H~drobiologia 135, 55 -60 (1986). O Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.

Brachionus calyciflorus Pallas as agent for the removal of E. coli in sewage ponds

M. T. Seaman', M. Gophen2, B. Z. Cavari2 & B. Azoulay2 Department of Zoology and Entomology, University of the Orange Free State, Bloemfontein 9300,

South Africa and Kinneret Limnological Laboratory, POB 345 Tiberias 14-102, Israel

Keywords: Brachionus calycijlorus, feeding, E. coli, Chlorella, sewage ponds

Abstract

Brachionus calyciforus (Pallas) is a common brachionid in sewage oxidation ponds. The uptake and as- similation of E. coli was optimal at concentrations of 2.7 - 6 . 9 ~ lo8 cells ml-' while assimilation coefficient per body weight of B. calyciflorus was found to be 10%.Ind.-' d-I. More than two eggs per individual were produced during 24 hours when brachionids were fed with a mixutre of E. coli (lo9 cells-ml-I) and Chlorella spp. (lo6 cells.ml-I). The nutritional value of the mixture of E. coli and Chlorella spp. was found to be higher than that of bacteria alone.

Introduction

One aim of sewage treatment is to produce water of a quality good enough to enter natural water courses. This often involves the reduction of nutrients, pathogenic components and organic load. The primary consumers in such a system play an important role in bacterial, algal and organic load reduction. In simple sewage purification sys- tems where treatment consists of an oxidation pond only, the function of consumers of bacteria (Gophen, 1979) and of organic matter in general becomes much more important. Such ponds in the Kinneret (Israel) watershed area have recently been studied, with an emphasis on the role of Daphnia magna as agent in the removal of E. coli from the water body (Hadas et al., 1982; Hadas et a/., 1983a; Hadas, et al., 1983b). Although D. magna was shown to be effective in reducing E. coli concentra- tion as well as other bacteria (Wynne & Gophen, 1981), it was present only during limited periods of the year. On the other hand the rotifer Brachionus calyciflorus occurred for longer periods in these ponds (Hadas, 1982) in concentrations usually be- tween 10 and 100 I-', but on at least one date it

reached a level of 12500 1-' in one of the inves- tigated ponds (Table 1).

As this species is reported to feed on small parti- cles in the size range of 0 to 18 pm (Pourriot, 1977), it is suspected of being an important bacterial feed- er. Furthermore, this species is common through- out the warm temperature world (Voigt, 1957) and is common in sewage ponds. Therefore, we decided to study the consumption, digestion and assimila- tion of bacteria by B. calycijlorus.

Materials and methods

Laboratory cultures were maintained at 26 "C under permanent diffused light in 200 ml glass beakers filled with feeding media less than half of their volume. B. calyciflorus was collected from sewage oxidation ponds.

B. calyciforus were fed on E. coli alone or mix- tures of E. coli and Chlorella spp. Effect of food content on the population dynamics was examined in 10 ml glass vials. Ten to twenty individuals were introduced into the medium and counted daily, medium was changed every 24 hours. Egg produc-

Table I . Numerical composition of the plankton and bacteria observed in the sewage oxidation pond (see text) on 27.12.1983 (Temp. - 13.3"C; Secchi disc - 0.22 m, Chlorophyll a - 717 pg I - ' ) .

Species No. 1-I

B. calyciflorus Asplanchna sp. Filinia sp. Cyclopoid nauplii Copepodites and copepoda Bacteria

tion was determined according to the method presented by Gilbert (1963) at 26 "C in permanent diffuse light.

Direct observation on food particle collection and gut passage processes were done under a microscope.

I4C labelkg study

Labelling of E. coli cells by 14C was carried out as described by Hadas et al. (1982) with the modifi- cation that 14C-glucose was used. To a culture of several thousands of B. calyciflorus in a volume of 100 ml, concentrated labelled E. coli was added. Two experiments were run: In one experiment, 1 dpm=9.77 x lo3 E. coli cells, at 2.0x108 cells ml-l, and in the other 1 dpm=1.12x104 cells at 1.7x108 cells ml-l. After appropriate time inter- vals, 50 to 100 B. calyciflorus individuals (mean length 150 to 160 pm) were withdrawn, carefully washed on a net and removed into a vial containing 0.5 ml formalin. Each sample was concentrated on an 8 pm millipore filter and washed with 100 ml deionised water. The animals were then washed off with deionised water into a petri-dish. In the first experiment described (Fig. 2), organisms were counted and pipetted individually into a microdepression glass slide and then transferred into scintillation vials. In the second experiment (Fig. 3), animals were filtered (20 pm nylon filter), washed with deionised water, then transferred to the scintillation vial and counted. Uptake of radio- active material by the braehionids was measured without sonication (Hadas et al., 1982). Controls of 0.25 ml water and filters without animals derived by the same process were taken.

Results and discussion

Computations of the population dynamics for the pond community of B. calyciflorus on 27.12.1982 using the method presented by Bottrell et al. (1976) indicated a high daily instantaneous birth rate value (b) of 0.21. According to Hutchin- son (1967) such a b value and temperatures ob- served in the pond can sustain a population if the life span is about 14 days. Nevertheless densities of potential predators of B. calyciflorus such as Asplanchna sp. and adult cyclopoids were rather high. Subsequently a month later, B. calyciflorus disappearend from the pond. It is likely that food conditions in the pond were optimal during Decem- ber. Identifiable contents of the guts of B. calyciflo- rus collected in the pond consisted of Chlamydo- monas sp., Euglena sp., Chlorella sp. and other small chlorophytes (13 pm diameter). This implies selection for small round chlorophytes as the entire phytoplankton in the pond included also Ankis- trodesmus sp. in similar concentrations as the other species. It is suggested that elongate Ankistrodes- mus were selectively excluded by B. calyciflorus. Chlorella sp. and bacteria (E. coli), both common in the oxidation pond, were therefore used as food sources in our experimental studies.

Feeding experiments

Particles up to 18 pm in diameter can be ingested by Brachionus calyciflorus (Pourriot, 1977). This author found that B. calyciflorus has food prefer- ences, which differ between populations. The strongest preferences have been shown to be for Eu- glenoides, Volvocales and Chlorococcales, and a slightly weaker preference for detritus, with or without bacteria.

Filtration rate in B. calyciflorus has been found by Erman (1962a) to remain constant up to concen- trations of about 106 Lagerheimia cells ml-' above which filtration rate decreased, and by Stark- weather, Gilbert & Frost (1979) to remain constant (0.5 p1 Ind-' h-') between Aerobacter aerogenes concentrations of 0.01 and 100 pg ml-l. Halbach and Halbach-Keup (1974) using Chlorella pyrenoi- dosa as food source found that filtration rate varied with food concentration and maximum filtration rate (3.4 p1 h-') occurred at 0.5 x lo6 cells ml-'. Starkweather & Gilbert (1977a) found a change in

filtration rate with Euglena gracilis concentration, it being 10 times lower than the bacterial rate men- tioned above (approximately 0.5 pl 1nd.-' h-') at food densities < 1.0 pg ml-I 'and 5 times higher between 50 and 100 pg ml-I.

Ingestion rates have also been found to increase with algal concentrations up to about 2 x lo6 cells ml-I and if the filtration rate is constant, ingestion rate is directly proportional to algal density (Pour- riot, 1977). Maximum ingestion rates have been found to be between 1000 and 4000 cells I d - ' h-' for Brachionus in algal concentrations of lo6 cells ml-I (Pourriot, 1977). It is suggested that diminution of ingestion rate at higher concentra- tions may be caused by toxic effects of the algae (Erman, 1962a, b; Halbach & Halbach-Keup, 1974) but is more likely due to a general regulation mech- anism of ingestion (Starkweather, 1980).

Measurements by direct observations of the rate of ingestion of bacterial cells by rotifer is practical- ly difficult because of the speed of ingestion, the small size and neutral colour of bacteria. However, the rate of ingestion of the algae, like Chlorella, was assessed by direct observation. Ingested cells of Chlorella by individual B. calyciflorus at 15 " C, were counted. Results indicated a rate of about 35 cells (1200 pm3) min-I from a culture of 2 x lo6 cells ml-I, i.e. filtration rate of about 1.05 p1 h-l, and at 20°C, 158+25 cells (5400 pm3 min-I, i.e. 4.75 pl h-I). Similar results were presented by Pourriot (1977) and Starkweather (1980).

At 15 "C and Chlorella concentration of 2x106 cells ml-I a mastax beat frequency of 28 min-I was recorded. This rose to 120 min-I at 25°C which is close to the maximum recorded for any of the tested individuals, both in algal and bacterial media (Fig. 1). As the mastax is driving the food into the intestine its beat frequency must be consid- ered proportional in some way to the ingestion rate of a specific food type.

Mastax beat frequency during feeding of E. coli was maximal when bacterial cell concentration was in the range of 2.7 x lo8 to 6 . 9 ~ lo8 E. coli cells ml-I although large individual variation was ob- served (Fig. 1). When E. coli concentration was lo9 cells ml-I mastax beat frequency dropped.

It is likely that filtration and ingestion rates with up to lo9 E. coli cells ml-' were directly related to food concentration. 2 . 7 ~ lo8 to 6 . 9 ~ lo8 E. coli cells ml-I (0.05 pm3 volume each) are equal in vol-

15

; la

E

f i

S

Fig. 1. Direct observation on individual B. calyciflorus: mastax beats per min at different E. coli cell conce&ations (cells ml-I) at 26 "C. Three individuals -1 ; I - - - 1 ; I -0-*-( ;) are presented. Standard deviations are shown for each value.

ume to 3.9x105 to 1.0x106 Chlorella cells ml-I (35 pm3 mean volume each) (Pollingher, unpubl. data). The ingestion rate of E. coli is limited rela- tive to filtration rate at the same food volume of al- gae (Pourriot, 1977). Erman (1962a, b) suggested that algal concentrations above lo6 cells ml-' in- hibited ingestion by its toxic effects. It is possible that bacteria have a toxic or other chemical effect on ingestion when their concentration in the medi- um is above maximum. A further point to consider at high ingestion rates and consequent rapid gut passage is the ability to assimilate food.

Results of uptake experiments with 14C-labelled E. coli cells as the food source are presented in Fig. 2. Two experiments were run and the results show close similarity. I4C uptake is linear for up to 300 min, cutting the Y-axis above 0. This implies that initially ingestion is not balanced by egestion and the raised intersection above 0 is equivalent to gut content. The line is further directly indicative of assimilation rate. As the line shows no tendence to curve, per cent respiration of assimilated 14C is probably constant. From the data presented in Fig- ure 2 it can be calculated that in the 60 min and 300 min experiments the equivalent of 811 E. coli cells (40.6 pm3) and 802 E. coli cells (43.1 pm3) 1nd.-I min-' respectively (X 41.8 pm3) was assimilated. Ingestion rate was no more than 100 pm3 (2000 E.

Ut""?..

Fig. 2. Uptake rate of labelled E. coli by B. calyciflorus at different bacterial concentrations: 1.7 x108 cells ml-I (- - -), (1 dpm=9.77xlOkeIls) , and 2 . 0 ~ 1 0 8 cells ml-1 (-) (1 dpm= 1.12 x 104 cells) both at 26 "C.

coli cells) min-I taken over at least the estimated ingestion time of 20 minutes (Starkweather & Gil- bert, 1977b; Seaman & Gophen, unpubl. results) which is a low rate compared to data presented by Dumont (1977) for Chlorella of over 1000 pm3 min-I. Assimilation rate was found to be relatively high (40%).

The average assimilation rate of 41.8 pm3 Ind. -I

min-I, is equivalent to 0.008% of wet body weight min-I, or about 12% d-I. It is too low for sustain- ing a healthy population of B. calyciforus, for which values of over 100% d-I are considered realistic (Dumont, 1977). However, these results are better than those observed for D. magna or other zooplankters feeding on E. coli or other bacteria (Gophen, 1977; Wynne & Gophen, 1981; Hadas et al., 1982) where assimilation in 5 x lo8 E. coli cells ml-I over 24 h is calculated to be less than 5% of body mass.

Similar experiments were undertaken with mix- tures of bacteria and algae. Three different E. coli concentrations were added (2.2 x lo7, 2.2 x lo8 or 2 . 2 ~ lo9 cells ml-I); in addition, E. coli ( 2 . 2 ~ los cells ml-I) and Chlorella (2.2x105 cells ml-I) were simultaneously added. Results (Fig. 3) suggested that Chlorella in the medium suppressed the inges- tion and possibly also the assimilation of E. coli by individual animals. The assimilation of E. coli at a concentration of 2 . 2 ~ lo7 cells ml-I was 46 cells min-I 1nd.-I after 120 min, which is about 6% of the rate of assimilation at a concentration of 2.0x108 cells ml-I (Fig. 2), or slightly less than would be expected if filtration rates had been

Fig. 3. Uptake rate of labelled E. coli (1 dpm= 1 . 6 ~ 104 cells) by B. calycSflorus at different concentrations of bacteria solely or mixed with Chlorella sp.: A - 2.2x107 E. coli cells ml-I (x) where average (-) is given and correlation (r2) was 0.94; 2 . 2 ~ lo8 E. coli cells ml-I ( ) and 2 . 2 ~ 108 E. coli cells ml-I mixed with 2 . 2 ~ 105 Chlorella sp. cells ml-I (+); B - 2 . 2 ~ 109 E. coli cells ml-I ( o ) , average is given, r2= -0.77.

equal. At a concentration of 2.2 x lo9 E. coli cells ml-I uptake was high initially and decreased with time. These results are in agreement with the results presented by Erman (1962a, b) on high algal con- centrations where he found that ingestion is in- hibited. After 120 min in 2.2x109 E. coli cells ml-I, the assimilation rate was only 400 E. coli cells min-I I d - ' which is only 0.5 times the value at 1.7 to 2.0x108 cells ml-I (Fig. 2) and showing signs of further decrease.

Reproduction rate and feeding efficiency

The factor most commonly implicated in mictic female production is density. At densities over 1 Ind. ml-I, mictic female proportion has been shown to rise (Gilbert, 1963), and the greater the mictic female production, the smaller is the poten- tial population increase in the short term, although in the long term the impact can be expected to be weak.

Most rotifers are superior to Cladocera in maxi- mum multiplication rate or intrinsic rate of in- crease (Erman, 1962a). A value of 0.64 per day has been found for B. calyciforus compared to 0.3 to 0.5 for most Cladocera. This may be consequence of the higher assimilation efficiency in B. calycifo- rus (up to 34%) compared to three species of Daphnia with values of 17 to 24% (Dumont, 1977). The lower assimilation efficiency of Cladocera also suggests that their faecal material contains resources that rotifers can digest.

Results of egg production determined after 24 h, are presented in Table 2. At a concentration of 1 x lo7 E. coli cells ml-I there was no egg extrusion during 24 h. There was a clear improvement in reproduction when E. coli cell concentration was elevated to 1 x lo8 cells ml-l, with further increase in concentration to lo9 cells ml-I, confirming feeding behaviour and uptake results (Fig. 2). When Chlorella was added to the medium together with E. coli cells the reproduction level increased dramatically. The value of Chlorella as a food or as a supplement to the bacteria is thus emphasized.

Conclusions

Optimal E. coli concentrations for filtration and ingestion by B. calyciflorus (1.35 to 3.45 g (wet wt) 1- I =2.7x108 to 6.0x108 cells ml-l) seem to be roughly the same as those of green algae (lo6 cells ml-I). However, actual ingestion of E. coli is shown to be very low. In contrast, assimilation rate relative to ingested food may be 40% which is high, possibly because of slow gut passage due to poor ingestion. Absolute assimilation relative to the bi- omass of individual rotifers is low (about 10% 1nd.-' d-l) in the presence of 2.0x108 E. coli cells ml-I which is also indicative of poor ingestion of E. coli.

Egg production in medium containing E. coli cells only, was relatively poor. At lo7 cells ml-l, no eggs were produced and between lo8 and lo9 cells ml-I an average of only 0.8 and 0.9 eggs per in- dividual were produced whereas more than 2 eggs 1nd.-I were produced when a mixture of lo6 E. coli cells ml-I and lo6 Chlorella cells ml-I was ad- ded.

The results indicate that B. calyciforus has meta- bolic difficulties when the food source is composed of E. coli alone even though its assimilation rate is high. Consequently a mixed diet of green algae and E. coli would seem to be better for reproduction.

In sewage ponds where a mixture of algae, micro- flagellates and bacteria coexist, the B. calyciforus population has best food conditions which enable it to reduce both bacterial and algal concentrations.

The rotifers, B. calyciforus have an advantage

Table 2. Egg production by females (mictic and amictic) of B. calyci~orus after 24 hours in different food media. Number of females with (1, 2 and 3 eggs) and without eggs and eggs per amictic female are presented.

Food media Egg numbers per female Total Amicitic Q Q eggs/ Q

Amictic Mictic

Repetition 0 1 2 3

2 . 7 ~ 10' cells ml-I, E. coli 1 15 2 13

2.7 x lo8 cells ml-l, E. coli 1 2 2 2

2 . 7 ~ 109 cells mlkl , E. coli 1 5 2 1

2.7 x lo9 cells ml-l, E. coli plus 1 0 2.5 x lo5 cells ml-', Chlorella 2 0

over other zooplankters in sewage ponds due to better survival under a wide range of dissolved oxy- gen, high pH and high organic concentrations (O'Brien & DeNoyelles, 1972). They also have been shown to be able to reproduce more quickly and, in most cases, to assimilate food more efficiently than crustaceans (Dumont, 1977). The present study has shown the poor nutritional value of E. coli for B. calycijlorus probably because of the low ingestion of the cells. However, the value of B. calyciforus in reducing the level of small algae, organic particles and bacteria, should be stressed. It is further pro- posed that B. calyciforus and other zooplankton could provide part of the nutritional requirements for fish introduced into wastewater systems which would lead to further reduction of the organic load in such systems. An improvement of the water quality while at the same time supplying a saleable commodity to offset management expenses, would then be achieved.

Acknowledgements

The South African Council for Scientific and In- dustrial Research, the Israeli National Council for Research and Development and the Israel Oceano- graphic and Limnological Research Ltd. are ac- knowledged for providing funds and facilities for this study. The University of the Orange Free State is thanked for giving the senior author leave to do this work.

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