Wastewater nutrient removal by marine microalgae cultured under ambient conditions in mini-ponds

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Pergamon 0273-1223(95)00502-1

War. Sci. Tech. Vol. 31. No. 12, pp. ISI-I60, 1995. Copyrigbt 0 1995 IAWQ Printed in Great Britain. AU rigbb lelerYed. 0273-1223I9S 59'SO + 0-00

WASTEWATER NUTRIENT REMOVAL BY MARINE MICROALGAE CULTURED UNDER AMBIENT CONDITIONS IN MINI-PONDS Rupert J. Craggs*, Valerie J. Smith** and Paul J. McAuley* • School ofBiological and Medical Sciences, Harold Mitchell Building, University ofSt Andrews. St Andrews. Fife. Scotland. UK •• School ofBiological and Medical Sciences. Gatty Marine Laboratory. University ofSt Andrews. St Andrews. Fife. Scotland, UK

ABSTRACT Two endemic strains of the marine miaualgal species Phaeodaclylum tricornutum (designated B2 and 84), previously isolated from a sewage outfall site in St Andrews Bay, Scotland, were cultured in 2().litre mini• ponds 10 detennine their ability to remove ammoniwn and orthopbosphate from wastewater diluted with seawater. These strains had been selected from 102 species for optimal nutrient removal and culture dommance in both hatch and continuous culture on wastewater under controlled environmental conditions. Wastewater (primary sewage efl1uent) was diluted 1:1 with sterile seawater and continuously added 10 algal cultures grown in an open greenhouse under ambient conditions. Nutrient concentrations in the diluted wastewater and in outflow from the cultures were measured daily. Both strains remained unialgal witb little change in biomass during the l4-day culture period and continuously removed >80% of ammonium from the wastewater. However, while strain B2 removed >80% of orthophosphate, there was a gradual accumulation of orthophosphate in tbe culture of strain B4. Measurement of nutrient concentrations in diluted wastewater and outflow from the continuous culture of strain B2 over 24 hours showed that at night nutrient removal dropped 10 a minimum of >70% for both ammonium and orthophosphate. These results indicate !be potential value of strain B2 for use in scaled-up treatment ponds.

KEYWORDS Ammonium; continuous culture; microalgae; mini-ponds; orthophosphate; Phaeodactylum tricornutum; primary sewage effluent. INTRODUCTION The growth of a particular algal species in culture is governed by a complex interaction of parameters such as temperature. light intensity, nutrients, salinity, pH, mixing, pre-adaptation, and culture apparatus (Goldman & Stanley, 1974; Richmond, 1983; Oswald, 1988). Therefore, under a given set of conditions, some algal species will be naturally more competitive than others (Birch & Bachofen, 1988). Continuous culture experiments of seawater mixed with secondary sewage effluent have revealed that only 1-4 of the species originally present in the seawater remain after two to three weeks culture in both IS-litre chemostats lSI

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exclusively dominated by a single microalgal species. usually of the division Bacillariophyceae (Goldman et al..1974). Preliminary research has isolated and screened a wide variety of microalgal species for their ability to treat wastewaters in monoculture under controlled environmental conditions. simulating to some degree the average ambient conditions of a temperate region to enable the selection of species best suited to these parameters (Craggs et at., submitted). Of 102 marine microalgal species tested for nutrient removal from wastewater diluted with seawater in 50 ml batch cultures under controlled conditions, 15 species were found to re~ove >90% of both ammonium and orthophosphate and remain in pure culture (Craggs et al., su~mltted). Two of these 15 best-treating species have been identified as endemic strains of the marine mlcroalga Phaeodactylum tricomutum. This alga has been reported to dominate seawater mixed with secondary sewage effluent in continuous cultures under laboratory conditions (Goldman & Stanley, 1974) and in large-scale outdoor pond cultures (Goldman & Ryther. 1976). The present study describes experiments designed to test the treatment ability of two endemic strains of this species in continuous culture under typical summertime conditions in a temperate region. MATERlALS AND METHODS Test al!:ae The two endemic strains of P. tricomutum (B2, 84) used in this study were isolated from the waters surrounding the wastewater outfall in St Andrews Bay using serial dilution (Guillard, 1985) and Pasteur pipette (Hoshaw & Rosowski 1985) methods. Unialgal stock cultures were maintained on Erd-Schreiber (E• S) medium (McLachlan, 1985), made with sterile seawater. in a controlled environmental incubator (Conviron SlOh) at 15 DC, 18-22~Em-2s-J PAR (Sylvania cool white fluorescent lamps). and 12h:12h photoperiod. Algal inocula were taken from stock cultures maintained in exponential growth by I: I dilution with E-S medium after seven days. Wastewater Unfiltered primary treated sewage effluent was obtained from the treatment plant at St Andrews. Fife, Scotland. To establish the typical inorganic loading of this effluent, weekly measurements of pH, temperature, conductivity, salinity and inorganic nutrient concentrations (nitrate N-N0 3-, ammonium N-NH4+ and orthophosphate P-P043-) were taken of the wastewater over the two-month period during which the continuous culture experiments were run. Parallel measurements were also made on the seawater used to dilute the primary effluent. The mean ± standard deviation of eight weekly measurements are given in Table I. For each experiment, primary sewage effluent was diluted I: I with seawater. Table I. Characteristics of primary treated effluent used in experiments and seawater used for dilution Parameter

pnmaq Ettluent

seawater

Values are means ± SD of eight weekly samples taken over the two month period in which experiments were made.

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Experimcmtal ilPparatus Continuous culture mini-ponds were constructed from 20-litre white polypropylene vats placed in a water• bath. An outflow tube was added to each mini-pond by cutting a hole in the side of the vat and fitting a small diameter glass tube through the hole. The connection was sealed with a gasket made of rubber tubing, and the desired depth of culture (30 em) was maintained by securing the external end of the tube at an angle using a retort stand and clamp. Diluted wastewater was pumped peristaltically (Watson-Marlow Ltd, Falmouth, Cornwall, UK; model 5028) to each mini-pond from a 1000 I fibreglass storage tank and flowed into each culture through a glass tube opening just above the bottom of the mini-pond. Mixing was accomplished with a plastic-coated stirring bar. Effluent flowed out of the culture through the outflow tube, from which 20ml samples were collected for analysis. When sampling was not in progress, effluent dripped into a waste pipe. A schematic diagram of a complete continuous culture unit is shown in Fig. I.

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Measurement of algal growth Algal cell density was determined from daily measurements of optical density (00) at 570 om using a platereader (Dynatech, Billingshurst, Sussex, model MRSOOO). Aliquots (300 ~I) of algal culture were pipetted into the flat-bottomed wells of a microtitre plate (Dynatech, M29A) in triplicate and measured against blank wells of I: I diluted wastewater.

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R. J. CRAGGS et aL

Analytical me3Surements Nutrient concenttations were measured against Milli-Q (M-Q) water blanks by standard colorimetric methods scaled down for use in 96-well microtitre plates. N-NH4+ was measured by the method of Parsons tt al. (1984) us~g stand:ards of 293.8 mmolm-3 (NH4>2S04' and read at 630 om (final volume 300 Ill). The samples were dIluted with M-Q water (1:4) prior to addition of reagents. N-NOf was determined by the m~thod of Snell (1981) using standards of 99.9 mmolm-3 (final volume 300 Ill). N-N0 3- was determined usmg Szechrome (NAS) reagent (Park Scientific Ltd. Northampton. UK) in a scaled-down reaction (final volume 27~ Ill). using 71.4 mmolm- 3 KN03 standards. P-P043- analysis was by the method of Henkel et al. (1988), uSIng 30.0 mmolm-3 K2HP04 standards. The assay was modified by substituting the polyvinyl alcohol for M-Q water and diluting the mixed reagents I: I with M-Q water before addition to the samples (final volume 250 Ill). The temperatures of wastewater and seawater were measured using a mercury in glass thermometer. while pH was determined using a combination pH electrode (Russell Ltd. Auchtermuchty. Fife, UK; Type CWL) read from a digital pH meter (Philips, UK; Type PW9409) and calibrated with pH 7 and pH 10 buffers. Conductance (siemens) was analysed using a conductance cell (Mullard Ltd. London. UK; Type E75911B) connected to a conductivity bridge (Mullard, Type E756). Salinity (o/oc) was calculated from the chloride ion concentration, which was measured with a direct reading digital chloride meter (Coming Ltd. Hallstead, Essex, UK; model 920). Continuous measurements of dissolved oxygen (DO) and temperature were made using an oxygen/temperature sensor (Kent Industrial Measurements Ltd. Chertsey, Surrey. UK; model 7131) read by a oxygen meter (Kent. model 7130). Ambient light intensity was measured continuously during the period of investigation using quantum photometer (Macam photometries Ltd. Livingston, Scotland; model QIOI-4) with the PAR sensor placed just below the water surface within a mini-pond without algae. Values are expressed lIS means ±SO of 6 measurements per day for 14 days culture. Operation The continuous culture unit was set up using sterile equipment Algae were pre-adapted to 1: I diluted wllStewater and the ambient culture conditions by adding 5 litres of algal culture (grown on E-S media) and S Iitres of wllStewater to a mini-pond and culturing for one week. To determine the dilution rate for the continuous culture experiments, a preliminary growth experiment was made by adding another 10 litres of I: I diluted wllStewater and making daily measurements of algal 00 570 over one week. The exponential doubling time (equal to the dilution rate) was calculated from a semilog (base 2) plot of algal 00 570 versus time, and was found to be about 0.2 d-I. Continuous culture experiments began when I: I diluted wastewater WllS pumped into the mini-pond at this dilution rate. Measurements of nutrients (N-NH4+ and P-P043-) and algal 00570 were made from 20 ml samples of algal culture taken daily from the outflow for 14 days. Samples (300 Ill) from each algal culture were pipetted in triplicate into the wells of a microtitre plate for centrifugation (26Og, 10 min). A1iquots of the supernatants were then pipetted into clean microtitre plates for nutrient determination. Only N-NH4+ concentration was measured due to its high concentration compared to all other N-sources in the primary effluent (Table I). Values are expressed as means ± SO of daily measurements over 14 days culture. Culture purity was confirmed through microscopic examination of each algal culture Ilt the time of nutrient measurement. The effect of diurnal variation in light intensity on nutrient removal by strain 82 was investigated. Concentrations of ammonium, nitrite, nitrate and orthophosphate, algal biomass (00 570> and physical parameters (temperature. DO and pH) of a mini-pond culture of the strain were measured at 2 h intervals over a 24 h period for analysis against light intensity. Values are expressed as means ± SO of measurements every 2 hover 24 h culture. RESULTS The nutrient concentrations in mini-pond cultures of two endemic strains (designated 82 and 84) of the marine microaIga P. tricornutum. grown in continuous 2D-litre mini-pond cultures under ambient conditions

Wastewater nutrient removal by marine miaoaJgae culture

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over 14 days. are shown in Figs. 2b. c; 3b. c. Both strains remained unialgal with little change in biomass (00570) during the culture period (Figs. 2a & 3a). indicating that cultures were in steady state. The nutrient concentrations of the influent diluted wastewater remained uniform throughout the period of analysis (400.4 ± 12.1 N-NH4+ and 57.5 ± 0.9 P,P043- for B2; 562.5 ± 8.0 N-NH4+ and 60.2 ± 1.0 p-P043. for B4). Strain B2 continuously removed >80% of ammonium and orthophosphate from the wastewater over the 14 day period (Figs. 2b. c). Strain 84 also removed >80% of ammonium. although orthophosphate gradually accumulated in the culture throughout the experiment, so that by day 14. the orthophosphate concentration of the outflow was 50% that of the influent wastewater (Figs. 3b. c). 0.6

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Ambient light intensities were similar for each strain. with a maximum incident light intensity of 2000J,1Em· 2s· 1 (Figs. 2d. 3d). On days of low light intensity (days 3-5 for strain 82. and days 10-11 for strain B4) (Figs. 2d. 3d). ammonium removal decreased. but was still >70% for both strains. When higher light intensities resumed. ammonium removal returned to 80% (Figs. 2b. & 3b.). There was no apparent change in orthophosphate concentration in the culture of strain B2 during the days of low light intensity. although an increllSC may have been offset by the decline in orthophosphate concentration of the influent diluted wastewater at this time (Fig. 2c.). By contrast orthophosphate removal by strain B4 was unaffected by the low light intensities (Fig. 3c.).

Wastewater nutrient removal by marine microalgae culture

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Physical parameters of the mini-pond cultures of both strains followed changes in light intensity and algal photosynthesis. Figure 4 shows the values for temperature, pH and dissolved oxygen in the culture of strain 82. Over the 14·day experimental period, the mean culture temperature was 20.5 ± 3.3 °C and ranged from 15.0 to 29.9 °C (Fig. 4a.). Generally, the pH was constant at around pH 9.1 ± 1.8; a maximum of pH 10.5 was reached during the day, although on days 3-5 when light intensities were low, pH decreased to 4.0 (Fig. 4b.). Dissolved oxygen concentration increased during daylight hou~ corresponding to algal photosynthesis, but declined during the night as respiration proceeded (Fig. 4c). Values exceeded 1250 mmolm- 3 O 2 in the late afternoon on most days. On days 3 and 5 when light intensity was low, values of all three paramete~ declined, but they recovered to their original levels on day 6 when higher light intensities resumed.

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DISCUSSION AND CONCLUSIONS

This study describes the abilities of two endemic strains of the marine microalga P. tricomutum (designated 82 and 84) to remove nutrients from diluted wastewater in 20-litre mini-pond cultures under ambient conditions over 14 days continuous culture. Both strains were found to remain in steady state and to remove high levels of nutrients from wastewater, although there were slight differences in treating ability between

Wastewalel' Dutrient removal by marine miaoalgae culture

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the two strains. The efficiency of nutrient removal shown by these algae is comparable to the efficiencies recorded for mixed algal populations by other workers (Dunstan & Menzel. 1971; Dunstan & Tenore. 1972) even though the nutrient concentrations in the present study are nearly twice those used by Dunstan & Menzel (1971) and over four times those used by Dunstan & Tenore (1972). The steady-state cultures of both strains of P. tricomutum examined in the present study were similar with respect to algal biomass and physical parameters. but exhibited differences in uptake of the two principal nutrients. ammonium and orthophosphate. In particular. orthophosphate tended to accumulate in the 84 culture but not in the culture of 82. Accumulation of orthophosphate is often indicative of nitrogen limitation (de la Noiie & 8asseres. 1989) and has been noted in earlier studies of continuous cultures of marine microalgae grown on seawater diluted wastewater (Dunstan & Menzel, 1971; Dunstan & Tenore. 1972). However, it is unlikely that nitrogen was limiting in the present study as the N:P atomic ratio of the diluted wastewater was considerably higher (20.6: 1) than the average for assimilation by marine microalgae (10:1; Dunstan & Menzel. 1971). Importantly, from the point of view of wastewater treatment, the accumulation of orthophosphate in the outflow of treatment ponds would be of little significance to marine eutrophication. since nitrogen is frequently the limiting nutrient for phytoplankton growth in coastal marine waters (Ryther & Dunstan. 1971). The continuation of high ammonium and orthophosphate removal overnight by strain 82 is in agreement with the findings of Marsot et al. (1992) who showed that at high cell densities. removal of N03 and N02 by P. tricornutum is unaffected by diurnal cycle. The continuous mini-pond culture system is a very useful apparatus for the determination of nutrient removal from diluted wastewater by microalgae species. Mini-ponds are inexpensive to set up, easy to operate and require little maintenance. Mini-pond systems to treat wastewater have also been used by Dunstan & Menzel (1971) although these had a volume of 15 litres and were artificially illuminated and cooled. The wastewater used by these workers was chlorinated secondary effluent, stored frozen before use and diluted 1:4 with filtered seawater. Here, we describe ponds which use fresh primary effluent, require no artificial illumination or temperature control and rely on a single microalga to reduce inorganic nutrients. Since both strains of P. tricomutum were effective in removing ammonium, they offer potential for the combined secondary and tertiary treatment of sewage before discharge into the sea. 82 is the most promising strain due to the added benefit of removing orthophosphate as well as ammonium. ACKNOWLEDGEMENTS The authors are particularly grateful to Dr Howard Fallowfield for his advice on experimental design, and Professor W. J. Oswald for his encouragement to undertake this research. We are indebted to the technical staff, especially Me Harry Hodge, for their assistance throughout this work. This investigation was supported by a Science and Engineering Research Council studentship. REFERENCES Birch. L. D. and Bachofen, R. ()988). Microbial production of Hydrocarbons. In: Biotu/ul()logy, Rebm. H. J. (cd.). Vol. 6b. Verlag Chemie. Weinheim. pp. 71-100 Craggs. R. J.. McAuley. P. J. and Smith. V. J. ()993). Batch culture screening of marine microalgal nutrient removal from primaty sewage efOuent Hydrobiologia (submitted). de la Nolle. J. and B~res, A. (1989). Biolre8lDlent of anaerobically digested swine manUle with microalgae. BioL Wastts.29. 17-31. DullSlaO. W. M. and Menzel, D. W. (1971). Continuous cultures of natural populations of phytopiankoon in dilute, treated sewage ernuent LimnoL OCtallOgr. 16.623-632. DullSlaO. W. M. and Tenore. K. R. (972). Intensive ouldoor culture of marine phylOplanklOn enriched with treated sewage ernuent Aquaculture. 1. 181-192Goldman. J. ~. and SlaOJey. H. I. (1974). Relative growth of different species of marine algae in wastewaler-seawalel' mixtures. ManM BIOlogy. 28. 17-25. Goldman. J. ~.• Tenore, K. R.. RythQ-. J. H. and Corwin. N. (1974). Inorganic nilrogen removal in a combined teniary treauncnt• manne aquaculture system. I. Removal efficiencies. Wat. Re$.. 8, 45-54. Goldman. J. C,. and Ry~r, J. H. (I976~. Waste reclamation ~ an integrated f~ chain system. In: Biological Corurol of Wattr PolllllUlfi. Tourbler. J. T. and Pierson. R. W. (cds). Um. of Pennsylvanl8 PreSS, Philadelpbia, PA, USA. pp. 197-214.

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