Assessment of bacterial production and mortality in Mediterranean coastal water

Estuarine,

Coastal

and Shelf

Science

(1988) 26,331-336

Assessment of Bacterial Production Mortality in Mediterranean Coastal

J. Vives-Rego”, Departamento Av. Diagonal Received

J. Martinez

de Microbiologia, 645,08028-Barcelona,

6Jul~v

and J. Garcia-Lara

Facultad de Biologia, Spain

1987 and in revisedfowl

and Water

5 October

Universidad

de Barcelona,

1987

seawater; bacteria; grazing

Keywords:

Bacterial production was estimated by thymidine incorporation using conversion factors estimated from the increase in bacterial abundance and thymidine incorporation in batch incubations. Grazing and bacterial mortality were estimated from the disappearance of the 3H-label of natural assemblages in filtered and unfiltered samples. The approach was also used to calculate bacterial production (about 0.2 x lo9 cells l- ’ h- ‘). Bacterial mortality was about 0.02 h- ’ and grazing represented 15-82”,, of the total mortality in a Mediterranean coastal water. Introduction The importance of the microbial food web in planktonic systems has only been recognized recently (Williams, 1981; Azam et al., 1983; Porter, 1984; Porter et al., 1985). Estimates of growth rates and change of bacterial biomass are essential to determine the contribution of bacteria to total heterotrophic activity in natural waters. Although there are several accepted methods of measuring bacterial biomass (Hobbie et al., 1977; King & White, 1977; Watson et al., 1977), important controversy over the methods ofmeasuring bacterial production still remains (Hagstrom et al., 1979; Fuhrman & Azam, 1980, 1982; Newell & Christian, 1981; Karl, 1981, 1982). Thymidine incorporation is the most frequently used method to estimate bacterial production (Pollard & Moriarty, 1984; Staley & Konopka, 1985). However, because total thymidine incorporation underestimates DNA synthesis, conversion factors must be used to calculate bacterial production from the rates of thymidine incorporation. As a result of the recent interest in marine microbial food webs, various techniques have been used to estimate grazing of bacteria by protozoa, although no one technique has been accepted as optimal (Servais et al., 1985; Wikner et al., 1986; Sanders & Porter, i986; Sherr et al., 1987). The rate of change of bacterial biomass in the water column is the result of the balance between bacterial production and mortality if dilution and sedimentation processes are insignificant. As the method proposed by Servais et al. (1985) can be used to approach total bacterial mortality and grazing, we have used it to estimate bacterial production; i.e. total bacterial production minus bacterial mortality. “Author

to whom

correspondence

should

be addressed.

331

0272-7714/88/030331

f06

$03.00/O

@ 1988 Academic

Press Limited

332

J. Viva-Rego et al.

We present in this paper estimates of bacterial production based on thymidine incorporation and growth-rate determinations. Bacterial mortality was assayed by following the disappearance of a previously 3H-labelled natural bacterial community (Servais et al., 1985). The data we report indicate that the methods we have used for estimating growth and mortality of marine bacteria are consistent and can be useful in the study of bacterioplankton dynamics. Material

and methods

Sampling

Seawater for all assayswas collected from Barceloneta, the natural beach of Barcelona, a Mediterranean city of high urban, industrial and agricultural activity. Three sampling points were used: water from the beach (CNB), water from 2 miles offshore (J-l), and water from a point situated at 90 miles offshore (J-2). Bacterial

counts

Enumeration of bacteria was carried out by epifluorescence, after acridine orange staining and filtration through 0.2~pm polycarbonate filters (Nuclepore), following the standard procedure of Hobbie et al. (1977). Bacterial

production

Bacterial production (in cells 1-i h-l) was estimated by the thymidine incorporation method of Fuhrman & Azam (1980, 1982) using 20-nM methyl-3H thymidine (80-90 Ci mmol- ‘, Amersham). Cold TCA-insoluble material was retained by filtration on 0.2~pm filters. Conversion factors were calculated by the following experiments: a filter-sterilized water sample was inoculated with water from the same habitat prefiltered by 2 pm to remove most grazers. Increasesin cell number were plotted against the total accumulated thymidine incorporation, which wasthe sumof the incorporations at intervals adjusted to keep the saturating concentration of thymidine (l-2 h) along the growth phase. From the samebatch, bacterial specific growth rates were estimated from the growth phaseusing the expression: p = In NZ2- In N,,/t2 - tl; where N is the bacterial number and t is the time elapsed between sampling. Bacterial production was also calculated by the expression P= pN; where P is production; p is the growth rate and N is the bacterial abundance. Bacterial

mortality

Bacterial mortality was assayedafter Servais et al. (1985) from previously 3H-thymidinelabelled natural assemblagesof bacteria and following the disappearanceof 3H-label in total cold TCA-insoluble material. The labelling was done by adding 3H-thymidine to a recently collected water samplewhich wasincubated at room temperature until the thymidine was totally exhausted. Mortality due to the bacteria grazed by protozoa was assessed comparing mortality in an unfiltered aliquot with respect to another filtered through 2 nm (Nuclepore). The flux of mortality was obtained by multiplying the specific rate of mortality by the bacterial abundance. Results Growth

rates a?ld bacterial

production

The conversion factor between thymidine incorporation and cell production from the three sampling points (Table 1) ranged from 1.38 to 2.42 ( x 10”) produced bacteria per

Production

axd mortalitzl

1. Dynamics

TABLE

333

in sea bacteria

of the bacterioplankton

from coastal

water Unaltered

Batch experiments

Habitat CNB

J-1 J-2

Regression line

rcz

E’= 3-6X158 Y=2.5X-O.O74 I’=O.874+0.504

Conversion coefficient x loYh

0.919 0.944 0.977

“r, Correlation index. “Cells produced per nmol lines.

TABLE

thymidine

242 2.42 1.38

(h’ ‘)

Bacterial abundance (bact. 10” l- ‘)

0.14 0.09 0.06

5.8 8.3 4.2

of thymidine

incorporated

2. Estimates of bacterial production incorporation and growth rates

Habitat CNB

J-1 J-2

Thymidine incorporation 0.1481 0.3049 0.0712

Growth

(in bacteria

samples Thymidine incorporation (nmol thy. 1 ’ h “1 0.0733 0.126 0.0516

calculated

from

the regression

10” l- ’ h- ‘) calculated

from

rate

0.8323 0.7611 0.2927

nmol of incorporated thymidine. The calculated growth rates in the studied area, declined with the distance from shore, but the variations in heterotrophic activity and bacterial numbers were independent of the distance from shore. The conversion factors varied from sample to sample, but the order of magnitude was about IO9 cells per nmol of thymidine incorporated into the cold trichloroacetic acid insoluble fraction. These conversion factors were used to estimate bacterial production in the coastal water from thymidine incorporation in unaltered water samples. Bacterial production was also estimated from u and Nas described in Materials and Methods. Table 2 shows the values of bacterial production obtained by both methods. Bacterial production estimated by these two methods differ within an order of magnitude from 2.4 to 5.6. The production estimates based on the thymidine incorporation were lower than estimates based on growth rate. Bacterial

mortality

Bacterial mortality (Table 3) ranged from 0.034 to 0.006 h- ’ and it appears that there is no significant variation with distance from shore. However, mortality caused by grazers retained by 2-urn filters, seems to increase significantly with distance to the shore. In the studied area, mortality due to the grazers which pass.%urn pores represent 12-90”,, of the total mortality. Net

bacterial

production

The net bacterial production calculated from total bacterial production estimated by thymidine incorporation and the flux of mortality (in cells per l- 1h- ‘) was 0.021 x lo9 for

334

J. L’iives-Rego et al.

TABLE 3. Bacterial

mortality Mortality

Habitat

Total

CNB

0.012AI.034 0~01&0~008 0~009-0~004

J-1 J-2

(h

’J

Grazing

Mortality due to grazing P,,)

0.003 0.013 0.008

12-15 80-82 82-90

Mortality (109cells1-’

flux hi ‘)

0.069-O. 197 0.066-O. 132 0.037-0.016

CNB, 0.172 x lo9 for J-l, and 0.04 x 10” for J-2. It seems that the flux of mortality represents an important part of total bacterial production, participating in an important extent (42-85”,,) in the elimination of the bacterial biomass in water column. In this process, grazing due to the Protozoa retained by 2-pm pores seems to be very important in low-polluted areas. Discussion The values that we have obtained for the conversion factors for calculating growth from thymidine incorporation are of the same order of magnitude as those reported previously, which ranged from 1.3 to 3 ( x 109) cells per nmol of thymidine (Fuhrman & Azam, 1980, 1982; Moriarty & Pollard, 1981, 1982; Kirchman et al., 1982; Bell et al., 1983; Staley & Konopka, 1985). These authors derived their estimates from theoretical calculations, rate of change in thymidine incorporation and correlation between thymidine incorporation and growth rate. An advantage of the batch experiments is that they permit growth at realistic ambient soluble substrate concentrations in the absence or near absence of grazing. However the changes in bacterial numbers and thymidine incorporation from unaltered samples were always smaller than those of filtered samples. The incubations in recipients and the effect of filtrations seem to remove the limiting factors which maintain bacterial populations and their activity at lower values in non-manipulated water. Recently, eucaryotic nanoflagellates measuring 1-2 pm in diameter have been reported (Porter et al., 1985). Our 2+m filtration cannot ensure the absolute absence of Protozoa. Therefore the reduction or absence of predators and the low initial bacterial population after reinoculation, could induce higher growth rates leading to larger cell size. The observed enhanced bacterial growth in water filtered through 2-pm filters can also be due to the dissolved organic matter derived from lysed cells on the filter. Estimates

of bacterial

production

in different

marine

areas

indicate

that

between

30 and

80”,, of the primary production is channeled through heterotrophic bacteria (Azam et al., 1983; HagstrGm, 1984; Williams, 1981; Lancelot & Billen, 1984). This channeling generates an important bacterial biomass that can be available for grazers. It has been suggested that Protozoa are the main predators, although some metazoan larvae (Fenchel, 1982u,b; Sherr et al., 1983) and Bdellovibrio (Shilo, 1984) may be also important. Our range of total mortality values (0.006-0.034 h- ‘) and grazing rates (0.003-0.013 h- ‘) are similar to values reported for the Belgian coastal zone of O-010-0.033 h-’ and [email protected] ‘, respectively (Servais et al., 1985). In the Mediterranean, using genetically marked minicells, Wikner et al. (1986), found bacterial predation values of 2-2.6 x lo4 bacteria ml- ’ h- ’ at bacterial densities of 0.93-0.95 x lo6 ml- I, which

Production

axd mortality

in sea bacteria

335

represent grazing values of 0.0215-0.0273 h- ‘. These grazing estimatesare higher than our values. The use of minicells of bacteria can facilitate the natural grazing and this is a possibleexplanation for the high grazing values observed by Wikner er al. (1986). Our data strongly suggestthat grazing due to Protozoa retained in 2-urn filters can be of importance in channeling bacterial biomassto higher trophic levels. Basedon our watercolumn results, one might tentatively conclude that during summer-autumn periods bacterial production in Barceloneta is about 0.2 x lo9 cells 1-l h- ‘; total bacterial mortality is about 0.02 h ’ and grazing accounts for 15%82”,,of the total mortality.

References Azam,

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