PLANKTON STUDIES IN A MANGROVE ENVIRONMENT

lU. BACTERIOLOGICAL ANALYSI~ OF WATERS IN CANANÉIA

(Received 13/ 8/ 1964)

Ko Watanabe & Miryam B. Kutner

Instituto Ocec nográfico da Universidade de São Paulo

SYNOPSIS

Watcrs Df the mangrove swamp at Cananéia were analysed bacterio-logically aiming the determination of the degree of bacterial contamination.

Aerobic counts varied significantly by season, tide and station with the highest of 13,000 and the lowest of 211ml while coliform counts showed significant variation only by station with the highest of 1,600 and the lowest of 3 per mI.

Pseudomonas-Achromobacter group comprised the highest percentage of the total flor a at the leveI of around 60% . The biochemical characteristic of the strains of this group are more saccharolytic than proteolytic, certa in-ly due to the great number of diatoms present.

INTRODUCTION

As a link which joins the inorganic and organic phases of the chemical cycle in the sea, bacteria play an important role in the nutritional relationship of marine organisms, hence in the productivity of the water (Waksman 1934; ZoBell 1959a, b; Sver-drup et alo 1960). However, little is known on the matter at present, especially about mangrove swamp waters. On the other hand, since the inshore waters can be utilized for mollusc cultures, sewage pollution arises as an important problem to be studied from the view-point of ensuring quality and hygiene of the pro-ducts (Fieger & Novak, 1961).

Previous papers (Teixeira & Kutner 1963; Teixeira, Tundisi & Kutner 1965) discussed in general some aspects of the bacterio-plankton in Cananéia. The present paper deals, more in detail with the quantitative as well as qualitative analysis of the man-grove swamp waters.

Publ. 209 dQ Inst. Ocea no da USP.

- 44 ~

MATERIAL AND METHODS

Water samples were. collected at Cananéia at three stations (Aroeira, Argolão and Ariri; for the exact position see article II of this series), four times, namely in January, April, July and October, 1962. Sampling of sub-surface layer was carried on with a 8-litre stainless steel plankton sampler. No sterilization was made but care was taken to handle it as clean as possible to avoid secondary contamination. Physical and chemical data of the sam-pIes at the collecting points are shown in Table I (see also article II of this series). Platings were ma de on the same day of sam-pling.

Total aerobic counts on meat-peptone-agar with 0.8 % salt (Difco) and coliform counts on violet-red-bile-agar (Difco) were performed. Incubation time was 4 days fôr the forme r and 2 days for the latter, at room temperature of about 20-30°C through the year. In both cases, electric power was not available.

Further floral analysis of microorganisms was ma de on the samples by isolating strains from the counting plates of total aero-bic counts and after a purification process, by observing the colony characteristics, and by mean of morphological studies, reactions to triple sugar iron agar (Difco), arrangement of flagella and resistance to 2.5 lU penicillin. Finally the microorganisms were identified as: Enterobacteriaceae, Aeromonas, Pseudomonas/ Vibrio, Achromobacter/ Alkaligenes, Flavobacterium, Micrococcus, B(willus, Corynebacterium and Actinomycetes. Some strains of Pseudomo-nas and Achromobacter group were submitted to further bio-chemical tests (Watanabe, 1960).

The results were treated statistically by the F test, regarding the whole design as 2 X 4 arrangement (tides X seasons) of 3 X 2 two way layout (stations X depths).

RESULTS

The bacterial counts are given (Table II) as well as the re-sults of F test applied on the data (Table lU).

Total aerobic counts varied significantly due to tide and sea-son at 1% leveI and by station at 5 % leveI. Thus it can be said that: 1) the bacteria were more abundant at low than at high tide though this varies with the position; 2) seasonally they were at their highest in January-April and lowest in July-October, and 3) that smaller but still significant differences were found among the stations 'with lowest (Ariri) or highest counts (Aroei-ra) when we calculated the means by excluding the extremely high counts of ten thousands at low t'ide on April 3, sampled dur-ing heavy rain.

TA

BL

E I

-

Ph

ysi

cal

and

ch

emic

al

dat

a of

th

e st

atio

ns

at

sam

pli

ng

d

ates

ST

AT

ION

I

TID

E

IDE~T

H I

S °/

00

T

oc

pH

1 12

0/1

/62

1 5

/4/6

2 1

19

/7/6

214

/10

/62

120

/1/6

2

1 5

/4/6

21

19

/7/6

2 1

4/1

0/6

21

20

/1/6

21

5/4

/62

119

/7/6

2/4

/10

/62

low

0.

0 3.

42

1.5

8 12

.15

8.26

25

.12

26.3

0 18

.98

22.1

2 -

--

7.55

3.

0 4.

20

1.1

6

12.0

2 7

.94

25

.10

26.1

3

19.3

0 22

.10

-

--

-A

roei

ra

hig

h

0.0

3.60

1

.84

8.

13

5.6

8 2

5.8

0

26.7

0 19

.09

22.4

0 -uo

± 3

.0

3.57

7

.07

14.0

0 5

.91

25.4

0 26

.65

19.0

8 22

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-7.

45

--

21/1

/62

2/4

/62

1

6/7

/62

11/1

0/6

2 /2

1/1

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1 2

/4/6

2 1

16

/7/6

2 /1

/10

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/21

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2 /

2/4

/62

1

-11

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-

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I lo

w

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95

10

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3 27

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27

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19

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5 -

8.20

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0.

0 21

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26.6

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24.7

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7.80

-

8.1

0

10.0

26

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31.1

1 28

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25.4

7 25

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26

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18.9

2 2

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24.5

1 27

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29.1

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8 26

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19.3

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98

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28

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3 30

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26.6

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8.15

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-.

T ABLE II - Total aerobic and coliform counts by tide, season, station and counts of inshore waters per ml of w~ter

-

High tide Low tide

Date Station Sampling

depth (m) Aerobic Coli Aeroblc I Coli

I mean mean mean mean I

I \ \ \ I 20/1 Aroeira O 3,600 96 7,400 160

3 12,000 1,600 9,200 240

21/1 Argolão O

\

110

I 11 590 16

10 420 12 13,000 260

19/ 1 Ariri O

\

100

I 15 320 26

10 150 8 600 8

5/4 Aroeira O

I 1,200

\

40 1,600 400 3 250 20 1,100 80

2/4 Argolão O

\

1,200

I 11 850 10

10 1,600 36 1,300 15

3 /4 Ariri O

\

1,100

1

81 74,000'1 18 10 570 . 6 34,000' 80

19/ 7 Aroeira O

1

860

1

13 720

1

13 3 960 21 710 52

16/7 Argolão O

1

21

I 12 77

1

8 10 36 25 1,003 42

1 1

. 1

18/7 Ariri. O 130 12 170 10 10 230 73 320 15

4 / 10 Aroeira - O

1

240

I 52 540

I 120

3 330 69 350 230

1 / 10 Argolão O

I 150 1 32 320 I 55 10 200 24 640 59

2/10 Arir! O

1

96

1

3 400

1

23 10 150 8 380 7 . Unusually high due to heavy rain at the time o.f sampllng.

T ABLE lI! - Analys1s of variance of the results shown in Table lI. (The treatment was made by transferring into logarithmic numbers)

Varlance Total ae,co bic

due to DF

ss

Tlde

\

1 2.75 Season 3 8.38 Statlon 2 2.58 Depth 1

I 0.31

Error 40 11.56 Total 47 25.58

• Slgnlficant at 1 % level. •• Slgnlfican t a t 5 % level.

I MS

I

I 2.75 2.79

I 1 .29 I 0.31

I I 0.29 -

Collform

F SS I MS I 9.49'

I 0.36 0.36

9.62' 1 .12 0.37 4 .45" 5 .42 2.71 1.04 0.19 0.19 - 8.79 I 0.22 - 15.88 -

F

1.63 1.68

11.23 0.86 --

r.l

Q .... E-< :r: t.!l .... :r: r.l Q .... E-< ~ O ...1

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22.2

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33

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11.1

44

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66.6

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85.6

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14.2

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16

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""" -:!

- 48-

Cülifürm cüunts shüwed significant variatiün ünly by statiün at 1 % leveI, highest at Arüeira, füllüwed by Argülãü and Ariri.

Table IV gives the generic distributiün üf the bacterial flüra o.f surface water. Pseudomonas and Achromobacter were the two. üf the highest in their percentages alI thrüugh . the year cümpris-ing abüut 40 % and 20 % respectively üf the tütal flüra, füllüwed by Corynebacterium and Micrococcus with equal cüntributiün üf abüut 5% . No. difference is nüticed between lüw and high tide.

Cümparing gram negative with gram püsitive prüpürtiün, mean percentage thrüugh the year shüwed 70 % für the fürmer and 30 % für the latter, a little higher für the fürmer at lüw tide. In July grÇlm püsitives were higher in the tütal flüra in büth tides.

MürphülügicalIy the düminant Pseudomonas-Achromobacter grüup shüwed rüd shape üf varied type and 'size, with münü pülar to. peritrichüus flagelli, generally with greyish, nürmal size, wet, entire translucent to. üpaque cülünies. .

In ünce case a strain showing the characteristics üf Chlamy-dobacteriales was isülated. Süme strains üf Micrücücci were lar-ger in size with thickened wall, granules and variable gram, as übserved 'by Kriss (1963).

As much as 21.4 % üf the tütal flüra showed yellüw to. bro.wn-ish cülo.red co.lo.nies. Only three strains üf green pigmented Pseu-domonas were iso.lated. Sverdrup (1960) states that abo.ut 70 % o.f marine bacteria are cülo.red as o.ppüsed to. 15 % o.f the terrestrial fo.rms. Thus the presentsamples are clo.sely related to. terrestrial fo.rms and certainly ,cüme Úüm the run-üff o.f lWater früm land to. the mangro.ve. This explains the great number o.f bacteria pre-sent after heavy rains.

T ABLE V - Some biochemical characteristics of the Pseudomonas-Achró-mobacter group isolated from the surface layer in October 1962, given as

percentage of total number of strain~ tested

'" I I I 'O 'O I 'O I OI I s:: 'O OI OI OI '" ... ... .~

- 49-

this group of water flora in the mangrove swamp is more sac-charolytic, probably due more to the great number of diatoms pre-sent, than to proteolytic strains. Most of them are nitrate reducers, very weak in Dulcitol fermentation, in the utilization in citrate as the only carbon source and in the production of acetyl-methyl carbinol (V.P. test).

DISCUSSION

As Miniussi (1959) pointed out, Aroeira has the most in-tricate water movement, receiving flows from three directions. This mechanical turbulence of tidal movement would be responsible for the highest aerobic count at this station, bringing up the abundant microorganisms and organic particles from bottom mud into upper water layers, supplying favorable conditions for bac-terial growth (Teixeira, Tundisi & Kutner 1965). On the other hand, the suppressing effects of sea-water on bacteria, as dilution, sedimentation, agglutination, osmotic pressure and biologically active organic matters (Sieburth -1962; J ones 1959) would decrease the contamination at Ariri and Argolão, that suffer a more direct influence of inflow from the sea.

The use of ~ not sterilized sampler in the present experiments is highly questionable, leading us to the confusing conclusion that there is novariation of bacterial counts in different depths, which disagrees with data obtained by other authors (Sverdrup, 1960).

Coliform counts had significant variation only by station. This is reasonable sincê the place of sewage discharge is the sole factor responsible for the contamination. However, the results obtained showed that the count at Aroeira is higher than at Ar-golão, which is closer to the town of Cananéia, the only large source of the coliforms in this region. This record does not agree with Rittenberg's (1956), which showed regularly higher .count closer to the sewage outfall. Nevertheless, this again might be explained by the same reasons indicated for the aerobics 01' may be due to local water currents.

Although the reliability of the number of coliform is dubious due to the lower temperature of incubation caused by the -difficulty in power supply, the counts are equal to Rittenberg's (1956) and the specific investigation of the isolates from the violet-red-bile agar proved most of them to be citrobacter spp. with some Esche-richia éoli. In few cases Aeromonas and Pseudomonas-like bac-teria were identified. In this connection the determinations of Escherichia coli and Enterococci, parallel to coliform would be-come important to give a clearer picture of the pollution situation.

Based on the information obtained, the most suitable area in this region for oyster culture from the hygienic point of view, would be found somewhere in the "Mar de Cubatão", nearer to

50 -

Ariri. The salinity condition for th~ mollusc growth would also be optimum there (Lima & Vazzoler, 1962).

The large majority of marine bacteria are gram negative. The phenomenon of low percentage of gram negative in July, the season of low bacterial contamination draw our interest to find out the cause. Scarcity of nutrients due to low bloom of plankton or low water temperature alone ca:Qnot fully explain the selectivity of species in the bacterial flora. In this respect it would be in-teresting to investigate the antibiotic action of sea-water specific to gram positives (Saz et al., 1963) in the plankton blooming season of J anuary-April.

On the whole the present data, although treated statistically, are insufficient to lead to definite conclusions and must be re-garded as a preliminary survey. 'The bacterial contamination in brac,kish water would be highly influenced by every day conditions of tide, weather, discharge of sewage, in addition to the factors analysed here. Therefore, more frequent samplings :within shorler intervals are necessary to obtain a more precise and detailed. picture of bacterial population of this mangrove region.

The authors aI'e grateful to Dr. Marta Vannucci and Mr. C. Teixeira for their advices and encouragement; to Misses Flavia Torgo and Eunice Maria dos Santos for their technical assistance and to the "Fundação de Amparo à Pesquisa do Estado de São Paulo" that supported partially this work.

RESUMO

As águas da reglao do mangue de Cananéia foram analisadas bacterio-làgicamente, com o objetivo de estabelecer seu grau de contaminação.

As contagens aeróbicas variaram significativamente nas várias épocas, locais e marés, com um máximo de 13.000 bactérias/ mI e um mínimo de 21/mI, ao passo que as contagens de coliformes mostraram variação signi-ficativa sàmente segundo os diversos locais com um máximo de 1.600 e um mínimo de 3/mI.

O grupo Pseudomo'lms/ Achromobacter abrangeu a mais alta percenta-gem da flora total com cêrca de 60 % . As características bioquímicas das linhagens dêste grupo são mais sacarolíticas do que proteolíticas, certamente devido ao grande número de diatomáceas presentes.

REFERENCES

FIEGER, E. A. & NOVAK, A. F . 1961. Microbiology of shellfish deteriora tion. In Borgstrom, G. (ed.),

Fish as food. New York, Academic Press, voI. 1, p. 585-598.

JONES, G. E . 1959. Biologically active substances in sea water. Paper prepared

for Int. oceanogr. Congr. 1.°,. New Yor.k, 27 p .

KRISS, A. E. 1963. Marine microbiology. London, Oliver and Boyd Ltd., p. 3,51.

LIMA, F. R . 1963.

MINIUSSI, r. 1959 .

51 -

& VAZZOLER, A. E. A. DE M. Sôbre O desenvolvimento das ostras e possibilidades da ostrei-cultura nos arredores de Santos. Bolm Inst. oceanogr. S Paulo, voI. 13, n .o 2, p. 3-20.

C. Propagação da onda de maré em tôrno da Ilha de Cananéia. Contrções . Inst. oceanogr. Univ. S Paulo, ser. Oceano física, n.o 2, 5 p .

RITTEN BERG, S. C. 1956. Studies on coliform bacteria discharged from the Hyperion out-

falI. Final report submitted to the Hyperion Engineers Inc. by the Univ. of Southern California, p. 1-51.

SAZ, A. K 1963.

et alo Antimicrobial activity of marine waters. r. Macromolecular nature of antistaphylococcal factor. Limnol. Oceanogr., Kansas, voI. 8, n.o 1, p . 63-67.

SIEBURTH, J . M. 1962. Biochemical warfare among the microbes of the sea. Honors

Lecture, Univ. of Rhode Island, 13 p.

SVERDRUP, H. U. et alo 1960. The oceans. Englewood Cliffs N. J. , Prentice Hall Inc., p. 908- .

920.

TEIXEIRA, C. 1963 .

TEIXEIRA, c., 1965.

& KUTNER, M. B. Plankton studies in a mangrove environment. ment of standing stock and ecological factors. nogr. S . Paulo, voI. 12, n .o 3, p. 101~124.

r. First assess-Bólm Inst. ocea-

T UNDISI, J. & K UTN ER, M . B . Plankton studies in a mangrove stock and some ecological factors. lo, voI. 14, n.O 1, p. 13-41.

environment. lI. Standing-Bolm Inst. oceanogr. S . Pau-

WAKSMAN, S . A. 1934. The role of bacteria in the cycle of life in the sea. Scient.

Mon., N ew York, vol. 38, p . 35-49.

W ATANABE, K. 1960. Bacterial flora of commercial fresh codfish. N ord. VetMed.,

Stockholm, voI. 12, p . 541-554.

ZOBELL, C. 1959b.

1959b .

E . Marine pollution problems in the southern California area. microbiology. N.Z. Dep. sci. industr. Res. Inf. Ser., n .o 22, p. 7-23.

Marine pollution problems in the southern California area. Transactions of the Second Seminar on Biological Problems in Water Pollution. Ohio, U.S. Public H ealth Service .