Indirect effects of chlorinated wastewater on bacteriovorous protozoa

Environmental Pollution (Series A) 34 (1984) 237 249

Indirect Effects of Chlorinated Wastewater on Bacteriovorous Protozoa

Sharon G. Berk & John A. Botts Department of Environmental Sciences, Clark Hall, University of Virginia, Charlottesville, VA 22903, USA

A BS TRA C T Comparisons were made among ciliate population growth on chlorinated Escherichia coli, irradiated E. coli, live Pseudomonasjsp. and irradiated Pseudomonas sp. No differences were observed in protozoan population growth when chlorinated E. coli, irradiated E. coli and irradiated Pseudomonas sp. were used, whereas significant differences resulted between live and irradiated Pseudomonas sp. as food. Food selectivity, determined by feeding experiments with chlorinated 14C-labelled E. coli and live Pseudomonas sp., showed that significant consumption of chlorinated coliforms occurred in the presence of live bacteria. To assess the extent to which dead bacterial cells may be available to bacteria-jeeders in water receiving chlorinated effluents, percentages of living (respiring) bacteria to total bacteria were determined for several sites downstream of an effluent and in a chlorine contact basin of the treatment plant. Populations upstream had a significantly higher percentage of respiring bacteria than those at the contact basin and a site 20 m downstream, but not those at a site 60 m downstream. Toxicity tests on upstream bacteria revealed significant decreases in the percentage of respiring bacteria after 24hours' exposure to water from both downstream sites, as well as water from the contact basin. These data indicate that dead bacterial cells can serve as food ]or ciliates, resulting in decreased ciliate production compared with growth on live bacteria. The treated effluent appeared to contribute dead cells by direct discharge of cells to the stream and via lethal toxicity to native stream bacteria.

237 Environ. Pollut. Set. A. 0143-1471/84/$03'00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain

238

Sharon G. Berk, JohnA. Bot~

INTRODUCTION Studies regarding the impact of chlorinated wastewater on biota in receiving waters have focused mainly on direct effects of chlorine residuals, whereas indirect effects have received little attention. Recently, Berk & Botts (1981) have shown that the ingestion of chlorinated coliforms by ciliated protozoa can result in significant decreases in protozoan population growth compared with the ingestion of living bacteria. Thus, chlorination of wastewater may have an indirect impact on populations of stream biota by affecting the quality of their food source. Protozoa are often neglected with respect to microbiological aspects of pollution studies; however, their importance in the cycling of nutrients, as links in aquatic food chains, and as tools in aquatic pollution assessment, has received more consideration (Johannes & Webb, 1970; Berk et al., 1977; Cairns, 1979; Porter et al., 1979; Stout, 1980). If the protozoa are able to avoid ingesting chlorinated bacteria, their populations may be maintained at normal levels, thereby allowing them to carry out their functions in an aquatic environment. The extent to which protozoa, especially suspension feeders, actively select food items is not clear. Coleman (1964) found no selection for native versus non-native bacteria for certain rumen bacteriovores and, although food preferences have been demonstrated in the carnivorous protozoa Stentor and Didinium (Berger, 1980; Rapport et al., 1972), filterfeeding ciliates were observed to ingest non-nutritive particles such as India ink, colloidal gold and polystyrene latex beads (Elliott & Clemmons, 1966; Weidenback & Thompson, 1974; Fenchel, 1980). The present investigation addresses three objectives: (1) To extend the study of Berk & Botts (1981) to include examination of protozoan growth rates on irradiated and chlorinated bacteria for the determination of the effects of dead, chlorinated or live coliforms and native bacteria on protozoan growth. (2) To determine whether suspension-feeding ciliated protozoa do not ingest chlorinated bacterial cells in the presence of live autocthonous bacterial cells. (3) To examine the effect of a chlorinated effluent on the respiration of native bacterial cells in receiving waters and the contribution of a sewage treatment plant to the numbers of dead bacteria, in order to assess the extent to which dead bacterial cells may be available to bacteria-feeders.

Effects of chlorinated wastewater on protozoa

239

METHODS

Population growth studies Population growth of the estuarine ciliate, Uronema nigricans, was monitored by direct cell counts using light microscopy for each of several feeding regimes. Comparisons were made among protozoan growth on chlorinated Escherichia coli (ATCC strain 25922), irradiated E. coli and irradiated Pseudomonas sp. strain 244, originally isolated from Chesapeake Bay, Md, and used to culture the ciliates in the laboratory for several years. In a separate experiment, growth was compared when the protozoa fed on irradiated Pseudomonas sp. versus live Pseudomonas sp. E. coli cells were cultured for 24 h in nutrient broth at 26 °C, washed by repeated centrifugation (Berk et al., 1977), resuspended in estuarine water and exposed for 30 min to 7.45 mg litre- ~ of chlorine in the form of NaOCI. Cells were washed free of chlorine residuals by centrifugation and resuspension in sterile water three times at 121 x g in a Sorvall RC-5B centrifuge. A toxicity test was performed on the final wash to determine whether residuals remaining caused any decrease in protozoan numbers. This procedure was described previously for mercury toxicity to these ciliates (Berk et al., 1978). For comparison of protozoan growth on dead cells killed with a minimum degree of chemical interference, washed suspensions of E. coli and Pseudomonas cells were exposed for 2h to 6°Co emitting 100-120 Gy min-1 at the nuclear reactor facility at the University of Virginia. All experiments were run in triplicate at 26°C. The optical density of all bacterial preparations was adjusted to yield equal numbers of bacteria, and subsamples were checked by direct counts with epifluorescent microscopy after staining with acridine orange. Equal numbers of protozoan cells were dispensed into suspensions of the bacteria to yield initial cell densities of 600 ciliates per millilitre, and approximately I x 10s bacteria per millilitre. Population growth of ciliates was monitored from direct cell counts in 50#1 subsamples or appropriate dilutions of 50 pl subsamples. An F-test was performed using slopes of exponential growth portions of growth curves, followed by a sequential variant of the Q test (Snedecor & Cochran, 1967) for least significant differences.

240

Sharon G. Berk, John A. Botts

Selectivity experiments For the examination of selectivity by the marine ciliate Uronema nigricans, E. co/i (ATCC strain 25922) was labelled by growth for 24 h in 50ml of nutrient broth (Difco) amended with 50#1 D-[u-t4c] glucose (New England Nuclear, 250 mCi mmol- 1). After the cells were washed by repeated centrifugation a 50 #l sample was checked for radioactivity. The Sampled

at

0 end 18 h

1

Sampled

at

18

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Sampled

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ml

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18 h

H2SO 4 +

0.1

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phenethylamine in I t r e p l

filtrate filter

0.t m l phenet hy I Imine

after

1 h

I

f liter

E.._~ ci....._. oI ~ ÷ Pseudom~as

Uronema

back~l~l

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÷

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Pseudornones

14CO2 produced by Pseudomones

Fig. 1. Experimental design for testing selectivity by the ciliate Uronema nigricans. 14Clabelled, chlorinated E. co/i cells were mixed with non-labelled, live Pseudomonas cells and live ciliate predators (Uronema). At 0 and 18 h (first column), differential filtration was used for the examination of radioactivity in the bacterial and ciliate fractions. A control treatment (second column) tested for changes in radioactivity of the bacterial mixture in the absence of ciliates. A third treatment (third column) tested for possible transfer of labelled carbon from the dead E. coli cells to live Pseudomonas. A CO 2 trap was employed to monitor any 14CO2 produced by the live bacteria.

Effects o f chlorinated wastewater on protozoa

241

cells were then killed by exposure to 7.45 mg litre- i chlorine in the form of NaOC1 for 45 min and subsequently washed as previously described. Pseudomonas was not labelled in the present study, but has been shown to serve as a good food source in previous feeding studies with Uronema nigricans (Berk et al., 1978). Three treatments were set up in triplicate, as shown in Fig. 1. In one treatment, each flask contained 1.0 ml of a washed suspension of nonchlorinated Pseudomonas cells, 1-0 ml of a washed, chlorinated E. coli suspension, 4 ml of a protozoan suspension and 6 ml of sterile estuarine water. At the time each flask was established, a 1.0ml sample of the mixture was passed through a Millipore filter of 5/~m pore diameter, which trapped the ciliates but allowed the bacteria to pass through. The filtrate was subsequently passed through a second filter of 0.22 #m pore diameter to collect the bacteria, and all filters were placed separately in liquid scintillation vials for examination of radioactivity. A significant decrease in radioactivity in the presence of protozoa would indicate that the coliforms were consumed, whereas selection against chlorinated cells would result in no decrease in radioactivity. Controls were established in triplicate to test for the loss of radioactivity in the absence of predation. The controls contained the same volumes of E. coli and Pseudomonas suspensions as the experimental flasks; however, 4 ml of sterile medium were added in place of the 4 ml of protozoan suspension. After incubation for 18 h, 1-0-ml samples were collected on a 0-22-#m filter. The initial counts are represented by the sum of radioactivity on the 5-/~m and 0.22-#m filters of the initial samples in the experimental flasks. A third treatment was used to test whether the radioactivity observed on the control filters could have derived from the incorporation of 14C from the dead E. coli into cells of Pseudomonas. If metabolism of 14Clabelled cells occurred in the control flasks, ~4CO2 would be produced. Therefore, the third treatment was designed to test for ~4CO2 production (Fig. 1). All counts were corrected for quench, and background counts were subtracted from all samples. Field survey and toxicity tests To examine the contribution of a treatment plant to the number of dead bacterial cells which may be potential sources of food to bacteriovores, the proportion of living to total bacterial cells at several sampling sites in a

242

Sharon G. Berk, John A. Botts

stream receiving treated sewage effluent was determined. Sampling stations at the Moores Creek Sewage Treatment Plant in Charlottesville, VA, were established 40 m upstream of the effluent source designated as station A, 20 m downstream (station DI), 60 m downstream (station D2) and in a chlorine contact basin (station E) prior to release to the stream. At each station, three samples were taken at 0.5 m depths using sterile containers or a modified J - Z sampler (ZoBell, 1941). The toxic effect of the treated effluent on native stream bacteria was examined by exposing bacteria from the upstream sample to filtered downstream effluents for 24 h. This was accomplished by filtering 10 ml of upstream samples through a field monitor of 0.22#m pore diameter (Gelman Acrodisc), followed by reverse filtration of 10ml of the downstream water. Controls were prepared by resuspending upstream bacteria in filtered upstream water. At the initial sampling and t = 24 h, 4-ml subsamples of resuspended bacteria were transferred to sterile vials and, as described below, incubated with acridine orange and 2-(piodophenyl)-3-(p-nitrophenyl)-5 phenyl tetrazolium chloride (INT) (Zimmerman et al., 1978), a compound which is reduced by cytochrome activity of living bacteria. Triplicate samples were taken at all stations. The samples were processed within 2 h of retrieval using the procedure of Zimmerman et al. (1978). A 4 ml volume of each sample was placed into sterile capped vials, and to each vial was added 0.4 ml of 0.2 ~ INT dye in phosphate-buffered saline solution. The prepared samples were incubated in the dark for approximately 30 min, after which cell respiration was stopped by adding 0.25 ml of 37 ~o formaldehyde. In preparation for microscopic examination, I ml of the fixed sample was filtered through a 0.22/tm Nucleopore filter prestained with Irgalin Black. The filter was carefully placed on a microscope slide precoated with a semi-solid gelatin solution (5 ~o gelatin and 0.05 ~o chrome alum in sterilised distilled H 2 0 ) and the gelatin was allowed to dry for 24 h. After presoaking in a pH 6.6 citrate buffer (1.2 m u citric acid and 48.8 mM Na citrate) for 3 min, the embedded filter was stained in an acridine orange solution (1:10000 in pH6.6 citrate buffer) for 7min. The filter was destained by successive immersion in a pH 6'6 citrate buffer for 6 min, a pH 5-0 citrate buffer (20.5 mM citric acid and 30.5 mM Na citrate) for 6 min, a pH 4.0 citrate buffer (33 mM citric acid and 17 mM Na citrate) for 10 min and sterilised distilled water for 2 min. All aqueous solutions used in the staining procedure were presterilised by filtration through a 0.2/am membrane filter. The filter was carefully removed from the slide, leaving

Effects of chlorinated wastewater on protozoa

243

bacterial cells embedded in the gelatin. A gelatin coating was applied immediately to seal the sample specimen. The prepared specimen was observed at 1000 x magnification using an epifluorescent Zeiss microscope equipped with a chromatic beamsplitting filter. The cytochemical product of INT reduction, INTformazan, can be identified by its distinctive red crystal. By altering epifluorescence and transmitted light, bacterial cells could be examined for the presence of red INT-formazan crystals. Using this technique, the ratio of respiring bacteria to the total number observed was determined. Total numbers of bacteria in stream stations and the total numbers of bacteria in toxicity test vials were determined using the acridine orange direct count method of Daley & Hobbie (1975). A 1 ml volume of the fixed sample was added t o 9 ml of sterilised distilled water and the sample was stained with acridine orange (Baker Chemicals, N J). After 3 min the sample was filtered through a 0-2/~m Nucleopore filter prestained with Irgalin black. The filter was mounted on a microscope slide and observed using epifluorescent microscopy. Data were analysed using one-way analysis of variance (ANOVA) and the Q method as described above to compare differences in per cent respiring cells among sampling stations.

RESULTS

Protozoan population growth A significant difference was observed between protozoan growth on live and irradiated Pseudomonas cells, growth on the living bacteria being greater (Fig. 2). Experiments using chlorinated E. coli, irradiated E. coli and irradiated Pseudomonas resulted in no significant difference among ciliate population growth curves (Fig. 3), thus indicating that the mode of achieving dead cells had no effect on ciliate growth. No division occurred in the absence of added food, as seen from results of control experiments. These data indicate that, although protozoa can derive nutritional benefit from dead cells, the yield is significantly decreased compared with population growth on living cells. The chlorine treatment of the bacteria, however, does not appear to be a key factor in causing ciliate population decreases, as there were no differences between growth on chlorinetreated and growth on irradiated bacteria. No mortality resulted from exposure of ciliates to the final wash from the chlorinated bacteria.

1tl

12

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8

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0

i 20

0

I 40

I 60

i 80

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Fig. 2. Population growth of Uronema nigricans fed live Pseudomonas ( 0 ) and irradiated Pseudomonas (,). Values plotted are averages of triplicate samples.

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Fig. 3.

Population growth of Uronema nigricans fed irradiated E. coli (,); irradiated Pseudornonas (A) and chlorinated E. co/i ( 0 ) . Values plotted are averages of triplicate samples.

Effects o f chlorinated wastewater on p r o t o z o a

245

TABLE 1 Radioactivity (DPM) in 1-ml Samples from Experimental and Control Flasks Containing Bacteria and Protozoa a (Results of experiments of Fig. 1) Sample

D P M at t= 0h

D P M at t = 18 h

Change f r o m initial D P M

Bacteria

2 022 _+ 56

283 _+ 36

- 1 739

Ciliates Control b Phenethylamine

40 + 7 2062 _+59

328 + 35 2 148 + 80 3.5 _+0.3

+288 +86 NAc

a Values for disintegrations per minute (DPM) are averages of triplicate samples + one standard error. b t = 0 h for controls is the sum of DPM in bacterial plus protozoan fractions in experimental flasks at t = 0. c Not applicable.

Selectivity experiments Results of selectivity experiments (Table 1) reveal that the chlorinated coliforms were readily consumed by protozoa in the presence of living native bacterial food sources. A significant decrease of 86.3 ~o in radioactivity (determined by a t test, p < 0.05) occurred after 18 h in the flasks containing protozoa, compared with no decrease in the controls without protozoa. Only 3-5 D P M above background were found in the 14CO2 trap per equivalent volume of bacterial suspension, i.e. 1.0ml, indicating no respiration of glucose derived from the chlorinated E. coli cells. A significant increase in radioactivity (p < 0"05) occurred in the Uronema sample; 15 ~o of the total radioactivity originally added was found there. No CO2 trap was used in the experimental flask, as respiration of ciliates was not pertinent to this study.

Field survey and toxicity tests Table 2 shows the percentage of respiring bacteria of the total number observed at each station, using the method of I N T reduction coupled with acridine orange staining. The upstream population was observed to have the highest percentage of respiring bacteria ( x = 3 2 - 0 + 6"5~o), this proportion being significantly higher than values for respiring cells at

246

Sharon G. Berk, John A. Botts TABLE 2

Bacterial Activity and Numbers in Water ReceivingTreated Sewage Effluent Station

Per cent respiring cells a

Number of cells (106 ml- 1 )b

A (upstream 40m) E (contactbasin) D 1 (downstream 20m) D E (downstream 60m)

32.0 + 6.5 1.5_+ 1.5 I 1.9 _+4-5 26.0 +_4.0

1-77 _+0.34 2.44 + 0.57 1.82 _+0.58 1.51 _+0.70

a Number of respiring cells determined from INT reduction/total number observed (mean _+ 1SE). b Total number of cells per miUilitre, determined using acridine orange direct count method (mean +_ l SE). stations E (1.5_+ 1.5%) and D 1 (11.9_+4.5%), but not those of D 2 (26-0 _+ 4.0%). At 60m below the effluent the bacterial population appears to recover with respect to the percentage of respiring bacterial cells in the population. The total bacterial counts per millilitre were all similar and within the same order of magnitude, suspended cells in the chlorine contact basin being slightly higher than at the other stations, but less than two-fold greater than any of the others (see Table 2). This suggests that there is no great input of dead cells to the stream from free bacteria suspended in the effluent basin.

g: m

ao-

A

E Station

[:)1

D2

Fig. 4. Changein per cent of upstream bacteria respiring after 0-5 hours (solidbars) and 24 hours (cross-hatched bars) exposure to water from each sampling station. Values are means of duplicate samples.

Effects of chlorinated wastewater on protozoa

247

The effect of the effluent on the respiration of native bacteria is shown in Fig. 4. Significant decreases in the proportion of living to total bacterial cells were observed for exposure to water from all downstream stations, whereas an increase in the numbers of living cells was observed in the control tests, exposed for 24 h to upstream water. The percentage change in the proportion of living to total cells was - 47.7 for cells exposed to the chlorinated effluent of station E, - 34.6 for cells exposed to the effluent of station D 1 and - 4 4 . 0 for cells exposed to the effluent of station D 2. An increase of + 48.7 ~ was recorded for cells exposed to water of station A, above the effluent source. The increased percentage of respiring cells in the controls was probably due to multiplication of healthy cells, a well known phenomenon of contained water samples (ZoBell, 1946).

DISCUSSION The results of the present investigation demonstrate that certain ciliated protozoa can utilise dead bacterial cells as a food source, although the resulting secondary productivity is decreased compared with growth on living bacterial food sources. This is consistent with previously published results using Uronema and E. coli (Berk & Botts, 1981). Chlorine treatment of bacteria did not appear to be a key factor in the suppression of ciliate population growth, as no differences were observed when either chlorinated or irradiated bacteria were provided as food. The mechanism of inhibition of population growth is as yet unknown; however, both irradiation and chlorination affect the structure of bacterial DNA molecules (Haas & Englegrecht, 1980). This may be important to the nutrition ofciliated protoza, since all species require purines, and several species cannot synthesise pyrimidines de novo (Elliott, 1973). Leakage of cytoplasmic contents from dead cells may also contribute to their decreased nutritional value. Since ciliates did not avoid ingesting chlorinated bacteria in the presence of living bacteria, populations of protozoa may be affected by non-discriminatory feeding in areas of streams where chlorine from effluents is no longer toxic to the protozoa, provided that the load of dead bacteria discharged is not large enough to offset the decreased yield resulting from feeding on dead cells. In the present study, the total numbers of free bacteria in the stream were not significantly different between any two sampling sites. Therefore, it is unlikely that the lowered

248

Sharon G. Berk, John A. Botts

nutritional value of the bacteria would be offset by an increased abundance of such food items. Effective flocculation and sedimentation treatment within the sewage treatment plant was probably responsible for the lack of large numbers of free bacteria discharged to the stream. Utilisation of attached bacteria in the stream cannot be disregarded, and further studies on the effects of attached bacteria to higher trophic levels are required. The fact that ciliate populations not only feed on killed bacteria, but also grow on them, may have an important influence on predator-prey models using microorganisms, particularly the mathematical models of Haas (1981), in which conditions for the simulation of predator-prey interactions assume that dead bacteria are irrelevant. For a simulation of chlorinated and non-chlorinated wastewater treatment, Haas (1981) assumed 3000-fold fewer bacterial prey in the chlorinated treatment. Our findings, however, revealed an approximate two-fold higher number of total bacteria in the chlorine contact basin than in the stream and, although dead, the bacteria could serve as prey items. Upon initial exposure to the chlorinated effluent, native stream bacteria were very sensitive, yet exposure to continuous discharge appeared to result in recovery of the bacterial populations with respect to the percentage of respiring cells. Selection for tolerant species or regrowth of chlorine-injured coliforms may have occurred; however, such changes in bacterial community structure were not monitored in the present study. Although the mode of killing bacteria does not appear to matter in the suppression of ciliate growth, chlorination of wastewater may have an effect by providing the ciliates with bacteria of altered food quality. Furthermore, dechlorination ofwastewater prior to discharge should not alleviate these indirect effects. Studies are in progress to assess the direct effects of effluents on ciliates in order to determine the point downstream of the discharge at which dead bacterial cells would exert an influence on the growth of higher trophic forms.

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Berk, S. G., Browlee, D. C., Colwell, R. R., Heinle, D. R. & Kling, H. J. (1977). Ciliates as a food source for marine planktonic copepods. Microbial Ecol., 4, 27-40. Berk, S. G., Mills, A. L., Hendricks, D. L. & Colwell, R. R. (1978). Effects of ingesting mercury-containing bacteria on mercury tolerance and growth rates of ciliates. Microbial Ecol., 4, 310 330. Cairns, J. Jr (1979). A strategy for use of protozoans in the evaluation of hazardous substances. In Biological indicators of water quality, ed. by A. James and L. Evison, Chapt. 6, 1-17. New York, John Wiley. Coleman, G. S. (1964). The metabolism of Escherichia coli and other bacteria by Entodinium caudatum. J. gen. Microbiol., 37, 209 23. Daley, R. J. & Hobbie, J. E. (1975). Direct counts of aquatic bacteria by a modified epifluorescence technique. Limnol. Oceanogr,, 20, 875-82. Elliott, A. M. (1973). Biology ofTetrahymena. Stroudsburg, PA, Hutchinson and Ross. Elliott, A. M. & Clemmons, G. L. (1966). An ultrastructural study of ingestion and digestion in Tetrahymena pyriformis. J. Protozool., 13, 311-23. Fenchel, T. (1980). Suspension feeding in ciliated protozoa: Functional response and particle size selection. Microbial Ecol., 6, 1 ! 1. Haas, C. N. (1981). Application of predator-prey models to disinfection. J. Water Pollut. Control Fed., 53, 378-86. Haas, C. N. & Englegrecht, R. S. (1980). Physiological alterations of vegetation microorganisms resulting from chlorination. J. Water Pollut. Control Fed., 52, 1976-89. Johannes, R. E. & Webb, K. L. (1970). Release of dissolved organic compounds by marine and freshwater invertebrates. In Organic matter in natural waters, University of Alaska, Sept. 2 4, 1968, ed. by D. W. Hood, 257-73. Porter, K. G., Pace, M. L. & Battey, J. F. (1979). Ciliated protozoans as links in freshwater planktonic food chains. Nature, Lond., 277, 563-5. Rapport, D. J., Berger, J. & Reid, D. B. W. (1972). Determination of food preference of Stentor coeruleus. Biol. Bull. Woods Hole Oceanogr. lnstn, 142, 103 9. Snedecor, G. W. & Cochran, W. G. (1967). Statistical methods. Ames, Iowa, lowa State University Press. Stout, J. D. (1980). The role of protozoa in nutrient cycling and energy flow. In Advances in microbial ecology, 4, ed. by M. Alexander, 1 50. New York, Plenum Press. Weidenback, A. L. S. & Thompson, G. A. Jr (1974). Studies of membrane formation in Tetrahymena pyriformis, VIII. On the origin of membranes surrounding food vacuoles. J. Protozool., 21, 745-51. Zimmerman, R., Iturriaga, R. & Becker-Birch, J. (1978). Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. environ. Microbiol., 36, 926-35. ZoBell, C. E. (1941). Apparatus for collecting water samples from different depths for bacteriological analysis. J. mar. Res., 4, 173 88. ZoBell, C. E. (1946). Marine microbiology. Waltham, MA, Chronica Botanica Co.