-
parative, they were able to give information about general
activity distributions. Not only was anaerobic respiration
involving sulfide production most pronounced in sands con¬
taining high amounts of sulfide, aerobic respiration was
also greatest in this type of sand. On the other hand.
the most rapid rates for autotrophic conversion of ammonia
to nitrite were not associated with sulfide-rich sands.
-3
Microbial activity.
INTRODUCTION
Although extensive examination of microbial populations
and activities in waters and sediments has been carried
out, there are many unanswered questions and unsolved prob¬
lems. Perhaps the least investigated aspect of microbial
activity has been the attempt to describe in situ nutrient
conversion rates. The most definitive studies up to the
present have concerned themselves with the heterotrophic
metabolism of organic nutrients such as glucose and acetate
(Uright and Hobbie, 1965; Harrison, Uright, and Morita, 1971).
Investigation of other important biogeochemical conversions
has proceeded much more slouly, especially in the marine
environment. Houever, recently interest in a variety of these
conversions has groun and a study of several conversions in
the sediments of Monterey Bay is currently underway (J. H.
Phillips, personal communication, 1972.).
This particular study was undertaken in an attempt
to measure microbial activity in beach sand, paying par-
ticular attention to its relationship to the commonly found
black sulfide layer. The primary difficulty encountered
in such a study is that no suitable procedure exists for
the direct measurement of microbial activity in the field.
Assay methods must be devised which allow for the measurement
of conversion rates under controlled laboratory conditions.
Hopefully, such methods will yield results which can be
considered good indices of the activities occurring in situ.
However, such an extrapolation can only be regarded as
-4
Wicrobial activity.
Uncertain at best. Nevertheless, a comparison of experimental
results obtained for different samples should give information
as to the relative amounts of microbial activity occurring
at different depths in the sand and with reference to the
black sulfide layer.
Conversions representative of three biogeochemical steps
were chosen for investigation. These were:
1)
Heterotrophic assimilation and conversion of
organic carbon or mineralization by respir-
ation.
(CH,O) + 0, - co, + H,O
2) Nitrification.
NHNO
3) Sulfate reduction.
50 — H,S
MATERIALS AND METHODS
Lollection and initial preparation of sand samples.
Sand samples were collected through the use of a poly-
vinylchloride, PVC, coring device. Cores 40 cm. in length
Uere obtained and used for analysis. Due to the large number
of analyses required per sand sample, work proceeded on only
one sample at a time.
Removal of sand from the coring device took place in
the laboratory, and the resulting core uas immediately divided
into four equal portions 10 cm. in length. Each portion
of the sample corresponded to 10 cm. in vertical core depth.
-5-
Microbial activity.
Each portion was slurried with 2.5 volumes of Artificial
Sea Uater (Maclead, Onofrey, and Norris, 1954) to homo¬
genize and alloued to settle. Supernatants were decanted
and saved.
Preparation of samples for incubation
2.0 m1. aliquots of the settled sand were transferred
to individual reaction flasks along with 5.0 ml. of the
appropriate supernatant containing fine suspended material.
Incubation of samples to be assayed for carbon miner-
alization was carried out in 60 ml. glass-stoppered reagent
bottles. 1.0 ml. of substrate at the desired concentration
was introduced into each bottle and air-saturated Artificial
Sea Uater, ASU, added until overflowing. Each bottle was
stoppered to create a closed aerobic system and inverted
tuice to mix substrate and sand.
Incubation of samples to be assayed for nitrification
was carried out in 50 ml. Erlenmeyer flasks. Air-saturated
ASU uas added to each flask to bring the total volume up to
29 ml. excluding sand volume. 1.0 ml. of substrate at the
desired concentration was added, and the solution swirled
to ensure complete mixing.
Samples to be assayed for sulfate reduction were incu-
bated in 30 ml. reagent bottles. Nitrogen-flushed ASU was
added to bring the total volume up to 29 ml. excluding sand
volume, and the bottles capped with rubber vaccine stoppers.
The bottles were flushed out with nitrogen gas to create
completely anaerobic conditions, and 1.0 ml. of substrate
-6-
Wicrobial activity.
at the desired contration added by means of a syringe. Each
bottle was inverted twice to ensure complete mixing.
luo replicates at each substrate concentration were
prepared. The concentration ranges utilized were as follous:
1) Carbon mineralization: 0, 10, 25, 50 po-at
sodium acetate-C/L.
2) Nitrification: 0, 25, 50, 100 ug-at NH,CI-N/L.
3) Sulfate reduction: 0, 100, 200, 400 uo-at
sodium acetate-C/L.
Incubation of samples.
Samples were incubated in the dark at a temperature
uhich varied from 14.8--15.5'C. Optimal incubation times
uere determined by experimentation and were as follows;
carbon mineralization, 30—-48 hrs.; nitrification, 124 hrs.:
sulfate reduction, 96 hrs. Zero time determinations were
made as uell.
lermination of incubation and chemical assays.
Uxygen consumption was measured as an index of acetate
mineralization. Dissolved oxygen was determined by means
of the Chesapeake Bay Institute's technique for the Uinkler
method (Carpenter, 1965) except for the follouing modi-
fications:
1) Concentration
of reagents.
MnSO 440
(2.15 M)
(12.5 N
Nach
(0.9 M)
KI
4250
(concentrated)
Na25203: H20
(0.01 N)
-7-
Microbial activity.
2) Termination of incubation.
Sand interferes with the fixation reac-
tion. To avoid this difficulty 48 ml.
of solution were draun up into a 50 ml.
syringe before being fixed. The fixed
solution was transferred to a 125 ml.
Erlenmeyer flask for titration.
Nitrite production was measured as an index of nitri-
fication. Incubation was terminated by Millipore filtration
and nitrite determined by the method of Strickland and
Parsons (Strickland and Parsons, 1968). The estimate of
precision was + 0.3 ug-at NO,-N/L., and linearity permitted
use of a factor of 19.7 x (0.D.).
Sulfide production was measured as an index of sulfate
reduction. Sulfide was determined by the method of Strick-
land and Parsons (Strickland and Parsons, 1968). The
estimate of precision uas + 1.7 ug-at H,S-S/L., and linearity
permitted use of a factor of 43.1 x (0.D.).
Calculation of conversion rates.
Rates were calculated using the following equation:
V/Liter of sand = C/T x 500 where,
V - reaction rate.
C = change in concentration, in ug-at/.,
of substance being assayed.
I - time of incubation in hours.
RESULTS
Data was obtained for tuo cores collected from the
Boatuorks Beach at Hopkins Marine Station. Core 1 was
-8
Microbial activity.
collected at 1300 hrs. on May 9°" at an approximate tidal
height of + 2.0 feet. Core 2 was collected at 900 hrs. on
May 18" at an approximate tidal height of + 2.5 feet.
Qualitative differences between the two cores were
observed. Core 1 had broun sand to a depth of 17 cm. At
this point the sand acquired a definite grayish tinge which
darkened with depth. Belou a depth of 30 cm., the sand was
dark gray in color. Core 2 was broun to a depth of 14 cm.
At this depth the sand had a slight grayish tinge which darkened wi
depth. Below a depth of 19 cm. the sand was dark gray except
for a distinct black band at 22--24 cm.
After homogenation with ASU to prepare for analysis,
the cores had the following coloration:
Core1
Core Depth
Core 2
0—-10 cm.
broun
broun
10—20 cm.
very light gray very light gray
20—-30 cm.
gray
black
30—40 cm.
ark gray
ark gray
Figures 1—-4 depict the results obtained from the assays
for oxygen consumption. In both cores the rates of oxygen
consumption shou an increase with increasing substrate
concentration, see figures 1 and 2. The rates of oxygen
consumption also increasecas a function of depth, see figures
3 and 4. To correct for non-biological consumption of oxygen
due to chemical oxidation (e.g., Fes, - Fe.0), a sample
sterilized under nitrogen was tested for oxygen consumption.
In this manner values for chemical oxidation were obtained
and corrected biochemical oxygen consumption rates could
-9
Microbial activity.
be computed. These corrected values, see figures 5 and 6.
substantiate the preliminary observations that oxygen con-
sumption rates show a general increase with depth and sub¬
strate concentration.
Although oxygen consumption rates shou a general increase
with depth, the primary correlation is uith sand color.
lable 1 compares the rates of oxygen consumption, at a
representative substrate concentration, with sand color in
the tuo cores and shous that higher rates seem to be correlated
with darker color. This relationship holds true even after
correction for chemical oxidation. Those portions of the
tuo cores having similar coloration have similar uncorrected
oxygen consumption rates.
Figures 7 and 8 show that the rate of nitrification
in all four portions of the two cores increases with sub-
strate concentration. Figures 9 and 10 indicate a decrease
in the rates of nitrification with depth. As with oxygen
consumption, the primary correlation appears to be with sand
color. Table 2 compares nitrification rates, at a repre-
sentative substrate concentration, with sand color in the
tuo cores and shous that lower rates seem to be correlated
with darker color. Similar rates are observed in those
portions of the tuo cores having similar coloration.
A time course experiment on sand from core 2 was run
to determine whether or not a significant lag or induction
period existed for the nitrification reaction. Figure 11
indicates that a 48—-72 hr. induction period was present.
-10-
Microbial activity.
Calculation of nitrification rates did not involve a cor-
rection for this lag period.
Results for sulfide production were obtained only from
core 2. Little of no sulfide production occurred below a
substrate concentration of 400 pg-at sodium acetate-C/t..
see figure 12. Data obtained for this substrate concentration
indicates a possible correlation of rate with sand color.
see figure 13.
DISCUSSION
The ubiquitous black sulfide-containing layer in beach
sand has been characterized as a region of lower oxidation¬
reduction potential and louer pH than overlying layers uhile
having high concentrations of metallic sulfides and a low
concentration or complete lack of oxygen (Baas Becking.
Uood, and Kaplan,1957; Perkins, 1957). It can be hypothesized
that these differences in physical and chemical conditions
will be reflected by differences in the microbial pop-
ulations uhich inhabit the different layers. If such variations
do in fact exist, differences in microbial activity should
be evident.
Although only a limited amount of data was collected
and analyzed during this study, it appears that significant
differences in microbial populations, as reflected by activity
data, do exist as a function of sand depth. These differences
are related to differences in sand color, an index of changing
physical and chemical conditions.
-11-
Microbial activity.
The results indicate a positive correlation between
the rate of oxygen consumption and darkness of sand coloration.
The higher rates observed in darkly colored sand indicate
that larger populations of heterotrophs capable of miner-
alization reside there despite the anaerobic conditions.
Not enough data was obtained for the sulfate reduction
reaction to conclude anything definite about reaction rate
Vs. sand color. There appears to be an increase in rate
with darkening sand color, possibly indicating a population
increase in sulfate reducing bacteria, but no definitive
statement as to rate can be made without further experimentation.
A negative correlation between rate of nitrification
and increasing darkness of sand was observed, see table 2.
This reflects a decrease in the population of nitrifying
bacteria and indicates that the conditions characteristic
of the black band are not favorable for these bacteria.
Data for reaction rate vs. substrate concentration can
be used to give an estimate of in situ activity and infor-
mation about the actual mechanisms of nutrient uptake
(Uright and Hobbie, 1966).
No saturation effect on the rate of nitrification was
observed for increasing substrate concentration, see figures
7 and 8. This seems to indicate an uptake mechanism governed
by simple diffusion (Uright and Hobbie, 1966). Houever,
a closer examination of figures 7 and 8 reveals that a linear
relationship doesn't exist between reaction rate and substrate
concentration in very dark sand. This may simply be a
-12-
Microbial activity.
reflection of differences in lag or induction periods or
an indication that a variety of uptake mechanisms are in-
volved.
Since oxygen consumption was used as an index of car¬
bon mineralization, no statement can be made about the actual
uptake mechanisms involved in this reaction.
Significant sulfate reduction was observed at only one
substrate concentration, see figure 12. Further experi¬
mentation at higher substrate concentrations would be
necessary before any statement could be make about the up-
take mechanisms involved.
The results obtained under controlled experimental
conditions during this study cannot be considered good
indices of actual rates in situ. For example, the lag or
induction period observed for the nitrification reaction
can be regarded as an indication that the assay method
devised does not allou measurement of the in situ rate,
uhich may be very lou. Only the establishment of exper¬
imental conditions brings forth the observed production
of nitrite. Similarly, objections can be raised to any
attempt at comparing laboratoryand in situ rates for the
carbon mineralization and sulfate reduction reactions.
Houever, if the results are considered as indicative
of changing microbial activity in relation to depth and/or
sand color, a general picture emerges. First of all, the
anaerobic conditions of the black sand layer may be largely
a reflection of the higher numbers of bacteria found there
-13
Microbial activity.
capable of aerobic respiration. The more rapid removal of
oxygen possible in such a highly populated region permits
sulfide production to proceed by allowing anaerobic respir-
ation to occur. Secondly, the high substrate threshold
observed for the sulfate reduction reaction may also re-
flect the large and active population of aerobic respiring
forms inhabiting the black sulfide layer. Even with lou
concentrations of available oxygen, aerobic forms may simply
leave little, of any, substrate available for the sulfate
reducers to utilize. Lastly, the activity distribution of
bacteria involved in the nitrification process differs from
those that are involved with mineralization of dissolved
organic carbon. These results suggest that the autotrophic
and heterotrophic micro-organisms have different vertical
distributions within beach sand.
SUMMARY
1) This study was an attempt to measure microbial
activity in beach sand with respect to the black sulfide-
containing layer. The conversions studied were carbon
mineralization by respiration, nitrification, and sulfate
reduction.
2) Changes in microbial activity with respect to depth
and substrate concentration were observed.
3) The changes in microbial activity for any particular
substrate concentration were primarily correlated with
differences in sand color, an index of changing physical
-14
Wicrobial activity.
and chemical conditions.
The rates of the carbon miner-
alization and the sulfate reduction reactions increased
Uith increasing darkness of sand coloration. The rate of
nitrification showed the opposite trend.
4) Conversion rates as determined under controlled
laboratory conditions were found not to be good indices
of in situ activity. However, since the results were
comparative, they were able to give insight as to relative
amounts of microbial activity at different depths in the
sand and with respect to the black sulfide layer.
5) The microbial activity patterns observed in this
study seem to indicate that the black sulfide-containing
layer is established and maintained actively by internally
based processes as well as passively by external influences.
-15
Microbial activity.
ACKNOULEDGMENTS
My sincere thanks go to the entire faculty, staff.
and students at Hopkins Marine Station for a most enjoyable
and rewarding experience. In particular, I would like to
acknouledge Mr. Will MoCarthy for his help with the Beckman
spectrophotometer and long hours of companionship in the
cold room, Mr. Delane Munson for his help in collecting
equipment, and finally Dr. John H. Phillips whose encour-
agement, guidance, and helpful criticism were invaluable
throughout the duration of this study.
-16
Microbial activity.
LITERATURE CITED
Baas Becking, L. G. M., Wood, E. J. F., and Kaplan, I. R.
1957. Biological processes in the estuarine environ-
ment, Xa. Koninkl. Ned. Akad. Wetenschap., Proc..
Ser. B, 60: 88—-95.
Carpenter, J. H. 1965. The Chesapeake Bay Institute tech-
nique for the Uinkler dissolved oxygen method.
Limnol. Oceanogr. 10(1): 141--143.
Harrison, M. 3., Uright, R. T., and Morita, R. Y. 1971.
Wethod for measuring mineralization in lake sediments.
Appl. Microbiol. 21: 698—-702.
Waclead, R. A., Onofrey, E., and Norris, M. E. 1954.
Nutrition and metabolism of marine bacteria. I. Survey
of nutritional requirements. J. Bact. 68: 680--686.
Perkins, E. J. 1957. The blackened sulphide-containing
layer of marine soils, with special reference to that
found at Uhitstable, Kent., Ann. Mag. Nat. Hist.
10: 25—35.
Strickland, J. D. H. and Parsons, T. R. 1968. A practical
handbook of seauater analysis. Fisheries Res, Board
of Canada, Bull. 167: 21--26, 41--44, 77—-80.
Uright, R. 3. and Hobbie, J. E. 1965. The uptake of organic
solutes in lake water. Limnol. Oceanogr. 9: 163--178.
Figure 1: Rates of oxygen consumption in core 1 as a
function of substrate concentration.
Core Depth
Sand Color
O—0
0—10 cm.
broun
+--4
10—20 cm.
very light gray
S—o
20—-30 cm.
gray
m--
30—-40 cm.
dark gray
40

2
Te
20


itffftitititien t.

-



o
20
30
HH
at Sodium Acetate-

—

6/1

H
0
Figure 2: Rates of oxygen consumption in core 2 as a
function of substrate concentration.
Sand Color
Core Depth
0—10 cm.
O—0
broun
+- --+
10—20 cm
very light gray
20—-30 cm.
ray

30—-40 cm.
dark gray
—---
10-


+t

20
ug-at Sodium Acetate
40
0/1
Figure 3: Rates of oxygen consumption in core 1 as a
function of sand depth and color.
Substrate Concentration
0
po-at sodium acetate-C/L.
+---+
10 pg-at sodium acetate-C/L.
—D
25 pg-at sodium acetate-C/L.
50 pg-at sodium acetate-C/L.
9-- -e
e



BROWN
60
SANO
VERY
LIGHT
GRAY
COLOR
GRAT
DARK GRAY

+30
20
0-10
H
tttsttet
20130
30140
CORETDEPTH (Cm.)

HHH.
Htt
HHHE
HI
Figure 4: Rates of oxygen consumption in core 2 as a
function of sand depth and color
Substrate Concentration
O—
0 po-at sodium acetate-C/L.
10 po-at sodium acetate-C/1.
+---+
25 pg-at sodium acetate-C/L.
S
0-- -0
50 ug-at sodium acetate-C/L.
BROWN
50

o
++

+30

20


o
010
COLOR
BLACK

10420
20130
COREHDEPTH (cm)
SAND
VERV
LIGHT
GRAV

DARKIGRAY
+30140
e
Figure 5: Corrected rates for biochemical oxygen consumption
in core 2 as a function of substrate concentration.
Sand Color
Core Depth
0—10 cm.
O
broun
very light gray
— — + 10—-20 cm.
20—-30 cm.
D—
black
-O
30—-40 cm.
dark gray
—-—-
40
—i20
+0+

H


430
40
20
iatSodiumAetate-.


50
Figure 6: Corrected rates for biochemical oxygen con-
sumption in core 2 as a function of sand depth
and color.
Substrate Concentration
O—
U pg-at sodium acetate-C/t.
10 yg-at sodium acetate-C/L.
+— —+
E
25 po-at sodium acetate-C/L.
•——e
50 pg-at sodium acetate-C/L.
brown
40


6
30

8
5
920
5
0-10
SAND
COLOR
very
black
light
gray

+
10120
20-30
DEPTH
(on
CORI
dark gray

HHH
30440
c
Table 1: Rates of oxygen consumption in cores 1 and 2
as a function of sand color.
1. Sand color.
2. Uncorrected rates for biochemical oxygen
consumption (ml. 0,/L. consumed/L. of sand/hr.).
3. Uncorrected rates for biochemical oxygen
consumption (ml. 0/L. consumed/L. of sand/hr.).
4. Corrected rates for biochemical oxygen
consumption (ml. 0,/L. consumed/L. of sand/hr.).
Substrate concentration - 50 pg-at sodium acetate-L/L
brown
very
light
gray
light
gray
gray
dark
gray
black
29.9 + 3.4
30.2 4 0.8
43.240.6
53.5 + 0.6
33.9+ 3.4
32.6+ 2.8
—
52.9 + 1.0
61.0 + 2.8
19.0 + 1.5
22.9+
2.8
251 + 1.0
31.3 + 2.9
Figure 7: Rates of nitrification in core 1 as a function
of substrate concentration.
Core Depth
Sand Color
broun
0—-10 cm
o—e
+—4
10—20 cm
very light gray
q--o
20—-30 cm.
gray
30—-40 cm.
O - - -0
dark ray
Ho
25
20
15
10

o
20
40
at NHC-
80
100
e
Figure 8: Rates of nitrification in core 2 as a function
of substrate concentration.
Core Depth
Sand Color
—
0—-10 cm.
broun
10—-20 cm.
very light gray
+———+
E
20—30 cm.
gray
O—— -O
30—-40 cm.
dark gray
5
20
15
10
5
40
60
at NHCEN
80
100
Figure 9: Rates of nitrification in core 1 as a function
of sand depth and color.
Substrate Concentration
O pg-at NH,CI-N/L.
25 pg-at NH,CI-N/L.
+—4

50 pg-at NH,CI-N/L.
—
100 pg-at NH,CI-N/L.
brown
25

+
20

+15

0
o

O
oo
H
LICOLORI
gray

n

+20-30
10420
COREHDEPTHET.)
SAND
veryt
light
gray
darkigray

30-40
H
e
Figure 10: Rates of nitrification in core 2 as a function
of sand depth and color.
Substrate Concentration
—0
O pg-at NH,CI-N/L.
25 pg-at NH,CI-N/L.
+—4
50 pg-at NH,CI-N/L.
—e
100 pg-at NH,CI-N/1.
brown
25
20


s
8
8


H
o

1

010
SANDE COLOR
veryt
black
ight
gray
20-30
DEPIH(cm.)
10120
CORE
dark gray
30-40
Table 2: Rates of nitrification in cores 1 and 2 as a
function of sand color.
1. Sand color.
2. Rates of nitrification (ug-at NO,-N/L.
produced/L. of sand/hr.) in core 1.
3. Rates of nitrification (ug-at NO,-N/L.
produced/L. of sand/hr.) in core 2.
Substrate concentration - 50 pg-at NH,CI-N/L.
brown
very
light
gray
light
gray
gray
dark
gray
black
10.5 + 0.8
11.5 + 1.4
1. + 0.8
7.8 + O.1
9.4 + 0.2
0.2 + 0.2
8.0 + 1.3
4.0 + 0.2
Figure 11:
Production of nitrite in two regions of core 2
as a function of inubation time.
Sand Color
Core Depth
E— 10—-20 cm.
very light gray
0— 20—-30 cm.
black
Substrate Concentration - 100 pg-at NH,CI-N/L.
O
10

24
96
72
120
48
Houtsof IIncubation
144
168
Figure 12: Rates of sulfide production in core 2 as a
function of substrate concentration.
Sand Color
Core Depth
O—
0—-10 cm
broun
+— —+ 10—-20 cm.
very light gray
—
20—-30 cm.
black
——
30—-40 cm.
ark gray
30
20

0

200 300
100
ug-at Sodium AcetateC
400
Figure 13: Rates of sulfide production as a function of
sand depth and color.
Substrate Concentration
O
U yg-at sodium acetate-C/L.
+-——4
100 ug-at sodium acetate-C/L.
G
200 jg-at sodium acetate-C/L.
————
400 pg-at sodium acetate-C/1.
brown
30

20
—
10
3 4

2
0-10
SAND
very
light
gray
10-20
CORE
COLOR
black
20-30
DEPTH(cm.)
dark gray
30-40