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GER BA M Deuxime Colloque International de Bactriologie marine CN RS , Brest, 1-5 octobrIFREMER, Actes de Colloques, 3, 1986, pp. 73-79

7ACTINOMYCETES OF THE BOTTOM SEDIMENTS OF VARIOUS SEAS

H. WEYLANDInstitut fur Meeresforschung Bremerhaven, Am Handelshafen 12,D-2850 BREMERHAVEN (FRG)

ABSTRACT- Occurrence, distribution and physiological characteristics of actinomycetes in the top layer sediments of various seas were stud ied. V iable counts were very low in open seasites.T he majority of the isolatescould be grouped to streptomycetes, Micromonosporae and nocardioforms. Streptomycetes are prerantly distributed in coastal and shelf regions. They exhibit relatively broad temperature and salinity and allow baroresistance. Micromonosporae are found in relation to the other taxa more frequently in dsediments. They grow better in media with low salt concentration and have a temperature optimum 30C.On the other hand they are very baroresistantallowing these organ isms to survive in the deep sea for longperiods. Nocard ioforms were found to form the major part of the sediment actinom ycetes in open sea arand monovalent cation dependence as well as a lower temperature optimum mark these organisms as nous bacteria. Since their maximal growth pressure is relatively low their distribution is limited up to abom. By numerical phenetic assay they could be distinguished from known nocardioforms and could be ato a new species of the genusRhodococcus : R.marinonascens.Key words : actinomycetes, bottom sediment, Atlantic Ocean,Rhodococcus marinonascens.

RSUM- La frquence, la distribution et les caractristiques physiologiques des actinomycetes de la cosuprieure des sdiments ont t tudies dans diffrents sites. En mer ouverte, le nombre d'actinomviables est trs faible. La majorit des isolais ont putre regroups dans les streptomycetes, Microm onosporaeet nocard ioform es. Les streptomy cetes sont prp ond ran ts dans les rgions ctires et sur le plateau conUs supportent une gamme de temprature et de salinit relativement large et sont barotolrants. Les Mnosporae sont trouvs plus frquemment dans les sdiments des eaux abyssales, en relation avec lestaxons. Ils se dveloppent mieux sur milieux faible concentration en sels et leur temprature optimd'environ 30C. Ils sont d'au tre p art trs baro tol ran ts, ce qui leur permet de survivre en milieu abyssal de longues priodes. Les nocardioformes constituent la majeure partie des actinomycetes prsents d

sdiments des ocans. Leur dpendance vis vis des sels et des cations monov alents, aussi bien que leutemprature optimale les indiquent comme organismes indignes. Puisque leur pression maximale de dpement est relativement faible, leur distribution est limite environ 2000 m de profondeur. Par anumrique des caractres phnotypiques, ils ont pu tre distingus des nocardioformes connus et classune nouvelle espce du genreRhodococcus : R. marinonascens.

Mois cls : actinomycetes, sdiments, ocan Atlantique,/?/io

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surveys give us some knowledge about the occurrence in oceanic sites (Zobell Kriss A.E. 1967, Weyland H. 1969, Walker J.D. and Collwell R.R. 1975, OkamO ka za kiT . 1978, Weyland H . 1981 a).In the course of our investigations on the bacterial populations of the sea seactinomycetes could be found repeatedly. These observations gave rise to mor

studies on occurrence, distribution, heterogeneity and activity status of actinomthe marine environment.MATERIAL AND METH ODS

Sediment samples were collected during various cruises of the F.R.V."AntonR. V ."Meteor" and R .V ."Polars tern" using van V een- and S hipek-grabs or Reicorers. From immediately prepared dilutions of the top sediment layers pour spread p late cultures were performed using a variety ofmedia.In mangrove areas usuallysmall boats and small van Veen-grabs were employed for sampling. In polar rein the Atlantic preliminaries and cultivations were performed at 1C as well asDetails of cultivation, counting and identification as well as of physiologicatemperature characteristics, cation dependence, salinity requirement, barotolerbaroresistance ared iscrib ed by H. Weyland (1981 a and 1981 b), by E. Helm ke1980 and1981) and by E. Helmke and H.Weyland (1984).

60*

30"

30"

6 0 '

FigureI : Investigated areas.

R E S U LT S A N DDISCUSSION

V arious sea regions were investigated (Fig .1).Results are given from areas of the Norwegian Sea north of the polar circle, off the Faeroe Islands, of the North Sea, of t

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Atlantic including an area of the M iddle A tlantic R idge, of the Biscay, of the Iberiaof the upwelling zone off NW -Africa, of the Lagun a de T erm inos , Gulf of Mexico,the Antarctic Sea.In table 1, some of these areas are quoted as illustrations of the am ou nt of actinomin relation to the total viable bacterial counts. Apart from the Antarctic Sea actincetes could be proved in the sedim ents of the majority of the stations of all areas. Buopen sea viable counts of the actinomycetes are usually low ranging a bout 100 cfu wet sediment. T hey are indicated per thousand of the total viable cou nts. T he m anexhibited the highest density of actinomycetes amongs t the areas investigated also their high salinity regions. In the sediments off NW-Africa the absolute numbeactinomycetes per ml sediment are comparatively low in spite of the fact that this asituated in a dust zone com ing from the Sah ara . U p till now actinomycetes could detected in the sediments of the Antarctic Sea. This may be due on the one hand absence of soil covered land and on the other hand to the Antarctic Ring Ocean whseperated from the neighboured oceans.

depthm

0-200

200-10001000-2000> 2 0 0 0

0-200

200-10001000-2000> 2 0 0 0

0-200

200-10001000-2000> 2 0 0 0

0-200

200-10001000-2000

> 20008

0-10

sampling sitestotal

16411416

27412

38

555

1087

8

10

Act pos.

cfu/ml sediment(mean values)

Bacteria

Norwegian Sea

14401416

918000394 0001060004 600

Antartic Sea

0

0

0

0

18 743 000347000279000162 000

North Sea

34

44

1637 000

665 000665 000

4 665 000

Off NW-Africa

6

8

7

8

246000203 000

13 800

4 800

Mangrove, Mexico

10 10

Act.

16929816463

0000

480

790790790

468585

46

16 651000

Act.V %

0.180.81.54

13.69

0.00.00.00.0

0.29

11.8811.8811.88

0.190.416.16

9.58

397 000

T able I : V iable counts of bacteria and actinom ycetes in the botto m sediments of different depth ra

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Actinomycetes in general are a large bacterial group with several genera andspecies. T he bulk of our isolates from the sea sedim ents could be classified ontaxonom ic groups : streptomycetes, M icrom onos porae and nocardioforms. Isbelonging to these taxa only constituted a minority (Weyland H.1981 b).

Area

Norwegian SeaNorth Atlantic

Off FaeroesBiscay

Iberian SeaOffNW-Africa

North SeaMangrove, Mexico

Antarctic seaAll regions

All regionexcept North Sea,

Antartic Seaand Mangrove

numberof

samplingsites

83214547

27221061025386

245

Streptomycetespersite

0.90.21.60.5

1.80.64.49.20.02.1

1.0

11 %

Micromonosporae

persite

3.4

1.01.97.2

3.63.14.63.60.03.6

3.6

3 9 %

Nocardioformspersite

5.511.47.12.1

0.60.60.70.40.04.7

4.7

5 0 %

allstrains

persite

9.812.610.69.8

6.04.39.7

13.20.09.3

9.3

100%

Table 2 : Isolated strains of the major actinomycete groups. Mean values per site.

The isolation of the strains were performed in such a way that the number of dted isolates largely reflects the proportions of the different taxa present on theculture pla tes. In table 2 the averaged values of the num ber of isolated s trains oactinomycete taxa are given per site examined. Summing up the data of open investigated the nocardioforms were the most frequently isolated group (50 %)by the M icrom ono sporae (39 % ). Streptomycetes have been found relativel(11%).For example in the north A tlantic5 sediment samples had to be examined in ordto isolate one streptomycete strain whereas in one sediment sample 11 nocastrains were yielded. With regard to the frequency of the streptomycetes, the but also the North Sea are standing out.

Differences of the distribution of the three recognized taxa between the variobecome m ore obvious by com paring the relative prop ortion s (F ig. 2). T he northlittle effected by terrestrial run off and dust, exhibit a high proportion of nocarIn other open sea areas remote from land the Micromonosporae predominate wthe North Sea and especially in the mangrove the streptomycetes are frequent

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i1.0

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100Z 80" 60? M^ 20

o loo

? K M* toif 20

r J J ml J i C i y I b t m n o f t

S t i N W - A F r i c j

u M i jN o r th S e * M i n g r o i t A n d r c t i cIL i

| J t l r t g i o n i\ f i l * * t P ' Honh Sl

" 1 i I *nd M' nr o ife S f n p t o i B y c t l H D M i c r o f l i o n o p o fi N o c j r d i o f o r m i

50 -40 -

70 -

50 -tO -20 -

Hotth Attantk

5

= 60S 40 -

: , n l ;

L

11

60 -4020 -

0- 20 0m 201- lOOm

j J l

K1-2tXOm I 200 0i r

llil

f t N W - A f r i c a

I . a l l l : - II.O-2 00m l 2OMO00m\ WOl-2O0fr

0 S t i p p l m y c e l nD Micromonospora Nocardiofofmi i n o M m p ! s t t i o m n o n o l a l M

Figure2 : Distribution of the majorF l g u r e 3 . P rop or t,o na l distribution of the actinomycete groupsactmomycete groups of the variousd e m o n d e p t h B a s e d ( h g r a t j o s o f i s o | a t sregions (in percent or the total number of actinomycetes isolated fromone area)

depen-

The sediments examined were hieved of course from different depths (Faeroe 2280 m, Norw egian Sea up to 3900 m, Atlan tic up to 4062 m, Biscay up to 4700 m, Sea up to 5510 m, off NW-Africa up to 3362 m, North Sea up to 670 m, Antarctic up to 4419 m). When con sidering the distrib ution of the isolates of the three actinomgroups in relation to various depth ranges - for each depth range the isolates of the are given in per cent - the following distribution pattern was found (arbitrary ranges) (Fig. 3): High portions of Microm ono sporae originated from deep sea sedimeAn increase with depth of the relative amounts of the Micromonosporae can be sHigh or fairly high amounts of nocardioforms could be encountered in sediments to about 2000 m water depth. In the deepest range the relative amount of nocardiowere found to be usually lower. T he strep tom yce tes were represented by the sedimshallow a reas. Even in the N orth Sea, for exam ple, a dom inan t position ofthe streptomycetes in sediment of the shallow range, was taken over by the Micromonosporae next deeper range (deep trench of the Skagerrak).

About the question, whether these distribution patterns are reflected by physiolocharacteristics, some studies were directed towards features that may indicate wgroup of these organisms is potentially able to metabolize and reproduce or cansurvive in the marine environment.The growth response to temperature of representatives of the three taxa are illusin figure.4, Streptomycetes and M icrom ono sporae showed temperature optima ator above 30C. On the other hand the marine noca rdioforms - in the meantime taxocally affiliated to the genusRhodococcus (H elmke E. and Weyland H.1984) - do not growat these tempe ratures. T heir optim um is about 20C. Considering the salinity requi(Fig. 5) these rhodoco cci behaved like typical m arine bac teria. T hey do not grow wsalt. Media with concentrations of75 % to 100 %seawater are optimal. Micromonosporae isolates are developing better in low sea salt concentrations. There is onlyresponse to increased salt concentrations by streptomycetes. But the completion olife cycle is favoured by the additions of some salt (Weyland H. 198) b).

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There was no significant effect of monovalent cations on the development of nosporae as well as of streptomycetes. However the growth of the marine rhdepends on the addition of monovalent cations. This effect could not be obseterrestrialRhodococcus- and Nocardia spp. (Helmke E. 1980).

As a further criterion the barotolerance of the organisms were determined (H198 ().T he best barotoleranc e with respect to growth rate and yield were exhibitethe nocardioforrn strains. Grow th rate and grow th yields of marine as well as Strains decreased only a little up to the maximal growth pressure. The maximpressure of the marine rhodococci were often relatively low. The Micromonosstreptomycetes examined were clearly more effected by pressures below theirgrowth pressure.T he pressure studies were supplemented with studies on the baroresistanc e inestim ate the survival ability of the different actinomycete taxa above the maximpressure (pub lication in prepa ration ). T he survival ability between these groupextrem ely. T he nocardioforrn strains were killed rap idly by pressures exceemaxim al grow th p ressure. Few of the streptomycetes tested survived high presspores as the survival stages. T hese strains belonged to the yellow colour serieswell as mycelium of the marine Micromonosporae survived long lasting treathigh hydrostatic pressure at low temperatures. This baroresistance shouldMicromonosporae to survive deep sea conditions and it corresponds with thehigh isolation counts from deep sea sediments.Summing up : the results of the physiological tests in connection with the didata givesom e indications on the potential abilities and possible role of actinomthe sea.T he streptom ycetes are most frequent in the coastal and shallow areas. T husually have broad salinity and temperature spectra. Reproduction may be asthe shallow sites. In high salinity mangrove areas we found streptomycetedominant actinomycete group.The Micromonosporae were most frequent encountered in deep sea sedimentone hand, concerning temperature and salinity, they are not well adapted toconditions on the other hand, regarding baroresistance, they are furnished wabilities to survive under deep sea condition s. T heir occurrence in the deep sea sa dormant status can be presumed.

Figure 4 : Examples of growth response of Figure 5 : Exam ples of growth responsethe majority of streptomy cete, Microm onos- the majority of streptomycete, Microm onporae and Rhod ococcus marinonascens iso- porae and Rhodococcus marinonascens lates to tem peratu re. lates to salinity.

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The non-sporeforming nocardioformsare found preponderantlyin the opensea up toabout 2000m. Their survival abilityat greater depth appearedto be relatively low.Buttheyare best adaptedfor reproductionin marine environmentsup to moderate depths.By a chemotaxonomicand numerical taxonomic assaythe majorityof the marinenocardioform isolates couldbe assignedto the genusRhodococcus and clearly differentiated from other known speciesof that genus. These organisms couldbe given speciesstatusand referringto the exclusive occurrenceof this taxonin the sea they were namedRhodococcus marinonascens (HelmkeE. and WeylandH. 1984). By this organismanindigenous marine actinomycete couldbe made evidentfor the first time.Its niche is stillunknown.

ACKNOWLEDGEMENTS

Sincere thanksare due to all the individualsand institutes who kindly supported me duringthe surveyof thedistributionof actinomycetesin the sedimentof varioussea areas. My gratitude also extendsto the scientificstaffs,officers and crewsof research vesselsfor all kindsof assistanceat sea. Special thanks is due to the staffof

the Bacteriology Sectionof the IfM Bremerhaven. This work was supportedby the Deutsche Forschungsge-meinschaft (DFG)and by the Bundesministerfur Forschungund T echnologie (BMFT ).

CROSS T .,1981.Aquatic actinomycetes: a critical surveyof the occurrence, growth and roleof actinomy cetes inaquatic habitats.J.Appl. Bacteriology 50 : 397- 423.HELMKEE., 1980.Dr. thesis, U niversitat Braunschweig, Federal Repub licof Germany,1980.HELMKEE., 1981. Growthof actinomycetes from marineand terrestrial origin under increased hydrostaticpressure.Zen tralbl. Bacteriol. Parasitenkd. Infektionskr. Hyg. Abt I. Suppl.II : 321-327HELMKEE. andWEYLAND,H., 1984.Rhodococcus marinonascens sp.nov., an actinomycete from the sea.Int. J.Syst. Bacteriol. 34 : 127- 138.

KRISSA.E., MISHUSTINAI.E., MITKEVICHN. ZEMSTOVAE.V., 1967. Microbial populations of oceans andseas.Edw. Arnold,Ltd. London.OKAMIY. and OKASAKIT., 1978. Actinomycetesin marine environments.In : Mordarski, M.and Jeljaszewicz,J., (eds.).Nocardia and Slreplomyces, Gustav Fischer, Stuttgart,pp 145- 151.WALKERJ .D.and COLLWELLR.R., 1975. Factors effecting enumerationand isolationof actinomycetes fromChesapeake Bayand southeastern Atlantic ocean sediments.Marine BiologyiO : 193- 201.WEYLANDH., 1969. Actinom ycetesin NorthSea and Atlantic ocean sediments.Nature 223, No. 5208: 858.

WEYLANDH., 1981 a. Distributionof actinomyceteson the sea floor.Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt.I Suppl. 11: 185- 193.WEYLANDH. 1981b. Characteristicsof actinomycetes isolated from marine sediments.Zentralbl. Bakteriol.Parasitenkd. Infektionskr. Hyg. Abt I suppl II. .309- 314.ZOBELLCE., 1946. Marine Microbiology.Chronica Botanica Co.Waltham Mass.

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