Download Isolation of Bacteria from the Corallum of Porites lobata (Vaughn

A M . ZOOLOGIST, 9:735-740 (1969).
Isolation of Bacteria from the Corallum of Porites lobata (Vaughn)
and Its Possible Significance
Louis H. DISALVO
Department of Zoology, Unixjersity of North Carolina,
Chapel Hill, N. C. 27514
SYNOPSIS. Bacteria were recovered from loci within skeletal regions of the glomerate
coral, Porites lobata. The origin of these bacteria is unknown, although areas of
discoloration suggest invasion from the substratum in the region of basal attachment.
Weakened areas of internal corallum contained from 10* to 105 bacteria per gram dry
weight as determined by plate counts on a peptone-agar medium. Some of the isolated
bacteria were capable of digesting chitin in vitro. This rinding suggested that the
mechanism for skeletal weakening might be bacterial breakdown of the organic matrix.
Absence of change from aragonitic to calcitic crystals from a discolored region
supported the contention that skeletal weakening was due to the breakdown of organic
matrix rather than dissolution of carbonate.
Results were obtained as part of a survey to determine the number and distribution
of bacteria in some coral reef environments.
Coral reefs are entire ecosystems whose
existence and perpetuity depend on the
buildup and re-arrangement of calcified
structures. In these diversified communities
specialized organisms biochemically construct skeletons from calcium, carbonate,
and small amounts of other ions removed
from the surrounding seawater. Concurrent with skeletal buildup, physical and
chemical forces, as well as the specialized
activities of boring organisms, fragment
and perhaps dissolve portions of secreted
skeletons. Two major groups of microscopically active organisms have been implicated in penetration and destruction of
calcareous reef substrates. These include
the clionid sponges (Goreau and Hartman, 1963; Neumann, 1966) and a diverse
taxonomic assemblage of "boring algae"
which penetrate limestone substrates (Purdy and Kornicker, 1958).
The activities of bacteria have sometimes been implicated in coral reef carbonate dynamics. Hypotheses were proposed by Purdy (1963) and Swinchatt
(1965) concerning bacteria-mediated breakdown and buildup of calcareous substrates.
This work was supported by the U.S.
Health Service (PHS5701 Es 0006104), the
Institute of Marine Biology, and the U.S.
Energy Commission through the Eniwetok
Biological Laboratory.
Public
Hawaii
Atomic
Marine
Additional hypotheses were reviewed and
augmented by Wood (1960), who admitted
to the almost total lack of knowledge about
bacteria on reefs.
BACTERIA ON CORAL REEFS
This report includes data collected during studies on the number, distribution,
and activities of bacteria in selected coral
reef environments. During exploratory research it was apparent that bacteria invaded coral skeletons. Casual splitting of glomerate coral heads during studies of Eniwetok Atoll in 1964 revealed discolored
(light brown) internal regions. These
regions did not have the appearance of the
typical boring algal bands, and the discolored skeleton was more friable than the
adjacent white skeleton. Peptone-agar cultures of scrapings from the discolored
regions yielded many bacterial colonies.
Similar cultures of scrapings taken from
adjacent intact skeletal areas of the same
head gave negative results. An additional
observation was that some penetration
paths of boring sponges appeared to follow
pre-existing streaks of discoloration into
deep regions of the corallum.
More recently I carried out research at
the Hawaii Institute of Marine Biology at
Kaneohe Bay, Oahu, during which I at735
736
Louis H.
FIG. 1. Corallum o£ Porites lobala split apart to
show internal altered region. Sites A, B, and C
represent zones sampled in the initial qualitative
bacterial recovery (Table 2) . Areas A and B are
light tan, C is gray-black.
tempted to verify and enlarge upon the
exploratory results using a single species of
coral. The only glomerate coral species
commonly found in Kaneohe Bay is Porites lobata
(Vaughn). These colonies
proved excellent for the studies because
different coralla when split apart showed
internal disturbances of varied intensity
and appearance. Figure 1 illustrates the
areas sampled in an internal decalcified
region. Most commonly these areas were
brown, although occasionally a gray-black
zone was found. The gray-black regions
were probably sites of sulfate reduction,
smelling weakly of H 2 S.
Bacterial invasion of coral skeletons undoubtedly depends on the quality and
quantity of nutrient material in the corallum. Wainwright (1963) described some
organic materials which might occur in
coral skeletons. He found a fibrillar chitinous matrix in the coral, Pocillopora damicornis as well as remnants of blue-green
boring algae, and a group of unidentified
organic filaments. Histochemical tests
showed that protein, polysaccharide, and
lipid moieties were present.
My initial experiment was conducted to
determine if the Porites skeleton contained alcohol-extractable substances which
would support the growth of bacteria in
vitro. The overall procedure included ex-
DISALVO
traction of powdered corallum, recovery ot
extracts, and preparation of an organic
matter-free liquid medium into which the
extract was placed as an organic enrichment. A bacterial inoculum was then injected into control and extract-enriched
media, and bacterial multiplication in
both media was periodically monitored by
bacteriological plating. Procedural details
are as follows. A 10-kg head of Porites
lobata was obtained from the reef and split
apart with a hammer and chisel. Superficial pieces of the head were successively
split off until fragments were obtained
from the interior which were at least 10 cm
away from any head surface and free ol
macroscopically visible borers. No algal
bands were visible in the sample. In all
subsequent handling procedures chromic
acid cleaned labware was used to prevent
organic contamination of the skeletal material. The material was ground in a mortar and pestle and dried at 60°C yielding a
weight of 173 g. The powdered skeleton
was then extracted in 100 ml absolute
ethyl alcohol for 2 hr at 60°C in a sealed
flask which was agitated at 0.5-hr intervals.
The alcohol was filtered through Whatman brand #1 paper and evaporated to
dryness in the oven at 60°C. The recovered
greenish-white material weighed 0.319 g
(0.184% of corallum extracted), and was
kept desiccated at room temperature until
used. An inorganic (control) medium was
prepared from seawater which had been
aged in the dark for six weeks and then
passed through a 0.45 ^ (pore size) Millipore filter. T o this water I added 0.05%
(NH 4 ) 2 SO 4 . Experimental medium was
prepared by enriching control medium
with 0.4% of the coral skeletal extract (4 g
1 - 1 ) . Five-ml aliquots of each of the two
media were placed in 75 X "> mm test
tubes, closed with cotton, and sterilized by
autoclaving. Each set of media was inoculated with approximately 1 X 1 ^ viable
bacteria from a seawater suspension of coral reef regenerative sediment (DiSalvo,
1969). All tubes were incubated in the
dark at 26°C and agitated by hand about
once every three days. One control tube
BACTERIA IN GLOMERATE CORAL HEADS
TABLE 1. Stimulation of bacterial growth by adding
coral skeletal extract to an inorganic liquid medium.
Colony count
Day of
incubation
Replicate
plate
No skeletal
extract
5
32
1
1
2
18
T-t
32
2
1
2
50
60
50
30
16
40
35
Skeletal
extract
ca.
ca.
ca.
ca.
ca.
400
1000
1000
600
600
150
180
and one enriched tube were harvested at
intervals recorded in Table 1. Duplicate
bacteriological platings were made using
ZoBell 2216e medium which contained
0.5% peptone, 0.1% yeast extract, 0.01%
FePO4, 1.5% agar, and aged seawater
qs (final pH = 7.5-7.8). A 0.1 ml aliquot
of a 1:100 dilution of each culture was
inoculated into Petri dishes (9 cm diameter) and pour-plates were made using
about 10 ml molten agar which had been
cooled to 40-45°C.
Table 1 demonstrates that the inoculated bacteria were able to utilize the coral
skeletal extract for growth, producing 5 to
20 times more colonies over a 4-week
period.
Concurrently, I made several attempts to
isolate bacteria from selected areas within
Porites lobata heads. Specific attempts
were made to culture chitinoclastic bacteria which might have been utilizing the
coral matrix as a nutrient source. In the
following experiments, coral heads weighing 2-4 kg were collected from the reef and
returned alive to the laboratory in seawater.
Each head was drained, dried of superficial water, and cleaved with a hammer and
chisel for sampling. Sampling zones within the head were chosen by arbitrary visual
inspection. The split coral face was immediately placed in a working chamber illuminated with a UV light prior to sampling to reduce the possibility of aerial
contamination. Sample scrapings were taken with a flame-sterilized microspatula and
rinsed into sterile seawater dilution
blanks. All dilution tubes were vigorously
737
agitated by hand for several minutes before plating or further dilution in order to
suspend as many bacteria as possible. After the rapid aseptic sampling had been
completed, additional sample scrapings
were obtained and observed microscopically for presence of boring sponge tissue.
Qualitative measurements. In the first
sampling, about 0.05 g skeletal scrapings
from each of three discolored areas were deposited in test tubes containing 1 ml of
sterile seawater. Well agitated 0.1-ml sample aliquots were pour-plated for total
counts in the peptone medium. Clearance
of paniculate chitin suspended in a mineral agar medium was the criterion used to
ascertain the presence of chitinoclastic
bacteria in the samples. The chitin-agar
medium contained 1.5% agar, 0.05%
(NH4)2SO4, 0.5% precipitated chitin particles, and aged seawater qs (Lear, 1963).
The head areas sampled were designated
"A", "B", and "C" (Fig. 1). Areas A and B
appeared light brown, while C was grayblack. The scrapings from areas A and B
were cultured under aerobic conditions,
and C was cultured anaerobically by using
a vacuum desiccator for a culture chamber.
The chamber was flushed repeatedly with
inert gas ("Q" gas, Nuclear Chicago
Corp.) after the inoculated plates were installed. Pyrogallol was included in the bottom of the desiccator to absorb residual
O2. All cultures were incubated in the
dark at room temperature (26°C). The
results are given in Table 2.
A second qualitative sampling was carried out as suggested by Odum and Odum
(1956), in which a bacteriological profile
of a Porites head was obtained. About 0.05
g of skeletal scrapings were taken from
each of seven sampling areas as listed in
Table 3. The scrapings were each initially
placed in 4.5 ml sterile seawater diluents.
Following procedures outlined above, 0.1
ml aliquots of each dilution were plated
for aerobic and anaerobic total counts as
well as for aerobic chitinoclast counts. The
results are listed in Table 3.
The above results show that bacteria
are present in discolored regions. No bor-
Louis H.
738
DISALVO
TABLE 2. Culture of aseptically sampled internal skeletal scrapings of Porites lobata to ascertain
presence of bacteria.
Colony counts
Sampling
zone
Appearance
of corallum
Replicate
plate
A
white-brown
B
white-brown
C*
gray-black
1
2
1
2
1
2
Peptone agar
(3 days)
ca.
ca.
ca.
ca.
ca.
ca.
600
600
600
800
800
800
Chitin agar
(12 days)
12
6
80
75
0
0
* Cultured under anaerobic conditions.
ing-sponge tissue was microscopically visible in the scrapings from these areas, suggesting that the bacteria were the sole
agents responsible for the degradative
effects on the corallum. The growth of
chitinoclasts suggested a possible role in
the breakdown of the chitinous coral matrix.
Quantitative measurements. An estimate
of bacteria per gram of skeleton was
obtained for an internal Porites region
affected by bacteria. An amount of
skeleton was recovered under aseptic conditions, and was ground in a heat-sterilized
mortar and pestle with 4.5 ml sterile seawater. A 1-ml aliquot of this ca. 10 ml of
slurry was diluted 1:100 and 1:1000 in sterile seawater, and the skeletal remains were
recovered by filtering onto Whatman #1
paper. The skeletal material was dried in
the oven at 60°C and weighed, yielding a
dry weight of 8.0 grams. One-tenth ml aliquots of the 1:100 and 1:1000 dilutions
were plated in peptone and chitin agar as
described above. The results of this determination are presented in Table 4.
A similar determination was performed
on Porites skeleton which showed incipient
invasion by boring sponge. A sample ol
sponge-invaded skeleton was obtained
using a sterile microspatula as described
above. The scrapings were placed in 4.5 ml
TABLE 3. Qualitative profile of a Porites lobata head with regard to the presence of total aerobic,
anaerobic, and chitinoclastic bacteria.
Colon}7 count
Description
of zone
Peptone
Eeplicate (aerobic)
7
plate
3
2
sub-polyp green band
3
green-brown band
10 mm sub-surface
It. brown area
20 mm sub-surface
center white area
1
2
1
2
It. brown band
20 mm from base
7
discolored zone,
5 mm from base
Blank plates for sterility tests
1
2
1
2
1
2
4
CO
5
I-l
CO
1
2
6
m = mycelium
2
0
0
0
0
0
0
0
0
0
1
1
14
17
0
0
5
25
25
10
1
2
4
3
0
lm
8
10
100
85
0
lm
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
5
2
0
0
Chitin
(aerobic)
7
3
nd
0
0
lm
0
0
0
0
nd
nd
nd
0
0
lm
0
0
0
0
nd
nd
r-l
polyp zone
Peptone
(anaerobic)
7
3
O
1
I-l
Sample
no.
0
1
0
1
0
3
0
0
0
0
medium
days incubated
739
BACTERIA IN GLOMERATE CORAL HEADS
TABLE 4. Relation between bacterial numbers and weight of corallum, Porites lobata, internal
discolored region.
Colony count
Medium
(incubation time)
Replicate
plate
Peptone (48 hr)
1
2
1
2
Chitin (100 hr)
Calculation:
Mean plate
count
(peptone)
(chitin)
12.5
18.5
1.25 X 108
ca. 300
ca. 300
17
20
Dilution
factor
1.25 X 3 04
1.25 X 103
sterile seawater and agitated, and 0.1 ml
aliquots were plated in duplicate in peptone agar. The skeletal scrapings were
recovered by filtration onto a small-tared
Whatman #1 paper. The dry weight of the
scrapings was 0.06 g. After 24 hr incubation the two replicate plates showed
counts of 30 and 40 colonies, representing
a minimum of 2.7 X 104 viable cells per
gram of sponge-infiltrated corallum.
Three replicate sample scrapings from a
light brown, interior, weakened zone of
Porites were assayed for crystal type by
X-ray crystallography (kindly performed
by Steven Smith, Univ. of Hawaii, Department of Oceanography). The results
showed all calcium carbonate to be in the
form of aragonite.
DISCUSSION
The present results provide the first evidence that bacteria are important in the
breakdown of coral skeletons, although implied evidence is found in the statement of
Sorby (1879, p. 71) that: ". . . powdered
coral, kept for weeks in water, loses organic matter and gives rise to such minute
particles that the water is like dilute
milk." My most important finding was that
bacteria isolated from the coral skeletons
were able to digest chitin suspended in
otherwise nutrient-free agar. The agar was
seldom attacked. Results of the crystal analysis showed no crystalline changes in the
weakened areas, indicating absence of
chemical decalcification. These results sug-
1.25 X10 4
10
15
2
1
Grams
skeleton
Colonies/
gram skeleton
8.0
8.0
1.95 X 10*
0.29 X 10*
gest that weakening of the corallum is due
to bacterial breakdown of the organic matrix. No X-ray crystallography was performed on gray-black regions of weakened
skeleton where chemical reactions accompanying sulfate reduction might be expected to convert aragonite to calcite (Revelle
and Fairbridge, 1957).
A survey which included the results
presented above showed that sediments in
proximity to the bases of coral skeletons
contained minimum counts of 107-108
bacteria per gram dry weight, of which an
estimated 10-20% were chitin-digesting species. Thus, there exists a large pool of
potential infectivity in contact with the
basal regions of most coral skeletons.
Studies on the environmental breakdown
of coral skeletons are complicated by the
several types of boring organisms which
are typically present and contributing to
acute skeletal defects. Further studies must
determine rates of bacterial activity, conditions which favor the sole presence of
bacteria in certain skeletal areas, and the
roles played by bacteria in the overall pattern of succession through which healthy
coralla are reduced to reef sediments.
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DISALVO
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