Download 04_Spradling - Rutgers University

RIOS 2004
The Effects of Containment on Oceanic Bacteria
Merry Spradling1, Gary Taghon2
1Arizona
State University, [email protected] 2Rutgers University
I. Abstract
0.030
0.15
mg N/mL
mg C/mL
Downplume 4°C
Downplume 20°C
Upplume 4°C
0.10
0.025
Downplume 4°C
0.020
Downplume 20°C
Upplume 4°C
0.015
Upplume 20°C
Upplume 20°C
0.010
0.05
0.005
0.000
0.00
5/28 5/29 5/30 5/31 6/1 6/2 6/3 6/4
5/28 5/29 5/30 5/31 6/1 6/2 6/3 6/4
Date
Date
Phosphorous Content
Bacterial Activity
0.45
0.035
0.40
nmoles MCA/mL/min
0.030
0.35
0.30
Downplume 4°C
0.25
Downplume 20°C
0.20
Upplume 4°C
0.15
Upplume 20°C
0.10
0.025
Downplume 4°C
0.020
Downplume 20°C
0.015
Upplume 4°C
Upplume 20°C
0.010
0.005
0.05
0.00
0.000
5/28 5/29 5/30 5/31 6/1 6/2 6/3 6/4
5/28 5/29 5/30 5/31 6/1 6/2 6/3 6/4
Date
Date
Date
5/28
5/30
6/1
6/4
Particulate C:N:P ratio
Upplume 4°C
Downplume 20°C Upplume 20°C
74:7:1
122:13:1
74:7:1
66:6:1
79:12:1
68:7:1
77:7:1
92:13:1
75:7:1
73:7:1
89:14:1
71:7:1
Downplume 4°C
122:13:1
80:12:1
89:13:1
81:13:1
Chlorophyll a
0.035
0.02
0.03
0.015
0.025
Downplume 4°C
0.02
Downplume 20°C
Upplume 4°C
0.015
Upplume 20°C
0.01
0.005
0.01
Downplume 4°C
Downplume 20°C
0.005
Upplume 4°C
Upplume 20°C
0
5/28 5/29 5/30 5/31 6/1 6/2 6/3 6/4
-0.005
0
5/28 5/29 5/30 5/31 6/1 6/2 6/3 6/4
-0.01
Date
Date
Bacterial Growth
4.00E+06
3.50E+06
3.00E+06
2.50E+06
2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
0
5
20
25
30
0.060
7.00
0.050
0.050
6.00
0.040
17-Jun
0.030
21-Jun
0.020
0.010
0.040
17-Jun
0.030
21-Jun
0.020
0.010
0.000
P (m g/m L)
0.060
N (m g)
C (m g)
P Content
N Content
C Content
dc
w /o
ucw
uc
w /o
5.00
17-Jun
4.00
21-Jun
3.00
2.00
1.00
0.000
dcw
dcw
Sam ple ID
dc
w /o
ucw
0.00
uc
w /o
dcw
ucw
d c w /o u c w /o
Sam ple ID
Sam ple ID
N Content
P Content
Isochrysis:
C Content
30.00
0.050
0.050
25.00
0.040
17-Jun
0.030
21-Jun
0.020
0.010
0.040
17-Jun
0.030
21-Jun
0.020
P(m g/m L)
0.060
N (m g)
0.060
0.000
0.000
d i w /o u i w
u i w /o
20.00
17-Jun
15.00
21-Jun
10.00
0.00
diw
d i w /o u i w
u i w /o
diw
uiw
d i w /o u i w /o
VI. Works Cited
Sam ple ID
Sam ple ID
Pavlova:
0.060
25.00
0.050
0.050
20.00
0.040
0.040
17-Jun
21-Jun
0.020
0.010
17-Jun
0.030
21-Jun
0.020
0.010
0.000
dp
w /o
upw
Sam ple ID
up
w /o
15.00
17-Jun
10.00
21-Jun
5.00
0.00
0.000
dpw
P (m g/m L)
0.060
N (m g)
C (m g)
P Content
N Content
C Content
0.030
The first experiment, which tried to relate bacterial growth to time, shows at first
a decline and then an increase in particulate C, N, and P. This was not surprising and
could be because of a number of explanations. The explanation that seems to be the best
is that there was a loss of predation. Another possible reason is that algae grew in the
flasks as well. However, these flasks were kept in the dark while incubating so that does
not seem to be a reasonable interpretation.
As for the difference in growth relating to the temperature difference, it appears
that, although contrary to intuition, these bacteria grow better in a colder environment.
If the explanation that the bacteria grew because of a loss of predation is accepted as
true, one could then also conclude that perhaps it is not that the bacteria acclimate better
to the 4°C environment but that instead the predators did not acclimate to the 4°C
environment and this allowed more bacteria to survive.
The results for the portion of the experiment considering the dissolved nutrients
were perplexing. It seems most likely that even though the algaes used as a C source
were supposed to be non-viable algae. The increase in C content in the flasks with no
dissolved C content something autotrophic must have been present. There were only 3
possible sources of algae though. One from the manufacture’s algae sold as non-viable.
Another possible source was from the filtered seawater used to dilute the algal paste.
The other possible source was the inoculate used from the upplume and downplume
water, but this water had been kept in the dark for several weeks.
Another problem with this experiment was the data from the spectroflorometer
for the chlorophyll a and phaeopigment measurements. These confusing results could
be due to the photomultiplier tube, which was newly installed to increase sensitivity to
the lower wavelengths where chlorophyll and phaeopigments are detected. When the
results were calculated they seemed to indicate that there was no chlorophyll when the
individual samples were considered. Yet, when one looked at the readings as a time
series there was a definite increase in the numbers.
Clearly this experiment, even though it had just begun, would need to be repeated
this time in the dark. This would hopefully provide more reliable results before
attempting to correlate growth to C:N:P ratios.
5.00
0.010
Sam ple ID
The upper middle graphs represent the data from the experiment run on the Hudson
River plume water for particulate C, N, and P content. From this data the C:N:P ratios
were calculated. A graph of the bacterial activity is also shown.
Obviously the upplume benthic water contains more of all the nutrients (C,N,P)
which isn’t surprising considering the amount of anthropogenic input in the Hudson
(Hetling 1999). What is more interesting is the apparent increase in particulate nutrients
over time for the water kept at 4°C, more so at least than the 20°C water.
15
Chaetocerous:
diw
IV. Results
10
Time (hrs)
C (m g)
Samples were collected from benthic upplume waters (latitude 40.41.142, longitude
74.01.713) and surface downplume waters (latitude 40.20.54, longitude 73.56.74) of the
Hudson River plume with a surface pump. Before half of each sample was stored at 4C
and 20C in a total of 36 flasks in the dark, 6 time (t)=0 samples were taken as a baseline.
Three repetitions were then filtered through Whatman GF/F filters in order to separate the
dissolved and particulate matter over time intervals of t=0, t=2, t=4, and t=7 days. These
samples were then analyzed for particulate C, N, P, chlorophyll, phaeopigments, and
bacterial activities. Samples were also later taken from latitude 39.45 longitude 74.23 with
niskins and kept at room temperature. Bacterial counts were preformed on these with time
intervals of t=0, t=1.5, t=3, t=18, and t=24 hours.
With the microbes leftover from the benthic upplume and surface downplume
Hudson plume waters, cultures were made with artificial seawater that included dissolved
nutrients. Then 12 flasks of 50mL artificial seawater were made up with half containing no
dissolved N or P and the other half containing 15mM NH4 and 6 mM PO4. One each of the
different flasks containing dissolved nutrients and containing no nutrients were then
inoculated with 1mL of a 1:100 solution of Chaetocerous (chae), Isochrysis (iso), and
Pavlova (pav) algeas. Measurements were then taken for the initial day and day 4.
# Bac/mL
III. Methods
The bacterial activity measurements also effectively support this explanation.
Bacterial activity is measured by how much a chemical called MCA (a food source) is
consumed by the bacteria. The bacterial activities for this experiment showed a large
increase for downplume 4°C over downplume 20°C. The same development is shown
for the upplume bacteria, though not as strongly.
The chlorophyll a and phaeopigments (decomposing algae) data, the middle
graphs, also support this trend. The chlorophyll a measurements are used in this case to
represent living algae where as phaeopigments represent dead algae. The phaeopigment
calculations for the downplume 4°C and 20°C were originally negative numbers that
were taken to mean 0 because a flask obviously can not have a negative number of
phaeopigments. The strong decreases in the chlorophyll a for downplume 20°C and
upplume 20°C could be interpreted as the bacteria eating the algae.
The bacterial growth measurements show that there is an increase in the bacteria
over time. The graph shows that the bacteria approximately double in numbers from t=3
to t=18 at room temperature.
The bottom middle graphs show the particulate C, N, and P content of the
microbes in the experiment conducted with the dissolved nutrients grouped by which
type of algae was used as the C source. The samples are identified as follows: The first
letter indicates d for downplume water sample microbes or u for upplume water sample
microbes. The second letter indicates which type of algae was the C source: c chae, i for
iso, or p for pav. The final identifying letter/s are either w for with dissolved nutrients or
w/o for without dissolved nutrients. Almost all of the measurements show a marked
increase in growth of the microbes in the flasks containing the dissolved nutrients.
V. Conclusions
Phaeopigments
Phaeopigments (µg/L)
In the study of oceanic microbes the storage and culturing are often necessary to
study many phenomena associated with them. These treatments present complications for
the interpretation and analysis of data.
The most forthcoming problem has to do with the effects of storage on these
samples. The question of whether or not the microbes grow, stay the same, or decline has
obvious effects on how samples are collected and treated. By various measurements it can
be determined if microbes from benthic upplume water and surface downplume water of
the Hudson River kept in different storage conditions (4C and 20C) vary as a function of
time.
Another problem is the consequences of the media nutrient concentrations on
growth and size. Kirchman (2000) points out that the growth medium ratio (C:N:P) has
consequences on the growth rates of bacteria. He goes on further to relate that bacteria
grown on richer media (laboratory cultures) are faster growing and larger that bacteria
grown in natural environments (Kirchman 2000). This causes a large surface area to
volume ratio difference severely affecting internal solute concentrations that in turn
influence the bacteria’s metabolism (Kirchman 2000). Another experiment was carried out
with the hope of eventually finding out what concentrations would best suit the bacteria by
determining the minimum amount of added dissolved nutrients needed through a sequence
of experiments hoping to correlate growth with different C:N:P ratios. Unfortunately due
to lack of time only the first phase of this project was completed where the two extremes
were compared, half the flasks were saturated with dissolved nutrients and the other half
had none.
0.035
0.20
mg P/mL
II. Introduction
Nitrogen vs Time
0.25
Chlorophyll a (µg/L)
Water from the Hudson River plume, benthic upplume and surface downplume, are
kept at two different temperatures (4°C and 20°C) in the dark. Various measurements are
thought to show an increase in bacteria that is greater for the 4°C water than the samples
kept at 20°C. Another experiment using benthic water at an open ocean site (latitude 39.45
longitude 74.23) was preformed to measure the bacterial numbers over a period of 24
hours. These measurements showed an approximate double in numbers between the times
of 3 hours and 18 hours. The increase in the bacteria is thought to be due to a decrease in
predation pressure brought on by the colder temperature.
Finally the leftover microbes from the upplume and downplume sites were used in
an experiment where half the flasks had dissolved nutrients and the other half did not. The
results did not come out very well as there must have been some contamination of some
sort. The possible source of this contamination is then discussed.
Carbon vs Time
dpw
dp
w /o
upw
Sam ple ID
up
w /o
dpw
upw
dp
w /o
Sam ple ID
up
w /o
Kirchman, David L. (2000) Uptake and Regeneration of Inorganic Nutrients by Marine Heterotrophic
Bacteria. Microbial Ecology of the Oceans. (ed. K.L. Kirchman), 261-288. Wiley-Liss, New
York.
Hetling, Leo; Norbert A. Jaworski and David J. Garretson. (1999). Comparison of nutrient input
loading and riverine export fluxes in large watersheds. Water Science and Technology. 39.12.
189-196.