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Environmental factors associated with proteolytic activity of estuarine bacteria
Pergamon Press
Life Sciancee Vol . 15, pp " 1425-1432 Printed in the O.S .A .
ENVIROrII~RTAL FACTORS ASSOCIATED WITH PROTEOLYTIC ACTIVITY OF ESTUARINE HACTERIAI Ronald &. Sizemore 2 and L. Harold Steveneon Department of Biology and Belle W. Baruch Institute for Marine Biology and Coastal Research University of South Carolina, Columbia, South Carolina 29208 (Received in final form 13 September 1974) SDMMARY The effects of various parameters on the e:tracellular hydrolysis of proteia by salt marsh bacteria were examined using an agar plate assay technique . Maxim:m activity vas observed at pH 8 and 18 C. Elimination of salts and nutrients from the growth media had little effect on activity ; vhile,incubation in a reduced oxygen atmosphere markedly restricted proteolysis by laboratory stocks and random isolates . The availability of oxygen appeared to be one of the important factors controlling extracellular protease production by salt marsh bacteria . Microorganisms have been implicated in the cyclic conversion of carbon, nitrogen, phosphorous, sulfur, iron, and other minerals (1) .
However, Johannes
(2) has suggested that the role of marine bacteria in the regeneration of nitrogen and phosphorous in the sea has been overemphasized . areas present a different type of ecosystem .
Many estuarine
Microorganisms that promote the
decomposition of organic material may be ecologically significant in areas of high nutrient concentratioae such as the detritus system of a salt marsh estuary.
Protein repreaeats a type of molecule that must be hydrolyzed before it
can contribute to the nutrition of bacteria and plants .
The ability of marine
bacteria to degrade protein macromolecules has been reported many times and is well known (3,4) .
Conversely, the effects of various parameters on protein de-
1Contribution No . 108 of the Belle W. Baruch Library in Marine Science 2Preaent address : Department of Microbiology, University of Maryland, College Park, Maryland 20742 1425
1426
Proteolytic Hacteria
Vol . 15, No .B
gradation by marine bacteria has received limited attention .
The objective of
the work reported in this presentation was to determine the effects of various parameters on protease production by heterotrophic estuarine bacteria . MATSRIALS AND METHODS Organisms .
A collection of proteolytic bacteria was obtained by randomly
selecting caseinolytic colonies that developed on dilution plates inoculated with estuarine water or sediment .
The samples were collected from the North
Inlet Estuary near Georgetow, South Carolina .
A complete description of the
sampling area and isolation techniques has been published elsewhere (5,6) . Culture media .
The effects of various parameters on bacterial protease
were tested using double-layer casein agar plates (7) .
This media consisted of
a lower indicator layer of milk-agar and en upper nutrient of, normally, Difco Marine Agar 2216 .
The standard technique involved spot inoculation of 6 case-
inolytic stock organisms on to each plate of assay media . was employed to guage protein degradation .
An arbitrary scale
Zero was used to designate no clear-
ing in the indicator layer, and 4 was used to indicate complete clearing of a 5 cm2 sector .
With the exception of the experiments done to determine the ef-
fect of temperature on activity, all cultures were incubated at 25 C . Media without added NaCl was prepared with 5 g peptone, 1 g yeast extract, and 20 g agar per liter of distilled water . using Marine Agar 2216 plus 200 g NaCl/1 .
High salinity plates were prepared In experiments designed to test the
effects of low nutrient concentration, the surface layer of screening plates wen prepared using only 2X agar in distilled water or 2X agar plue 3 .5X NaCl in distilled water .
The effect of pH on activity was examined using a aeries of
double-layer plates prepared by adjusting the pH of the Marine Agar 2216 from 5 to 10 with either HC1 or NaOH . Anaerobic Effects .
Double-layer assay plates were spot inoculated and
placed in BHL Gaspak Anaerobic jars . Division of Becton, Dickinson and Co .)
(Baltimore Biological Laboratory, A duplicate set of platen that remained
Vol . 15, No . 8
Protaolytic Bacteria
exposed to the atmosphere served as controls .
1427
The BBL mathylann blue anaerobic
indicator strip was used to estimate tha reduction potential . RBSIILTS Fifty-eight caseinolytic cultures were naintainad as laboratory stocks . Thirty-seven ware isolated from avatar and the remainder from sediment .
Approxi-
mataly one-half were gram position, 20x were spherical, and 71x had pigmentation other than white.
The Frasar (8) method of detecting gelatin hydrolysis re-
vealed that 56 of the caseinolytic stock cultures could also degrade gelatin . 4
F FO V F OW O Q:n.
The effect of temperature on the proteolytic activity of the stock cultures after 4 and 8 days of incubation on proteolytic indicator plates . Protaolytic activity was eoaluated according to the arbitrary scale described in the Materials and Methods . The average proteolytic activity after 4 and 8 days of incubation at various temperatures ie illustrated in Fig . 1.
Greater activity was evident at 25
and 30 C after 4 days of growth ; however, after 8 days, the maximum activity was observed in cultures growing at 18 C .
Maximum growth was observed at 37 C.
Thn affect of media pH on the enaymatic activity of the stock cultures after 4 and 8 days of growth is illustrated in Fig. 2. when the growth medium was at a pR of 8.
Maximum hydrolysis occurred
1428
Proteolytic Bacteria
Vol . 15,
No .
8
sF F O
O F
O WF O s d
FIG. 2 The effect of pH on the proteolytic activity of the stock cultures after 4 and 8 days of incubation on proteolytic indicator plates . The responses of the proteolytic stock organisms to alterations in nutrient and salt concentrations, and depletion of oxygen are illustrated in Fig . 3. Strong activity represents 3 and 4 on the arbitrary scale .
Most of the stocks,
70x, demonstrated strong activity when cultivated on marine agar double-layer plates, which served as the control or reference media .
c 90
W H N W H N~
CONTROL
ANAER061C
NO SALT
NO NUTRENTS NO 9ALT
HIRN SALT
NA NA 1AIA SA
NA NÂ A~ SA
N8 NA 1IrA SA
NO NUTRIENTS
2O
K O v.
0 z W~ V K
â 20
FIG. 3 A comparison of the proteolytic activity of tures when grown under various conditions . organisms exhibiting no growth (NG), growth weak activity (WA) and strong activity (3A)
caseinolytic stock culThe percentage of the but no activity (NA), is illustrated .
Vol . 15, No . 8
142 9
Proteolytic Bacteria
Incubation in a reduced oxygen atmosphere reduced both growth and proteolysis with only lOX of the organisms demonstrating strong activity .
Case-
inolytic organisms grown on double-layer plates that lacked added nutrients in the upper growth layer showed little reduction in activity when compared to the control media .
None of the cultures required added salt for growth .
How-
ever, about 30X of the organisms did not demonstrate activity without added salt and this substrate resulted in a 50X decrease in the percentage of bacteria showing strong proteolytic activity .
When both salt and nutrients were
left out of the upper layer, about 15X of the isolates failed to grow, but about 40X of the cultures still demonstrated strong activity .
Moat of the
isolates were capable of growth when the upper nutrient layer was supplemented with 23 .5X NaCl, and most of those were proteolytic .
However, in contrast to
the control cultures, about half demonstrated only weak activity . The harked reduction of proteolytic activity noted when the stock cultures were subjected to anaerobic conditions, prompted an investigation of organisms capable of surviving and functioning under fluctuating oxygen tension . ment samples were diluted and plated on marine agar .
Sedi
Following 8 hr of expo-
sure to the air, the plates warn placed in anaerobic jars and incubated for 7 days .
Every colony that developed on these plates vas transferred to repli-
cats double-layer casein agar plates .
Oae set of the assay plates was incuba-
Led aaaerobically and the other set aerobically .
Following 8 days of incuba-
tion, the plates were eaamiaed for roans of clearing around each structure . The results of four experiments employing samples obtained from various sediment locations are summarised is Table 1 .
In every case, a conaietently
higher percentage of the facultative organisms hydrolysed casein aerobically . It was also noted that the zones of clearing in the protein indicator layer were much larger oa the aerobic plates than on plates incubated at a reduced oxygen tension .
When the organisms obtained from an area covered only during
1430
Vol . 15, No .
Proteolytic Bacteria
strong spring tides were grown aerobically, 75x were proteolytic .
This repre-
sents the highest percentage of proteolytic organisms found in any sample tested during this study. TABLE 1 The Effect of Anaerobiosis of Proteolytic Activity of Bacteria Obtained from Estuarine Sediment
Sampling location
Aerobic Incubation * G P Z
Anaerobic Incubation G P
x
Spring Tide Zone (Feb . 1970)
68
51
75
94
27
29
Intertidal Zone Sample #1 (Feb . 1970)
35
13
37
51
2
4
58
22
38
75
5
7
66
17
26
82
3
4
Sample #2
(May 1970)
Submerged Zone (Feb . 1970)
*G represents the number of colonies growing and P represents the number of colonies demonstrating proteolytic activity . Values are eapreased to the nearest percent . DISCUSSION An assortment of physical variables may regulate the growth, enzyme product ion, and activity of proteolytic bacteria .
Emery and Steveneon (9) have
stated that estuarine organisms need to be euryhaline ; however, an estuarine population may be a mixture of marine, terrestrial, and fresh water bacteria (10) .
Salinity changes would aeverly affect some of these organisms.
The
experimental organisms used in this study grew well under low salinity condiboas and showed only a 15x drop in the total percentage of organisms showing proteolytic activity .
Nigh salinity reduced, but did not eliminate, the acti-
vity of the stock cultures .
Although the upper layer of the teat plates were
prepared with 23 .52 NaCl, the effective concentration was probably lower due to diffusion of salt into the milk indicator layer.
8
Vol . 15, No . 7
143 1
Proteolytic Bacteria
The salinity and nutrient concentrations involved in these experiments were not exact .
Interpretation of experiments with double-layer plates was hampered
by the possibility of diffusion of salts and nutrients between the two layers . Phrthermore, enriched media have been shown to contain some ions (11) and organic components may interact with some canons (12) .
The experiments with low
nutrient double-layer plates were similarly affected by the possibility of diffusion of nutrients between the two layers .
Nevertheless, the results suggest
that the data on proteolysis obtained on enriched media in the laboratory were valid for the bacteria that normally live in a relatively nutrient-poor environment . The availability of oxygen appears to be an important factor controlling The activity of both the stock cultures and
proteolysis by sediment bacteria .
the facultative organisms used in this study was markedly reduced in the ab sence of oxygen .
However, the strict anaerobes were not examined .
ence of oxygen was also noted by Sizemore et al .
The influ-
(6) when they demonstrated
that the levels of "cell-free" proteolysic enzymes were lowest in sediment obrained from anaerobic zones .
Similarly, Sugahara, Sugiyama, and Rawai (13)
reported a greater than 100-fold decrease in the number of gelatin liquefiers in the sand of an experimental sea water aquarium when the system was changed from an 89X to a 6X oxygen saturation . altered very little by this change .
The total number of heterotrophs was
These observations suggest that proteo-
lyric activity may be limited under natural anaerobic conditions in sediments . Limited evidence indicates that the activity in the gut of some marine invertebrates may present a somewhat different picture .
Anaeromonas proteolytica ,
which was isolated from the gut of a wood-boring crustacean, Li.mnoria tripunc tats , by Merkel et al . (14), demonstrated very strong proteolysis when cultured in anaerobic jars .
1432
Proteolytic Hacteria
Vol . 15, No . 8
ACKNOWLEDGEMENT This research was supported in part by grant number NG-33-72 from the United States Department of Commerce aad by the Belle W. Baruch Coastal Research Institute . REFERENCES 1.
A. E . KRISS, Symposi~.en on M~ine MiorobioZogy, (Ed . : C . H. Oppenheimer), pp . 493-499 C . C. Thomas, Springfield (1963) .
2.
R. E. JOHANNES, Advances in Microbiology of the Sea, (Ed . : M. R . Droop and E . J . F . Wood), pp . 203-213 Academic Press Inc ., New York (1968) .
3.
T . M. SKERMAN, Symposiwn on Marine MicrobioZogy, pp . 685-698 C . C. Thomas, Springfield (1963) .
4.
E . E. ZOBELL, Marine MicrobioZogy, Chronica Botanica Co ., Whatam (1946) .
5.
F . J. VERNBERG, Belle W. Baruch Librcmy in Marine Science. Ida. 1 Estuarine Microbial Ecology, (Ed. : L . H. Stevenson and R. R. Colwell), pp . 3-8 University of South Carolina Press, Columbia (1973) .
6.
R. K. SIZEMDRE, L. H. STEVENSON, and B. H. HEHELER, Belle W. Baruch Library in MEu~tine Science. No . 1 Estuarine MicrobiaZ Ecology, (Ed. : L . H. Stevenson and R. R. Colwell), pp . 133-143 University of South Carolina, Columbia (1973) .
7.
R. K. SIZEt~RE and L. H. STEVENSON, App l. Microbial. 20 991-992 (1970) .
8.
W. C . FRAZIER, J. Infect Dtis . 38 302 (1926) .
9.
K. 0. EMERY and R. E. STEVENSON, Treatise on Marine Ecology and Paleoecology, Vo Z. 1 ., (Ed . : J . M. Hegpath), pp . 673-693 Ecology Geol . Soc. Amer . Mem 67 (1957) .
(Ed . : C. H . Oppenheimer),
10 .
J. M. HEDGPATH, Treatise tin Marine Ecology and Paleoecology, Vol. 1 ., (Ed. : J . M. Hedgpath), pp . 693-729 Ecology Geol . Soc . Amer . Mem 67 (1957) .
11 .
R. A. MACLEOD, BacterioZ. Rev. 28 9-23 (1965) .
12 .
E. R. GONYE and G. E . JONES, BeZZe W. Baruch Library tin Martine Science No . 1. Eatr~ine Microbial Ecology, (Ed. : L . H. Stevenson and R. R . Colwell), pp . 243-257 University of South Carolina, Columbia (1973) .
13 .
I . SUGAHARA, M. SUGIYAMA, and A . KAWAI, United States - Japan Seminar on Effects of the Ocean Environment on MiicrobiaZ Activities, (Ed . : R. Y . Morita aad R. R. Colwell), pp . 327-340 University Park Press, Baltimore (1974) .
14 .
J. R. MERREL, E. D. TRAGANZA, B . B. MUKHF.RJEE, T. B. GRIFFIN, and J. M. PRESCOTT, J. BacteriaZ. B4 1227-1233 (1964) .
Life Sciancee Vol . 15, pp " 1425-1432 Printed in the O.S .A .
ENVIROrII~RTAL FACTORS ASSOCIATED WITH PROTEOLYTIC ACTIVITY OF ESTUARINE HACTERIAI Ronald &. Sizemore 2 and L. Harold Steveneon Department of Biology and Belle W. Baruch Institute for Marine Biology and Coastal Research University of South Carolina, Columbia, South Carolina 29208 (Received in final form 13 September 1974) SDMMARY The effects of various parameters on the e:tracellular hydrolysis of proteia by salt marsh bacteria were examined using an agar plate assay technique . Maxim:m activity vas observed at pH 8 and 18 C. Elimination of salts and nutrients from the growth media had little effect on activity ; vhile,incubation in a reduced oxygen atmosphere markedly restricted proteolysis by laboratory stocks and random isolates . The availability of oxygen appeared to be one of the important factors controlling extracellular protease production by salt marsh bacteria . Microorganisms have been implicated in the cyclic conversion of carbon, nitrogen, phosphorous, sulfur, iron, and other minerals (1) .
However, Johannes
(2) has suggested that the role of marine bacteria in the regeneration of nitrogen and phosphorous in the sea has been overemphasized . areas present a different type of ecosystem .
Many estuarine
Microorganisms that promote the
decomposition of organic material may be ecologically significant in areas of high nutrient concentratioae such as the detritus system of a salt marsh estuary.
Protein repreaeats a type of molecule that must be hydrolyzed before it
can contribute to the nutrition of bacteria and plants .
The ability of marine
bacteria to degrade protein macromolecules has been reported many times and is well known (3,4) .
Conversely, the effects of various parameters on protein de-
1Contribution No . 108 of the Belle W. Baruch Library in Marine Science 2Preaent address : Department of Microbiology, University of Maryland, College Park, Maryland 20742 1425
1426
Proteolytic Hacteria
Vol . 15, No .B
gradation by marine bacteria has received limited attention .
The objective of
the work reported in this presentation was to determine the effects of various parameters on protease production by heterotrophic estuarine bacteria . MATSRIALS AND METHODS Organisms .
A collection of proteolytic bacteria was obtained by randomly
selecting caseinolytic colonies that developed on dilution plates inoculated with estuarine water or sediment .
The samples were collected from the North
Inlet Estuary near Georgetow, South Carolina .
A complete description of the
sampling area and isolation techniques has been published elsewhere (5,6) . Culture media .
The effects of various parameters on bacterial protease
were tested using double-layer casein agar plates (7) .
This media consisted of
a lower indicator layer of milk-agar and en upper nutrient of, normally, Difco Marine Agar 2216 .
The standard technique involved spot inoculation of 6 case-
inolytic stock organisms on to each plate of assay media . was employed to guage protein degradation .
An arbitrary scale
Zero was used to designate no clear-
ing in the indicator layer, and 4 was used to indicate complete clearing of a 5 cm2 sector .
With the exception of the experiments done to determine the ef-
fect of temperature on activity, all cultures were incubated at 25 C . Media without added NaCl was prepared with 5 g peptone, 1 g yeast extract, and 20 g agar per liter of distilled water . using Marine Agar 2216 plus 200 g NaCl/1 .
High salinity plates were prepared In experiments designed to test the
effects of low nutrient concentration, the surface layer of screening plates wen prepared using only 2X agar in distilled water or 2X agar plue 3 .5X NaCl in distilled water .
The effect of pH on activity was examined using a aeries of
double-layer plates prepared by adjusting the pH of the Marine Agar 2216 from 5 to 10 with either HC1 or NaOH . Anaerobic Effects .
Double-layer assay plates were spot inoculated and
placed in BHL Gaspak Anaerobic jars . Division of Becton, Dickinson and Co .)
(Baltimore Biological Laboratory, A duplicate set of platen that remained
Vol . 15, No . 8
Protaolytic Bacteria
exposed to the atmosphere served as controls .
1427
The BBL mathylann blue anaerobic
indicator strip was used to estimate tha reduction potential . RBSIILTS Fifty-eight caseinolytic cultures were naintainad as laboratory stocks . Thirty-seven ware isolated from avatar and the remainder from sediment .
Approxi-
mataly one-half were gram position, 20x were spherical, and 71x had pigmentation other than white.
The Frasar (8) method of detecting gelatin hydrolysis re-
vealed that 56 of the caseinolytic stock cultures could also degrade gelatin . 4
F FO V F OW O Q:n.
The effect of temperature on the proteolytic activity of the stock cultures after 4 and 8 days of incubation on proteolytic indicator plates . Protaolytic activity was eoaluated according to the arbitrary scale described in the Materials and Methods . The average proteolytic activity after 4 and 8 days of incubation at various temperatures ie illustrated in Fig . 1.
Greater activity was evident at 25
and 30 C after 4 days of growth ; however, after 8 days, the maximum activity was observed in cultures growing at 18 C .
Maximum growth was observed at 37 C.
Thn affect of media pH on the enaymatic activity of the stock cultures after 4 and 8 days of growth is illustrated in Fig. 2. when the growth medium was at a pR of 8.
Maximum hydrolysis occurred
1428
Proteolytic Bacteria
Vol . 15,
No .
8
sF F O
O F
O WF O s d
FIG. 2 The effect of pH on the proteolytic activity of the stock cultures after 4 and 8 days of incubation on proteolytic indicator plates . The responses of the proteolytic stock organisms to alterations in nutrient and salt concentrations, and depletion of oxygen are illustrated in Fig . 3. Strong activity represents 3 and 4 on the arbitrary scale .
Most of the stocks,
70x, demonstrated strong activity when cultivated on marine agar double-layer plates, which served as the control or reference media .
c 90
W H N W H N~
CONTROL
ANAER061C
NO SALT
NO NUTRENTS NO 9ALT
HIRN SALT
NA NA 1AIA SA
NA NÂ A~ SA
N8 NA 1IrA SA
NO NUTRIENTS
2O
K O v.
0 z W~ V K
â 20
FIG. 3 A comparison of the proteolytic activity of tures when grown under various conditions . organisms exhibiting no growth (NG), growth weak activity (WA) and strong activity (3A)
caseinolytic stock culThe percentage of the but no activity (NA), is illustrated .
Vol . 15, No . 8
142 9
Proteolytic Bacteria
Incubation in a reduced oxygen atmosphere reduced both growth and proteolysis with only lOX of the organisms demonstrating strong activity .
Case-
inolytic organisms grown on double-layer plates that lacked added nutrients in the upper growth layer showed little reduction in activity when compared to the control media .
None of the cultures required added salt for growth .
How-
ever, about 30X of the organisms did not demonstrate activity without added salt and this substrate resulted in a 50X decrease in the percentage of bacteria showing strong proteolytic activity .
When both salt and nutrients were
left out of the upper layer, about 15X of the isolates failed to grow, but about 40X of the cultures still demonstrated strong activity .
Moat of the
isolates were capable of growth when the upper nutrient layer was supplemented with 23 .5X NaCl, and most of those were proteolytic .
However, in contrast to
the control cultures, about half demonstrated only weak activity . The harked reduction of proteolytic activity noted when the stock cultures were subjected to anaerobic conditions, prompted an investigation of organisms capable of surviving and functioning under fluctuating oxygen tension . ment samples were diluted and plated on marine agar .
Sedi
Following 8 hr of expo-
sure to the air, the plates warn placed in anaerobic jars and incubated for 7 days .
Every colony that developed on these plates vas transferred to repli-
cats double-layer casein agar plates .
Oae set of the assay plates was incuba-
Led aaaerobically and the other set aerobically .
Following 8 days of incuba-
tion, the plates were eaamiaed for roans of clearing around each structure . The results of four experiments employing samples obtained from various sediment locations are summarised is Table 1 .
In every case, a conaietently
higher percentage of the facultative organisms hydrolysed casein aerobically . It was also noted that the zones of clearing in the protein indicator layer were much larger oa the aerobic plates than on plates incubated at a reduced oxygen tension .
When the organisms obtained from an area covered only during
1430
Vol . 15, No .
Proteolytic Bacteria
strong spring tides were grown aerobically, 75x were proteolytic .
This repre-
sents the highest percentage of proteolytic organisms found in any sample tested during this study. TABLE 1 The Effect of Anaerobiosis of Proteolytic Activity of Bacteria Obtained from Estuarine Sediment
Sampling location
Aerobic Incubation * G P Z
Anaerobic Incubation G P
x
Spring Tide Zone (Feb . 1970)
68
51
75
94
27
29
Intertidal Zone Sample #1 (Feb . 1970)
35
13
37
51
2
4
58
22
38
75
5
7
66
17
26
82
3
4
Sample #2
(May 1970)
Submerged Zone (Feb . 1970)
*G represents the number of colonies growing and P represents the number of colonies demonstrating proteolytic activity . Values are eapreased to the nearest percent . DISCUSSION An assortment of physical variables may regulate the growth, enzyme product ion, and activity of proteolytic bacteria .
Emery and Steveneon (9) have
stated that estuarine organisms need to be euryhaline ; however, an estuarine population may be a mixture of marine, terrestrial, and fresh water bacteria (10) .
Salinity changes would aeverly affect some of these organisms.
The
experimental organisms used in this study grew well under low salinity condiboas and showed only a 15x drop in the total percentage of organisms showing proteolytic activity .
Nigh salinity reduced, but did not eliminate, the acti-
vity of the stock cultures .
Although the upper layer of the teat plates were
prepared with 23 .52 NaCl, the effective concentration was probably lower due to diffusion of salt into the milk indicator layer.
8
Vol . 15, No . 7
143 1
Proteolytic Bacteria
The salinity and nutrient concentrations involved in these experiments were not exact .
Interpretation of experiments with double-layer plates was hampered
by the possibility of diffusion of salts and nutrients between the two layers . Phrthermore, enriched media have been shown to contain some ions (11) and organic components may interact with some canons (12) .
The experiments with low
nutrient double-layer plates were similarly affected by the possibility of diffusion of nutrients between the two layers .
Nevertheless, the results suggest
that the data on proteolysis obtained on enriched media in the laboratory were valid for the bacteria that normally live in a relatively nutrient-poor environment . The availability of oxygen appears to be an important factor controlling The activity of both the stock cultures and
proteolysis by sediment bacteria .
the facultative organisms used in this study was markedly reduced in the ab sence of oxygen .
However, the strict anaerobes were not examined .
ence of oxygen was also noted by Sizemore et al .
The influ-
(6) when they demonstrated
that the levels of "cell-free" proteolysic enzymes were lowest in sediment obrained from anaerobic zones .
Similarly, Sugahara, Sugiyama, and Rawai (13)
reported a greater than 100-fold decrease in the number of gelatin liquefiers in the sand of an experimental sea water aquarium when the system was changed from an 89X to a 6X oxygen saturation . altered very little by this change .
The total number of heterotrophs was
These observations suggest that proteo-
lyric activity may be limited under natural anaerobic conditions in sediments . Limited evidence indicates that the activity in the gut of some marine invertebrates may present a somewhat different picture .
Anaeromonas proteolytica ,
which was isolated from the gut of a wood-boring crustacean, Li.mnoria tripunc tats , by Merkel et al . (14), demonstrated very strong proteolysis when cultured in anaerobic jars .
1432
Proteolytic Hacteria
Vol . 15, No . 8
ACKNOWLEDGEMENT This research was supported in part by grant number NG-33-72 from the United States Department of Commerce aad by the Belle W. Baruch Coastal Research Institute . REFERENCES 1.
A. E . KRISS, Symposi~.en on M~ine MiorobioZogy, (Ed . : C . H. Oppenheimer), pp . 493-499 C . C. Thomas, Springfield (1963) .
2.
R. E. JOHANNES, Advances in Microbiology of the Sea, (Ed . : M. R . Droop and E . J . F . Wood), pp . 203-213 Academic Press Inc ., New York (1968) .
3.
T . M. SKERMAN, Symposiwn on Marine MicrobioZogy, pp . 685-698 C . C. Thomas, Springfield (1963) .
4.
E . E. ZOBELL, Marine MicrobioZogy, Chronica Botanica Co ., Whatam (1946) .
5.
F . J. VERNBERG, Belle W. Baruch Librcmy in Marine Science. Ida. 1 Estuarine Microbial Ecology, (Ed. : L . H. Stevenson and R. R. Colwell), pp . 3-8 University of South Carolina Press, Columbia (1973) .
6.
R. K. SIZEMDRE, L. H. STEVENSON, and B. H. HEHELER, Belle W. Baruch Library in MEu~tine Science. No . 1 Estuarine MicrobiaZ Ecology, (Ed. : L . H. Stevenson and R. R. Colwell), pp . 133-143 University of South Carolina, Columbia (1973) .
7.
R. K. SIZEt~RE and L. H. STEVENSON, App l. Microbial. 20 991-992 (1970) .
8.
W. C . FRAZIER, J. Infect Dtis . 38 302 (1926) .
9.
K. 0. EMERY and R. E. STEVENSON, Treatise on Marine Ecology and Paleoecology, Vo Z. 1 ., (Ed . : J . M. Hegpath), pp . 673-693 Ecology Geol . Soc. Amer . Mem 67 (1957) .
(Ed . : C. H . Oppenheimer),
10 .
J. M. HEDGPATH, Treatise tin Marine Ecology and Paleoecology, Vol. 1 ., (Ed. : J . M. Hedgpath), pp . 693-729 Ecology Geol . Soc . Amer . Mem 67 (1957) .
11 .
R. A. MACLEOD, BacterioZ. Rev. 28 9-23 (1965) .
12 .
E. R. GONYE and G. E . JONES, BeZZe W. Baruch Library tin Martine Science No . 1. Eatr~ine Microbial Ecology, (Ed. : L . H. Stevenson and R. R . Colwell), pp . 243-257 University of South Carolina, Columbia (1973) .
13 .
I . SUGAHARA, M. SUGIYAMA, and A . KAWAI, United States - Japan Seminar on Effects of the Ocean Environment on MiicrobiaZ Activities, (Ed . : R. Y . Morita aad R. R. Colwell), pp . 327-340 University Park Press, Baltimore (1974) .
14 .
J. R. MERREL, E. D. TRAGANZA, B . B. MUKHF.RJEE, T. B. GRIFFIN, and J. M. PRESCOTT, J. BacteriaZ. B4 1227-1233 (1964) .