- Email: [email protected]
Wastewater nutrient removal by marine microalgae grown on a corrugated raceway
Pergamon PII: S0043-1354(96)00093-0
Wat. Res. Vol. 31, No. 7, pp. 1701-1707, 1997 © 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0043-1354/97$17.00+ 0.00
WASTEWATER N U T R I E N T REMOVAL BY MARINE MICROALGAE GROWN ON A C O R R U G A T E D RACEWAY RUPERT J. CRAGGSI'~@, PAUL J. McAULEY t and VALERIE J. SMITH 2. ~Harold Mitchell Building and 2Gatty Marine Laboratory, School of Biological and Medical Sciences, University of St Andrews, St Andrews, Fife KY16 9TH, Scotland
(Received July 1995; accepted in revised form April 1996) Abstract--Two marine microalgal isolates from a sewage outfall site in St Andrews Bay, Scotland were cultured on corrugated raceways (2.5 m long, 0.2 m wide) to determine their ability to remove ammonium and orthophosphate from wastewater diluted with seawater. The isolates (SA91B33, preliminarily identified as Phaeodactylum tricornutum, and SA91CY1, Oscillatoria sp.) both have surface-adherent properties and were selected from 102 isolates for optimal nutrient removal and culture dominance in both batch and continuous culture on wastewater under controlled environmental conditions. Wastewater (primary sewage effluent)was diluted 1: 1 with sterile seawater and continuously added to raceway algal cultures grown under ambient conditions. Nutrient concentrations in the diluted wastewater influent and in the effluent from the raceways were measured daily. Both isolates remained unialgal during the four month culture period and continuously removed 100% of ammonium and orthophosphate from the wastewater. Nitrite and nitrate levelsin both influent and effluentwere negligible.Measurement of influent and effluentnutrient concentrations over 24 h showed ammonium and orthophosphate removal remained unaltered during the diurnal cycle. These results indicate the potential for using microalgal species grown on raceway ,;urfaces for wastewater treatment. © 1997 Elsevier Science Ltd
Key words--ammonium, marine, microalgae, orthophosphate, raceway, wastewater
INTRODUCTION It is generally recognized that microalgae play an important role in the self-purification of natural waters (Soeder, 1980). Because microalgae use solar energy to supply oxygen required for aerobic degradation and recycle the nutrients responsible for eutrophication into potentially valuable biomass (de la Noiie and De Pauw, 1988; Oswald 1988), they may offer an inexpensiw,• alternative to conventional forms of tertiary wastewater treatment. The design, construction and operation of microalgal wastewater treatment systems has been influenced by two major factors. First, the need for adequate mixing to maintain efficient treatment (de la Noiie and De Pauw, 19:g8), and second, the difficulty of separating the microscopic algal biomass from the treated effluent efficiently and economically in order to complete the process (Oswald, 1988). Thorough mixing of microalgal ponds ensures homogeneous conditions by avoiding sedimentation *Author to whom all correspondence should be addressed. [Tel.: (01334) 463 368; Fax: (01334) 463 366; Email: [email protected]]. tPresent address: EEHSL, University of California at Berkeley, 1301 $o. 46th St., Richmond, CA 94804, U.S.A.
of algal cells and increasing the efficiency of light utilization in the culture. Mixing also prevents thermal stratification and the occurrence of nutrient and pH gradients, supersaturation of oxygen and the depletion of carbon dioxide at the pond surface, and anaerobic conditions on the bottom (Richmond, 1986; Oswald, 1988). Various systems have been designed to enhance the mixing of algal mass cultures, including paddlewheels in shallow circulating raceways (Oswald, 1988), air-water lifts (Laws et al., 1983) or mixing boards in rectangular ponds (Materassi et al., 1984), and pumps to recirculate the culture in sloping culture units (Roubicek et al., 1985). More recently, systems have been designed which circulate the algal culture within closed plastic photobioreactors with a high surface area to volume ratio (Richmond et al., 1993). Although high levels of treatment may be achieved using these types of apparatus, the costs of construction and operation severely restrict their economic use in microalgal wastewater treatment systems. In addition, a major cost in operation of these systems is that of removal of microalgae from the treated effluent. Since the BOD of the algal biomass in the effluent from many microalgal treatment ponds is usually above the discharge standard for natural water bodies (Ryther, 1983), the
1701
1702
R. J. Craggs et al.
algal biomass must be removed before the effluent is discharged. By their very nature, microalgae are small (>20/xm). This, coupled with the facts that culture densities are relatively low (2-6 g m-3), most species have a specific gravity slightly greater than that of water, and many have a strongly negative charge on their surface which keeps them dispersed, makes harvesting difficult and costly (Oswald, 1988). Various harvesting methods available, including floatation, sedimentation, precipitation, centrifugation, filtration and flocculation, have been extensively reviewed. The most successful techniques are centrifugation, filtration and flocculation (Mohn, 1988), but incompatibility between efficiency of the harvest methods and their cost-effectiveness restricts their application in microalgal wastewater treatment (Benemann et al., 1980). Although immobilization of algae in beads of carageenan or alginate offers a novel and elegant way to recover the algal biomass, because the beads sediment in a few seconds and can be used repeatedly (de la Notie et al., 1992), the cost of the immobilization substrate may be prohibitive for use in large-scale systems. An alternative approach is to capitalize on natural characteristics of the microalgae in the design of treatment apparatus. We have identified two endemic isolates of microalgae which continually remove high concentrations of ammonium and orthophosphate from 1:1 diluted wastewater, and which possess the ability to aggregate and adhere to the culture apparatus (Craggs, 1994). Surface adherence is a useful property in two respects. First, there is no need to separate the algal biomass from the pond effluent, since the algae remain attached to the surface of the culture apparatus, leaving a treated effluent which is virtually algal free. Second, by inclining the surface of the apparatus, wastewater will trickle through the adhered algal biomass without the need for any additional mechanical mixing or recirculation. An apparatus designed specifically for these adherent species, with a high surface area to volume ratio, may therefore require lower capital investment and lower operational costs. In this paper, we describe the construction and operation of such an apparatus for wastewater treatment by two marine microalgal isolates, and monitoring of efficiency of removal of nutrients over 16 weeks.
MATERIALS AND METHODS
Algae Two species of marine microalgae, SA91 B33 (preliminarily identified as a colonial strain of the oval morphotype of Phaeodactylum tricornutum) and SA91CY 1 (Oscillatoria sp.) were both isolated from a plankton tow in April 1991, St Andrews Bay, Fife, Scotland, and were selected from 102 isolates for optimal nutrient removal and culture dominance in both batch and continuous culture on wastewater under controlled environmental conditions (Craggs, 1994). Both isolates were maintained in 11 continuous cultures with a 2d residence time at 13-18°C in ambient light supplemented by three mercury vapour lamps (Trulite
400W, MBF/U, GEC, London, U.K.), 12:12 light:dark, 150-170/zmol photon m--' s-L Cultures were grown on seawater diluted 1 : I with primary sewage effluent collected from the effluent of the St Andrews treatment facility, and provided suspensions of microalgae in exponential growth phase suitable for seeding the raceways. Apparatus Corrugated raceways were constructed from four 20 cm wide strips of clear corrugated polyethylene sheeting. The strips were joined by overlapping two corrugations of the higher strip over two corrugations of the lower strip and sealing with silicone sealant (Vallance Ltd, Birmingham, England). Side walls of clear plastic sheet were attached and sealed with the sealant to produce a water tight continuous raceway 2.5 m long. The apparatus was enclosed in a wooden frame for support. The corrugation increased the surface area of the raceway and formed discrete microponds. Upon inclination of the raceway at a slight angle (5°), medium added at the top overflowed from one micro-pond to the next; this slowed the flow of wastewater down the raceway and reduced problems of desiccation by increasing the holding capacity. Each raceway contained 78 micro-ponds and had a total holding capacity of 2.1 1 and surface area of 0.6 mL The raceway was protected from rain and falling debris by a glass cover, which was raised to facilitate ventilation. Influent and effluent tubes passed through the end walls of the raceway. Wastewater used to test efficiency of nutrient removal was unfiltered primary sewage effluent from St Andrews treatment facility, primarily from domestic sources, diluted 1:I with sterile seawater. Average nutrient composition and physical properties of the effluent are given in Craggs et al. (1994). The diluted wastewater was pumped peristaltically (Watson-Marlow Ltd, Falmouth, England; model 502S) to each raceway from a 10001 fibre-glass storage tank and dripped from the influent tube into the first micro-pond. Medium trickled from one micro-pond to the next and finally out of the raceway into a collection vessel, through the effluent tube. A schematic diagram of a complete continuous corrugated raceway culture unit is shown in Fig. 1. Operation For one week, each raceway (one for each isolate) was seeded daily with 500 ml of algal culture. At the start of the treatment period (day 0), the wastewater was added continuously at a pumping rate of 41day -~ (0.5 day residence time). The raceways were run as continuous cultures for 16 weeks (July 14th-November 2nd 1993), during which maximum ambient temperatures varied from 23°C to 4°C. Twenty millilitre samples of the 1:1 diluted wastewater influent and corrugated raceway effluent were collected daily at 2.00 pm for measurement of nutrient concentrations (ammonium, nitrite, nitrate and orthophosphate), pH and microscopic examination. Nutrient concentrations were measured against Milli-Q (M-Q) water blanks by standard colorimetric methods scaled down for use in flat-bottomed 96-well microtitre plates (Dynatech, M29A, Billingshurst, Sussex, England) with a plate-reader spectrophotometer (Dynatech, MR5000). Three 300 #1 aliquots of each of the influent and effluent samples were pipetted into the wells of a plate for centrifugation (260g, 10 min). The required volumes of supernatant were then pipetted into clean micotitre plates for nutrient measurement. N-NH4 + was measured by the method of Parsons et al. (1984) using standards of 293.8mmolm -3 (NH,)2SO4, and read at 630nm (final volume 300/~1). The samples were diluted with M-Q water (1:4) prior to addition of reagents. N-NOz- was determined by the method of Snell (1981) using standards of 99.9 mmol m -3 (final volume 300/~1). N-NO3- was determined using Szechrome (NAS) reagent (Park Scientific Ltd,
Microalgal wastewater nutrient removal Northampton, England) in a scaled down reaction (final volume 275/A), using 71.4mmol m-3 KNO3 standards. P-PO43- analysis was by the method of Henkel et al. (1988), using 30.0 mmol m -3 K_~HPO4standards. The assay was modified by substituting the polyvinyl alcohol for M-Q water and diluting the mixed reagents 1: 1 with M-Q water before addition to the samples (final volume 250/~1). Medium pH was determined using a combination pH electrode (Russell, type CWL) read from a digital pH meter (Philips, type PW9409) and calibrated with pH 7 and pH 10 buffers. The purity of the cultures of the two microalgal isolates was checked using a compound microscope (Nikon Labophot) with x 10 and x40 objectives. Light intensity
Ambient light intensity was measured continuously over the period of investigation using a quantum photometer (QI01-4; Macam Phc,tometrics Ltd., Livingston, Scotland) with a PAR sensor. RESULTS The influent and effluent nutrient concentrations during the first 35 days of operation of the corrugated raceways using tile two isolates, SA91CY1 and SA91B33, grown under ambient conditions, are shown in Fig. 2. For both isolates, the reduction in effluent nutrient concentrations to zero corresponded to the growth of a dense algal culture on the surface of the raceways. ]isolate SA91CY1 took longer to establish a uniforra covering than SA91B33, which may account for the higher initial nutrient removal by the latter. Once established, raceway cultures of both isolates totally removed ammonium and orthophosphate from the seawater:wastewater mix, although complete removal of ammonium was achieved before that of orthophosphate. An overnight blockage in the influent tube of diluted wastewater between culture
1703
days 25 and 26 caused both floways to partially dry out and some of the algal cultures to die. This resulted in an increase of the orthophosphate concentration of the effluents between days 26 and 29 (Fig. 2(b)), although the ammonium concentration of the effluents remained unchanged (Fig. 2(a)). During the 14 day period (days 36-50) when detailed measurements were taken, influent nutrient concentrations remained uniform at 497.7 + 60.7 mmol N m -3 and 76.2 + 4.9 mmol P m -3, and both species continued to remove 100% of ammonium and orthophosphate (Table 1). The mean pHs of the effluent from the raceway cultures were 9.8 + 0 . 2 and 9.7 +0.1 for SA91CY1 and SA91B33, respectively, over the 14 days of detailed measurement, although that of the influent was only 7.6 _ 0.1 (Table 1). To determine whether N-NH~ ÷ was actually removed from the culture and not converted to nitrite and nitrate, the concentrations of these nutrients were also measured. Nitrite concentration (2.8 + 0.9 mmol N m -3) in the influent was less than 1% of the N-NH4 ÷ concentration (497.7 + 60.7 mmol N m-3), while nitrate was absent. Nitrite was also removed by the raceway cultures while nitrate remained absent (Table 1). To determine the effect of diurnal variation in light intensity on nutrient removal, nutrient concentrations in the diluted wastewater influent and effluent from the two corrugated raceways were measured over the 24 h diurnal cycle (Fig. 3 and Table 2) 48-49 days after initial seeding. Both ammonium and orthophosphate removal remained at 100% over the light:dark cycle (Fig. 3(a) and (d)). Nitrite and nitrate concentrations were much lower
3
\
Fig. 1. Schematic diagram of a complete continuous culture unit. 1: diluted wastewater supply tank; 2: peristaltic pump; 3: wastewater inflow;4: corrugated raceway; 5: algal culture within micro-pond; 6: glass rain cover; 7: outflow; 8: treated wastewater.
1704
R . J . C r a g g s et al.
a.
. - - T
0
5
T
T
10
15
. . . . .
T
T
T
T
20
25
30
35
,,~ 200 b. 150
.
100
*~'~N~
0 0
5
10
15
20
25
30
35
Incubation T i m e (d) Fig. 2. (a) A m m o n i u m
a n d (b) o r t h o p h o s p h a t e c o n c e n t r a t i o n s in the 1:1 d i l u t e d w a s t e w a t e r influent
( • - • ) and effluents of the corrugated raceways of isolate SA91B33 (•-Ill) and isolate SA91CY 1 ( • - O ) over 35 days culture. Nutrient values are means ___s.d. of triplicate samples. The s.d. may be too small to be seen.
than that of ammonium, and showed little change over the period of measurement, with changes in the effluent reflecting those of the influent (Fig. 3(a)-(c)). Ammonium and orthophosphate removal remained at 100% during operation of raceway cultures of the two isolates for a further 8 weeks, and microscopical examination showed that the raceways remained unialgai. During this time, the concentrations of nitrite and nitrate relative to ammonium were less than 1%. DISCUSSION
This paper describes the abilities of two endemic marine microalgal isolates, a cyanobacterium (SA91CY1) and a diatom (SA91B33), to remove nutrients from diluted wastewater in a specially designed corrugated raceway under ambient summer and autumn conditions. Both isolates were found to remain in monoculture and to continuously remove all the ammonium and orthophosphate from wastewater over the 16 weeks of the experiment,
demonstrating the stability of the algal culture on the raceway surfaces. Although a control raceway without an algal culture was not operated in this study, the reduction of the nutrient concentrations in the effluent from the raceways during the establishment of cultures of both isolates (Fig. 2) indicated that nutrient removal was due to the microalgae. Nutrient removal by the raceway algal cultures was probably not just by uptake and assimilation into algal biomass. The pH of the raceway effluents was high ( > 9.5) which results from bicarbonate uptake for photosynthesis when the algae are carbon limited. Ammonium may have been volatilized and orthophosphate precipitated, both of which are known to occur at elevated pH (>8.5) (Soeder and Hegewald, 1988). It is unlikely that ammonium was nitrified to nitrite and nitrate, since neither of their effluent concentrations increased from the influent values and the aerobic nature of the photosynthetic algal biomass would have probably prevented denitrification of the nitrate to nitrogen gas from occurring. However, further research is required
Table I. Percentage removal of ammonium, nitrite and orthophosphate, and effluent pH, from corrugated raceways of two marine microalgal isolates on 1: I diluted wastewater. Values are means _+ s.d. over 14 days of culture (n = 14). Influent concentrations were 497.7 4- 60.7 mmol m -3 N-NH4 +, 2.8 _+ 0.9 mmol m-3 N-NO2-, 76.2 + 4.9 P-PO4~-, and influent pH was 7.6 + 0.1 (range 7.73-7.50) Algal isolate
% N-NH4 + removal
% N-NO~removal
% P-P043removal
Mean + s.d.
pH Range
SA91CYI SA91B33
100.0 + 0.4 100.0 _+ 0.2
51.7 + 10.7 82.9 _+ 7.1
99.4 -I- 0.8 100.0 + 0.3
9.8 + 0.2 9.7 + 0.1
10.0--9.4 9.9-9.5
a.
,001 0 0
4
8
12
16
20
24
2 310
b.
0
4
8
12
16
20
24
10'1
c.
6 4
2
t
0
.
4
I
8
12
16
20
:1 Or
~
i' 1 0
24
J 4
8
12 16 Incubation Time (h)
I,r---. 20
Fig. 3. (a) Ammonium, (b) nitrite, (c) nitrate and (d) orthophosphate concentrations in the 1:1 diluted wastewater influent ( 0 - @ ) and effluents of the corrugated raceways of isolate SA91 B33 ( l - i ) and isolate SA91CYI ( 0 - 0 ) in relation to light intensity (e) over 24 h culture. Nutrient values are means _ s.d. of triplicate samples. The s.d. may be too small to be seen. 1705
24
1706
R.J. Craggs et al.
on the mechanisms of nutrient removal by the raceway algal culture. The sequence of nutrient removal in the raceways, with ammonium removed before the orthophosphate, is representative of high-rate ponds, which function as nitrogen-limited systems (Weissman et al., 1978). The increase in the orthophosphate concentration of the effluents following the partial drying out of the two cultures (Fig. 2) may have been due to release from the dead portion of the algal culture, while any released ammonium was possibly assimilated by the remaining algal biomass, since nitrogen was limited. The efficiency of nutrient removal of established raceway cultures of both isolates (100%) is comparable to the efficiencies recorded for algal cultures by other workers (Robinson et al., 1988; Megharaj et al., 1992), even though the influent nutrient concentrations in the present study were over twice those used by these authors. Further, except when the inflow pipe was temporarily blocked, there was no reduction in removal of orthophosphate by the raceway cultures over the course of this study, although this has been consistently noted in earlier studies of continuous cultures of marine microalgae grown on seawater diluted with wastewater (Dunstan and Tenore, 1972; Craggs, 1994) and in continuous cultures of freshwater microalgae grown in artificial medium (Robinson et al., 1988; Megharaj et al., 1992). Although the ability to assimilate ammonium in the dark has been shown to vary from species to species (Eppley et al., 1971), the continued high rates of ammonium and orthophosphate removal by both isolates during the dark period of the daily light:dark cycle is in agreement with more recent findings that removal of nitrate, nitrite and orthophosphate can be unaffected by the light:dark cycle at high culture densities (Nalewajko and Lee, 1983; Marsot et al., 1992). One of the main problems for the mass culture of microalgae is contamination by unwanted species (Richmond, 1986). The cultures of both isolates remained unialgal in open continuous culture under ambient conditions, suggesting dominance over the algal species which naturally occur in wastewater. These two endemic marine isolates of St Andrews Bay could possibly have been tolerant of or adapted to the salinities of estuarine conditions which may have given them a competitive advantage over freshwater species occurring in the wastewater (Craggs et al., 1994). Many conditions govern the competitive advantage of one species over another Table 2. Percentage removal o f a m m o n i u m , nitrite and orthophosphate from corrugated raceways o f two marine microalgal isolates on 1:1 diluted wastewater over 2 4 h ( n = 12). The influent concentrations were 575.1 __. 16.3 m m o l m -3 N-NH:, 1.5 + 0.4 m m o l m-3 N - N O c , 86.6 5:6.0 P-PO43Algae isolate
% N-NH4 + removal
% N-NO2removal
% P-PO4 3removal
SA91CY1 SA91B33
100.0 _ 0.1 100.0 _ 0.1
16.9 + 40.0 50.8 5:20.0
98.3 + 2.5 100.0 + 0.2
(Goldman et al., 1982). Although the results of laboratory studies investigating competition between species are variable (Dortch, 1990) and there is some debate as to whether laboratory studies are applicable to the field (Grover, 1991), the present research has shown these two isolates to be dominant in culture under all experimental conditions from small-scale batch cultures to large-scale open continuous cultures (Craggs, 1994). Both bacillariophyceaen and cyanophyceaen species have been found to dominate phytoplankton blooms in marine waters and mass cultures (Marshall and Orr, 1927; Torzillo et al., 1986). The ability of these marine species to remain in unialgal culture may enable a microalgal wastewater treatment process to operate more efficiently by optimizing conditions for the growth of a particular species. Nutrient removal (100%) by both isolates was higher when cultured on raceways in this study than when these and other isolates were cultured in small ponds using the same influent (44.2-98.8%) (Craggs, 1994; Craggs et al., 1995). This may have been due to a number of complementary factors. There was little wash-out in raceway cultures, because the algae adhered to the raceway surface, while algal biomass was continuously lost in the outflow of the continuous cultures in ponds. Raceways have a higher surface area than pond systems, thus continuously exposing a higher proportion of the culture to light. Furthermore, the raceways were operated in a linear configuration so that treated and untreated wastewater never came into contact. The high nutrient removal achieved by the isolates cultured on the corrugated raceways demonstrates the potential advantage of designing microalgal treatment systems to suit particular algal species, rather than relying on a mixed assemblage of microalgal species with variable nutrient removal rates to grow up which occurs with most systems in use today (Oswald, 1988). Corrugated raceways have two further advantages which reduce the operational costs of the system. Raceways do not require mechanical mixing and may enable simple and economic separation of the algal biomass from the treated effluent by simply scraping it from the drained surface. Although sloping culture units have previously been used for microalgal culture (Roubicek et al., 1985), they have not been operated as linear systems using adherent microalgal species. Efficient nutrient removal by marine microalgal systems could be used to reduce the concentrations of eutrophication-causing nutrients discharged to coastal waters. CONCLUSIONS
(1) Two endemic isolates of cultures of marine microalgae, grown in continuous culture on raceways, were able to remain in unialgal culture and to treat wastewater during summer and autumn conditions in Scotland.
Microalgal wastewater nutrient removal (2) Removal of a m m o n i u m and orthophosphate from a !:1 wastewater:seawater mix was complete and continuous, antt was not affected by the diurnal cycle. (3) By exploiting the adherent properties of these isolates, the corrugated raceway microalgal treatment system does not require mixing, and may enable simple mechanical harvesting of the algal biomass. (4) This study indicates the potential for the use of marine microalgae for nutrient removal from wastewaters even in temperate areas. Acknowledgements--This work was supported by a Science and Engineering Research Council studentship to RJC, and by the Royal Society. The authors are particularly grateful to Dr Howard Fallowfield and Professor W. J. Oswald for their encouragement and advice during this research. We are indebted to the technical staff, especially Mr Harry Hodge, for their assistance throughout this study, and to Andrew Whiston for his help with algal identification.
REFERENCES
Benemann J. R., I~oopman B. L., Weissman J. C., Eisenberg D. and Goebel R. (1980) Development of microalgae harvesting and high-rate pond technologies in California. In Algae Biomass: Production and Use (Edited by Shelef G. and Soeder C. J.), pp. 457-495. Elsevier Biomedical Press, Amsterdam. Craggs R. J. (1994) Wastewater Nutrient Removal by Marine Microalgae. Ph.D. Thesis, University of St Andrews. Craggs R. J., McAuley P. J. and Smith V. J. (1994) Batch culture screening of marine microalgal nutrient removal from primary sewage effluent. Hydrobiologia 288, 157-166. Craggs R. J., Smith V. J. and McAuley P. J. (1995) Wastewater nutrient removal by marine micro-algae cultured under ambient conditions in mini-ponds. Wat. Sci. Technol. 31, 151-160. De la Nolle J. and De Pauw N. (1988) The potential of microalgal biotechnology: a review of production and uses of microalgae Biotech. Adv. 6, 725-770. De la Noiie J., Laibert6 G. and Proulx D. (1992) Algae and waste water. J. Appl. Phycol. 4, 247-254. Dortch Q. (1990) Review of the interactions between ammonium and nitrate uptake in phytoplankton. Mar. Ecol. Prog. Ser. 611, 183-201. Dunstan W. M. and Tenore K. R. (1972) Intensive outdoor culture of marine phytoplankton enriched with treated sewage effluent. Aquaculture 1, 181-192. Eppley R. W., Rogers J. N. and McCarthy J. J. (1971) Light/dark periodicity in nitrogen assimilation of the marine phytoplankters Skeletonema costatum and Coccolithus huxleyi in N-limited chemostat culture. J. Phycol. 2, 150-154. Goldman J. C., Azov Y., Riley C. B. and Dennett M. R. (1982) The effect of pH in intensive microalgal cultures. II. Species competition. J. Exp. Mar. Biol. Ecol. 57, 15-24. Grover J. P. (1991) Dynamics of competition among microalgae in variable environments: experimental tests of alternative models. Oikos 62, 231-243. Henkel, R. D., Vandeberg, J. L., Walsh, R. A. (1988)
1707
A microassay for ATPase. Anal. Biochem. 169, 312-318. Laws E. A., Terry K. L., Wickman J. and Chalup M. S. (1983) A simple algal production system designed to utilize the flashing light effect. Biotechnol. Bioeng. 25, 2319-2336. Marshall S. M. and Orr A. P. (1927) The relation of the plankton to some chemical and physical factors in the Clyde Sea area. J. Mar. Biol. Assoc. 14, 837-868, Marsot P., Mouhri K. and Cembella A. D. (1992) Assimilation du nitrate en cycles diurnaux par Phaeodactylum tricornutum. Plant Physiol. Biochem. 30, 665-673. Materassi R., Tredici M. R., Milicia F., Sili C., Pelosi E., Vincenzini M., Torzillo G., Balloni W., Florenzano G. and Wagener K. (1984) Development of a production size system for the mass culture of marine microalgae. In Energy from Biomass (Edited by Palz W. and Pirrwitz D.), pp. 150-158. D. Reidel Publ. Co., Dordrecht. Megharaj M., Pearson H. W. and Venkateswarlu K. (1992) Removal of nitrogen and phosphorus by immobilized cells of Chlorella vulgaris and Scenedesmus bijugatus isolated from soil. Enzyme Microb. Technol. 14, 656-658. Mohn F. H. (1988) Harvesting of micro-algal biomass. In Micro-algal Biotechnology (Edited by Borowitzka M. A. and Borowitzka L. J.), pp. 395-414. Cambridge University Press, Cambridge. Nalewajko C. and Lee K. (1983) Light stimulation of phosphate uptake in marine phytoplankton. Mar. Biol. 74, 9-15. Oswald W. J. (1988) Large-scale culture systems (engineering aspects). In Micro-algal Biotechnology (Edited by Borowitzka M. A. and Borowitzka L. J.), pp. 357-394. Cambridge University Press, Cambridge. Parsons T. R., Yoshiaki M. and Lalli C. M. (1984) A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford. Richmond A. (1986) Outdoor mass cultures of microalgae. In CRC Handbook of Microalgal Mass Culture (Edited by Richmond A.), pp. 285-329. CRC Press Inc., Boca Raton, Florida. Richmond A., Boussiba S., Vonshak A. and Kopel R. (1993) A new tubular reactor for mass production of microalgae outdoors. J. Appl. Phycol. 5, 327-332. Robinson P. K., Reeve J. O. and Goulding K. H. (1988) Kinetics of phosphorus uptake by immobilized Chlorella. Biotechnol. Lett. 10, 17-20. Roubicek R. V., Patton J. T., McCorkle J. H. and Rakow A. L. (1985) Novel Pond Designs. In Algal Biomass Technologies: An Interdisciplinary Perspective (Edited by Barclay W. and Mclntosh R.), pp. 218-221. Ryther J. H. (1983) The Evolution of Integrated Aquaculture Systems. J. Worm Maricult. Soc. 14, 473-484. Snell, F. D. (1981). Photometric and Fluorometric Methods of Analysis: Non-metals, pp. 585-586. John Wiley & Sons, New York. Soeder C. J. (1980) Massive cultivation of microalgae: results and prospects. Hydrobiologica 72, 197-209. Soeder C. J. and Hegewald E. (1988) Scenedesmus. In Micro-algal Biotechnology (Edited by Borowitzka M. A. and Borowitzka L. J.), pp. 59-84. Cambridge University Press, Cambridge. Torzillo G., Pushparaj B., Bocci F., Balloni W., Materassi R. and Florenzano G. (1986) Production of Spirulina biomass in closed photobioreactors. Biomass 11, 61-74. Weissman J. C. Eisenberg D. M. and Benemann J. R. (1978) Nutrient removal from wastewater. Biotechnol. Bioeng. Symp. 8, 229-316.
Wat. Res. Vol. 31, No. 7, pp. 1701-1707, 1997 © 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0043-1354/97$17.00+ 0.00
WASTEWATER N U T R I E N T REMOVAL BY MARINE MICROALGAE GROWN ON A C O R R U G A T E D RACEWAY RUPERT J. CRAGGSI'~@, PAUL J. McAULEY t and VALERIE J. SMITH 2. ~Harold Mitchell Building and 2Gatty Marine Laboratory, School of Biological and Medical Sciences, University of St Andrews, St Andrews, Fife KY16 9TH, Scotland
(Received July 1995; accepted in revised form April 1996) Abstract--Two marine microalgal isolates from a sewage outfall site in St Andrews Bay, Scotland were cultured on corrugated raceways (2.5 m long, 0.2 m wide) to determine their ability to remove ammonium and orthophosphate from wastewater diluted with seawater. The isolates (SA91B33, preliminarily identified as Phaeodactylum tricornutum, and SA91CY1, Oscillatoria sp.) both have surface-adherent properties and were selected from 102 isolates for optimal nutrient removal and culture dominance in both batch and continuous culture on wastewater under controlled environmental conditions. Wastewater (primary sewage effluent)was diluted 1: 1 with sterile seawater and continuously added to raceway algal cultures grown under ambient conditions. Nutrient concentrations in the diluted wastewater influent and in the effluent from the raceways were measured daily. Both isolates remained unialgal during the four month culture period and continuously removed 100% of ammonium and orthophosphate from the wastewater. Nitrite and nitrate levelsin both influent and effluentwere negligible.Measurement of influent and effluentnutrient concentrations over 24 h showed ammonium and orthophosphate removal remained unaltered during the diurnal cycle. These results indicate the potential for using microalgal species grown on raceway ,;urfaces for wastewater treatment. © 1997 Elsevier Science Ltd
Key words--ammonium, marine, microalgae, orthophosphate, raceway, wastewater
INTRODUCTION It is generally recognized that microalgae play an important role in the self-purification of natural waters (Soeder, 1980). Because microalgae use solar energy to supply oxygen required for aerobic degradation and recycle the nutrients responsible for eutrophication into potentially valuable biomass (de la Noiie and De Pauw, 1988; Oswald 1988), they may offer an inexpensiw,• alternative to conventional forms of tertiary wastewater treatment. The design, construction and operation of microalgal wastewater treatment systems has been influenced by two major factors. First, the need for adequate mixing to maintain efficient treatment (de la Noiie and De Pauw, 19:g8), and second, the difficulty of separating the microscopic algal biomass from the treated effluent efficiently and economically in order to complete the process (Oswald, 1988). Thorough mixing of microalgal ponds ensures homogeneous conditions by avoiding sedimentation *Author to whom all correspondence should be addressed. [Tel.: (01334) 463 368; Fax: (01334) 463 366; Email: [email protected]]. tPresent address: EEHSL, University of California at Berkeley, 1301 $o. 46th St., Richmond, CA 94804, U.S.A.
of algal cells and increasing the efficiency of light utilization in the culture. Mixing also prevents thermal stratification and the occurrence of nutrient and pH gradients, supersaturation of oxygen and the depletion of carbon dioxide at the pond surface, and anaerobic conditions on the bottom (Richmond, 1986; Oswald, 1988). Various systems have been designed to enhance the mixing of algal mass cultures, including paddlewheels in shallow circulating raceways (Oswald, 1988), air-water lifts (Laws et al., 1983) or mixing boards in rectangular ponds (Materassi et al., 1984), and pumps to recirculate the culture in sloping culture units (Roubicek et al., 1985). More recently, systems have been designed which circulate the algal culture within closed plastic photobioreactors with a high surface area to volume ratio (Richmond et al., 1993). Although high levels of treatment may be achieved using these types of apparatus, the costs of construction and operation severely restrict their economic use in microalgal wastewater treatment systems. In addition, a major cost in operation of these systems is that of removal of microalgae from the treated effluent. Since the BOD of the algal biomass in the effluent from many microalgal treatment ponds is usually above the discharge standard for natural water bodies (Ryther, 1983), the
1701
1702
R. J. Craggs et al.
algal biomass must be removed before the effluent is discharged. By their very nature, microalgae are small (>20/xm). This, coupled with the facts that culture densities are relatively low (2-6 g m-3), most species have a specific gravity slightly greater than that of water, and many have a strongly negative charge on their surface which keeps them dispersed, makes harvesting difficult and costly (Oswald, 1988). Various harvesting methods available, including floatation, sedimentation, precipitation, centrifugation, filtration and flocculation, have been extensively reviewed. The most successful techniques are centrifugation, filtration and flocculation (Mohn, 1988), but incompatibility between efficiency of the harvest methods and their cost-effectiveness restricts their application in microalgal wastewater treatment (Benemann et al., 1980). Although immobilization of algae in beads of carageenan or alginate offers a novel and elegant way to recover the algal biomass, because the beads sediment in a few seconds and can be used repeatedly (de la Notie et al., 1992), the cost of the immobilization substrate may be prohibitive for use in large-scale systems. An alternative approach is to capitalize on natural characteristics of the microalgae in the design of treatment apparatus. We have identified two endemic isolates of microalgae which continually remove high concentrations of ammonium and orthophosphate from 1:1 diluted wastewater, and which possess the ability to aggregate and adhere to the culture apparatus (Craggs, 1994). Surface adherence is a useful property in two respects. First, there is no need to separate the algal biomass from the pond effluent, since the algae remain attached to the surface of the culture apparatus, leaving a treated effluent which is virtually algal free. Second, by inclining the surface of the apparatus, wastewater will trickle through the adhered algal biomass without the need for any additional mechanical mixing or recirculation. An apparatus designed specifically for these adherent species, with a high surface area to volume ratio, may therefore require lower capital investment and lower operational costs. In this paper, we describe the construction and operation of such an apparatus for wastewater treatment by two marine microalgal isolates, and monitoring of efficiency of removal of nutrients over 16 weeks.
MATERIALS AND METHODS
Algae Two species of marine microalgae, SA91 B33 (preliminarily identified as a colonial strain of the oval morphotype of Phaeodactylum tricornutum) and SA91CY 1 (Oscillatoria sp.) were both isolated from a plankton tow in April 1991, St Andrews Bay, Fife, Scotland, and were selected from 102 isolates for optimal nutrient removal and culture dominance in both batch and continuous culture on wastewater under controlled environmental conditions (Craggs, 1994). Both isolates were maintained in 11 continuous cultures with a 2d residence time at 13-18°C in ambient light supplemented by three mercury vapour lamps (Trulite
400W, MBF/U, GEC, London, U.K.), 12:12 light:dark, 150-170/zmol photon m--' s-L Cultures were grown on seawater diluted 1 : I with primary sewage effluent collected from the effluent of the St Andrews treatment facility, and provided suspensions of microalgae in exponential growth phase suitable for seeding the raceways. Apparatus Corrugated raceways were constructed from four 20 cm wide strips of clear corrugated polyethylene sheeting. The strips were joined by overlapping two corrugations of the higher strip over two corrugations of the lower strip and sealing with silicone sealant (Vallance Ltd, Birmingham, England). Side walls of clear plastic sheet were attached and sealed with the sealant to produce a water tight continuous raceway 2.5 m long. The apparatus was enclosed in a wooden frame for support. The corrugation increased the surface area of the raceway and formed discrete microponds. Upon inclination of the raceway at a slight angle (5°), medium added at the top overflowed from one micro-pond to the next; this slowed the flow of wastewater down the raceway and reduced problems of desiccation by increasing the holding capacity. Each raceway contained 78 micro-ponds and had a total holding capacity of 2.1 1 and surface area of 0.6 mL The raceway was protected from rain and falling debris by a glass cover, which was raised to facilitate ventilation. Influent and effluent tubes passed through the end walls of the raceway. Wastewater used to test efficiency of nutrient removal was unfiltered primary sewage effluent from St Andrews treatment facility, primarily from domestic sources, diluted 1:I with sterile seawater. Average nutrient composition and physical properties of the effluent are given in Craggs et al. (1994). The diluted wastewater was pumped peristaltically (Watson-Marlow Ltd, Falmouth, England; model 502S) to each raceway from a 10001 fibre-glass storage tank and dripped from the influent tube into the first micro-pond. Medium trickled from one micro-pond to the next and finally out of the raceway into a collection vessel, through the effluent tube. A schematic diagram of a complete continuous corrugated raceway culture unit is shown in Fig. 1. Operation For one week, each raceway (one for each isolate) was seeded daily with 500 ml of algal culture. At the start of the treatment period (day 0), the wastewater was added continuously at a pumping rate of 41day -~ (0.5 day residence time). The raceways were run as continuous cultures for 16 weeks (July 14th-November 2nd 1993), during which maximum ambient temperatures varied from 23°C to 4°C. Twenty millilitre samples of the 1:1 diluted wastewater influent and corrugated raceway effluent were collected daily at 2.00 pm for measurement of nutrient concentrations (ammonium, nitrite, nitrate and orthophosphate), pH and microscopic examination. Nutrient concentrations were measured against Milli-Q (M-Q) water blanks by standard colorimetric methods scaled down for use in flat-bottomed 96-well microtitre plates (Dynatech, M29A, Billingshurst, Sussex, England) with a plate-reader spectrophotometer (Dynatech, MR5000). Three 300 #1 aliquots of each of the influent and effluent samples were pipetted into the wells of a plate for centrifugation (260g, 10 min). The required volumes of supernatant were then pipetted into clean micotitre plates for nutrient measurement. N-NH4 + was measured by the method of Parsons et al. (1984) using standards of 293.8mmolm -3 (NH,)2SO4, and read at 630nm (final volume 300/~1). The samples were diluted with M-Q water (1:4) prior to addition of reagents. N-NOz- was determined by the method of Snell (1981) using standards of 99.9 mmol m -3 (final volume 300/~1). N-NO3- was determined using Szechrome (NAS) reagent (Park Scientific Ltd,
Microalgal wastewater nutrient removal Northampton, England) in a scaled down reaction (final volume 275/A), using 71.4mmol m-3 KNO3 standards. P-PO43- analysis was by the method of Henkel et al. (1988), using 30.0 mmol m -3 K_~HPO4standards. The assay was modified by substituting the polyvinyl alcohol for M-Q water and diluting the mixed reagents 1: 1 with M-Q water before addition to the samples (final volume 250/~1). Medium pH was determined using a combination pH electrode (Russell, type CWL) read from a digital pH meter (Philips, type PW9409) and calibrated with pH 7 and pH 10 buffers. The purity of the cultures of the two microalgal isolates was checked using a compound microscope (Nikon Labophot) with x 10 and x40 objectives. Light intensity
Ambient light intensity was measured continuously over the period of investigation using a quantum photometer (QI01-4; Macam Phc,tometrics Ltd., Livingston, Scotland) with a PAR sensor. RESULTS The influent and effluent nutrient concentrations during the first 35 days of operation of the corrugated raceways using tile two isolates, SA91CY1 and SA91B33, grown under ambient conditions, are shown in Fig. 2. For both isolates, the reduction in effluent nutrient concentrations to zero corresponded to the growth of a dense algal culture on the surface of the raceways. ]isolate SA91CY1 took longer to establish a uniforra covering than SA91B33, which may account for the higher initial nutrient removal by the latter. Once established, raceway cultures of both isolates totally removed ammonium and orthophosphate from the seawater:wastewater mix, although complete removal of ammonium was achieved before that of orthophosphate. An overnight blockage in the influent tube of diluted wastewater between culture
1703
days 25 and 26 caused both floways to partially dry out and some of the algal cultures to die. This resulted in an increase of the orthophosphate concentration of the effluents between days 26 and 29 (Fig. 2(b)), although the ammonium concentration of the effluents remained unchanged (Fig. 2(a)). During the 14 day period (days 36-50) when detailed measurements were taken, influent nutrient concentrations remained uniform at 497.7 + 60.7 mmol N m -3 and 76.2 + 4.9 mmol P m -3, and both species continued to remove 100% of ammonium and orthophosphate (Table 1). The mean pHs of the effluent from the raceway cultures were 9.8 + 0 . 2 and 9.7 +0.1 for SA91CY1 and SA91B33, respectively, over the 14 days of detailed measurement, although that of the influent was only 7.6 _ 0.1 (Table 1). To determine whether N-NH~ ÷ was actually removed from the culture and not converted to nitrite and nitrate, the concentrations of these nutrients were also measured. Nitrite concentration (2.8 + 0.9 mmol N m -3) in the influent was less than 1% of the N-NH4 ÷ concentration (497.7 + 60.7 mmol N m-3), while nitrate was absent. Nitrite was also removed by the raceway cultures while nitrate remained absent (Table 1). To determine the effect of diurnal variation in light intensity on nutrient removal, nutrient concentrations in the diluted wastewater influent and effluent from the two corrugated raceways were measured over the 24 h diurnal cycle (Fig. 3 and Table 2) 48-49 days after initial seeding. Both ammonium and orthophosphate removal remained at 100% over the light:dark cycle (Fig. 3(a) and (d)). Nitrite and nitrate concentrations were much lower
3
\
Fig. 1. Schematic diagram of a complete continuous culture unit. 1: diluted wastewater supply tank; 2: peristaltic pump; 3: wastewater inflow;4: corrugated raceway; 5: algal culture within micro-pond; 6: glass rain cover; 7: outflow; 8: treated wastewater.
1704
R . J . C r a g g s et al.
a.
. - - T
0
5
T
T
10
15
. . . . .
T
T
T
T
20
25
30
35
,,~ 200 b. 150
.
100
*~'~N~
0 0
5
10
15
20
25
30
35
Incubation T i m e (d) Fig. 2. (a) A m m o n i u m
a n d (b) o r t h o p h o s p h a t e c o n c e n t r a t i o n s in the 1:1 d i l u t e d w a s t e w a t e r influent
( • - • ) and effluents of the corrugated raceways of isolate SA91B33 (•-Ill) and isolate SA91CY 1 ( • - O ) over 35 days culture. Nutrient values are means ___s.d. of triplicate samples. The s.d. may be too small to be seen.
than that of ammonium, and showed little change over the period of measurement, with changes in the effluent reflecting those of the influent (Fig. 3(a)-(c)). Ammonium and orthophosphate removal remained at 100% during operation of raceway cultures of the two isolates for a further 8 weeks, and microscopical examination showed that the raceways remained unialgai. During this time, the concentrations of nitrite and nitrate relative to ammonium were less than 1%. DISCUSSION
This paper describes the abilities of two endemic marine microalgal isolates, a cyanobacterium (SA91CY1) and a diatom (SA91B33), to remove nutrients from diluted wastewater in a specially designed corrugated raceway under ambient summer and autumn conditions. Both isolates were found to remain in monoculture and to continuously remove all the ammonium and orthophosphate from wastewater over the 16 weeks of the experiment,
demonstrating the stability of the algal culture on the raceway surfaces. Although a control raceway without an algal culture was not operated in this study, the reduction of the nutrient concentrations in the effluent from the raceways during the establishment of cultures of both isolates (Fig. 2) indicated that nutrient removal was due to the microalgae. Nutrient removal by the raceway algal cultures was probably not just by uptake and assimilation into algal biomass. The pH of the raceway effluents was high ( > 9.5) which results from bicarbonate uptake for photosynthesis when the algae are carbon limited. Ammonium may have been volatilized and orthophosphate precipitated, both of which are known to occur at elevated pH (>8.5) (Soeder and Hegewald, 1988). It is unlikely that ammonium was nitrified to nitrite and nitrate, since neither of their effluent concentrations increased from the influent values and the aerobic nature of the photosynthetic algal biomass would have probably prevented denitrification of the nitrate to nitrogen gas from occurring. However, further research is required
Table I. Percentage removal of ammonium, nitrite and orthophosphate, and effluent pH, from corrugated raceways of two marine microalgal isolates on 1: I diluted wastewater. Values are means _+ s.d. over 14 days of culture (n = 14). Influent concentrations were 497.7 4- 60.7 mmol m -3 N-NH4 +, 2.8 _+ 0.9 mmol m-3 N-NO2-, 76.2 + 4.9 P-PO4~-, and influent pH was 7.6 + 0.1 (range 7.73-7.50) Algal isolate
% N-NH4 + removal
% N-NO~removal
% P-P043removal
Mean + s.d.
pH Range
SA91CYI SA91B33
100.0 + 0.4 100.0 _+ 0.2
51.7 + 10.7 82.9 _+ 7.1
99.4 -I- 0.8 100.0 + 0.3
9.8 + 0.2 9.7 + 0.1
10.0--9.4 9.9-9.5
a.
,001 0 0
4
8
12
16
20
24
2 310
b.
0
4
8
12
16
20
24
10'1
c.
6 4
2
t
0
.
4
I
8
12
16
20
:1 Or
~
i' 1 0
24
J 4
8
12 16 Incubation Time (h)
I,r---. 20
Fig. 3. (a) Ammonium, (b) nitrite, (c) nitrate and (d) orthophosphate concentrations in the 1:1 diluted wastewater influent ( 0 - @ ) and effluents of the corrugated raceways of isolate SA91 B33 ( l - i ) and isolate SA91CYI ( 0 - 0 ) in relation to light intensity (e) over 24 h culture. Nutrient values are means _ s.d. of triplicate samples. The s.d. may be too small to be seen. 1705
24
1706
R.J. Craggs et al.
on the mechanisms of nutrient removal by the raceway algal culture. The sequence of nutrient removal in the raceways, with ammonium removed before the orthophosphate, is representative of high-rate ponds, which function as nitrogen-limited systems (Weissman et al., 1978). The increase in the orthophosphate concentration of the effluents following the partial drying out of the two cultures (Fig. 2) may have been due to release from the dead portion of the algal culture, while any released ammonium was possibly assimilated by the remaining algal biomass, since nitrogen was limited. The efficiency of nutrient removal of established raceway cultures of both isolates (100%) is comparable to the efficiencies recorded for algal cultures by other workers (Robinson et al., 1988; Megharaj et al., 1992), even though the influent nutrient concentrations in the present study were over twice those used by these authors. Further, except when the inflow pipe was temporarily blocked, there was no reduction in removal of orthophosphate by the raceway cultures over the course of this study, although this has been consistently noted in earlier studies of continuous cultures of marine microalgae grown on seawater diluted with wastewater (Dunstan and Tenore, 1972; Craggs, 1994) and in continuous cultures of freshwater microalgae grown in artificial medium (Robinson et al., 1988; Megharaj et al., 1992). Although the ability to assimilate ammonium in the dark has been shown to vary from species to species (Eppley et al., 1971), the continued high rates of ammonium and orthophosphate removal by both isolates during the dark period of the daily light:dark cycle is in agreement with more recent findings that removal of nitrate, nitrite and orthophosphate can be unaffected by the light:dark cycle at high culture densities (Nalewajko and Lee, 1983; Marsot et al., 1992). One of the main problems for the mass culture of microalgae is contamination by unwanted species (Richmond, 1986). The cultures of both isolates remained unialgal in open continuous culture under ambient conditions, suggesting dominance over the algal species which naturally occur in wastewater. These two endemic marine isolates of St Andrews Bay could possibly have been tolerant of or adapted to the salinities of estuarine conditions which may have given them a competitive advantage over freshwater species occurring in the wastewater (Craggs et al., 1994). Many conditions govern the competitive advantage of one species over another Table 2. Percentage removal o f a m m o n i u m , nitrite and orthophosphate from corrugated raceways o f two marine microalgal isolates on 1:1 diluted wastewater over 2 4 h ( n = 12). The influent concentrations were 575.1 __. 16.3 m m o l m -3 N-NH:, 1.5 + 0.4 m m o l m-3 N - N O c , 86.6 5:6.0 P-PO43Algae isolate
% N-NH4 + removal
% N-NO2removal
% P-PO4 3removal
SA91CY1 SA91B33
100.0 _ 0.1 100.0 _ 0.1
16.9 + 40.0 50.8 5:20.0
98.3 + 2.5 100.0 + 0.2
(Goldman et al., 1982). Although the results of laboratory studies investigating competition between species are variable (Dortch, 1990) and there is some debate as to whether laboratory studies are applicable to the field (Grover, 1991), the present research has shown these two isolates to be dominant in culture under all experimental conditions from small-scale batch cultures to large-scale open continuous cultures (Craggs, 1994). Both bacillariophyceaen and cyanophyceaen species have been found to dominate phytoplankton blooms in marine waters and mass cultures (Marshall and Orr, 1927; Torzillo et al., 1986). The ability of these marine species to remain in unialgal culture may enable a microalgal wastewater treatment process to operate more efficiently by optimizing conditions for the growth of a particular species. Nutrient removal (100%) by both isolates was higher when cultured on raceways in this study than when these and other isolates were cultured in small ponds using the same influent (44.2-98.8%) (Craggs, 1994; Craggs et al., 1995). This may have been due to a number of complementary factors. There was little wash-out in raceway cultures, because the algae adhered to the raceway surface, while algal biomass was continuously lost in the outflow of the continuous cultures in ponds. Raceways have a higher surface area than pond systems, thus continuously exposing a higher proportion of the culture to light. Furthermore, the raceways were operated in a linear configuration so that treated and untreated wastewater never came into contact. The high nutrient removal achieved by the isolates cultured on the corrugated raceways demonstrates the potential advantage of designing microalgal treatment systems to suit particular algal species, rather than relying on a mixed assemblage of microalgal species with variable nutrient removal rates to grow up which occurs with most systems in use today (Oswald, 1988). Corrugated raceways have two further advantages which reduce the operational costs of the system. Raceways do not require mechanical mixing and may enable simple and economic separation of the algal biomass from the treated effluent by simply scraping it from the drained surface. Although sloping culture units have previously been used for microalgal culture (Roubicek et al., 1985), they have not been operated as linear systems using adherent microalgal species. Efficient nutrient removal by marine microalgal systems could be used to reduce the concentrations of eutrophication-causing nutrients discharged to coastal waters. CONCLUSIONS
(1) Two endemic isolates of cultures of marine microalgae, grown in continuous culture on raceways, were able to remain in unialgal culture and to treat wastewater during summer and autumn conditions in Scotland.
Microalgal wastewater nutrient removal (2) Removal of a m m o n i u m and orthophosphate from a !:1 wastewater:seawater mix was complete and continuous, antt was not affected by the diurnal cycle. (3) By exploiting the adherent properties of these isolates, the corrugated raceway microalgal treatment system does not require mixing, and may enable simple mechanical harvesting of the algal biomass. (4) This study indicates the potential for the use of marine microalgae for nutrient removal from wastewaters even in temperate areas. Acknowledgements--This work was supported by a Science and Engineering Research Council studentship to RJC, and by the Royal Society. The authors are particularly grateful to Dr Howard Fallowfield and Professor W. J. Oswald for their encouragement and advice during this research. We are indebted to the technical staff, especially Mr Harry Hodge, for their assistance throughout this study, and to Andrew Whiston for his help with algal identification.
REFERENCES
Benemann J. R., I~oopman B. L., Weissman J. C., Eisenberg D. and Goebel R. (1980) Development of microalgae harvesting and high-rate pond technologies in California. In Algae Biomass: Production and Use (Edited by Shelef G. and Soeder C. J.), pp. 457-495. Elsevier Biomedical Press, Amsterdam. Craggs R. J. (1994) Wastewater Nutrient Removal by Marine Microalgae. Ph.D. Thesis, University of St Andrews. Craggs R. J., McAuley P. J. and Smith V. J. (1994) Batch culture screening of marine microalgal nutrient removal from primary sewage effluent. Hydrobiologia 288, 157-166. Craggs R. J., Smith V. J. and McAuley P. J. (1995) Wastewater nutrient removal by marine micro-algae cultured under ambient conditions in mini-ponds. Wat. Sci. Technol. 31, 151-160. De la Nolle J. and De Pauw N. (1988) The potential of microalgal biotechnology: a review of production and uses of microalgae Biotech. Adv. 6, 725-770. De la Noiie J., Laibert6 G. and Proulx D. (1992) Algae and waste water. J. Appl. Phycol. 4, 247-254. Dortch Q. (1990) Review of the interactions between ammonium and nitrate uptake in phytoplankton. Mar. Ecol. Prog. Ser. 611, 183-201. Dunstan W. M. and Tenore K. R. (1972) Intensive outdoor culture of marine phytoplankton enriched with treated sewage effluent. Aquaculture 1, 181-192. Eppley R. W., Rogers J. N. and McCarthy J. J. (1971) Light/dark periodicity in nitrogen assimilation of the marine phytoplankters Skeletonema costatum and Coccolithus huxleyi in N-limited chemostat culture. J. Phycol. 2, 150-154. Goldman J. C., Azov Y., Riley C. B. and Dennett M. R. (1982) The effect of pH in intensive microalgal cultures. II. Species competition. J. Exp. Mar. Biol. Ecol. 57, 15-24. Grover J. P. (1991) Dynamics of competition among microalgae in variable environments: experimental tests of alternative models. Oikos 62, 231-243. Henkel, R. D., Vandeberg, J. L., Walsh, R. A. (1988)
1707
A microassay for ATPase. Anal. Biochem. 169, 312-318. Laws E. A., Terry K. L., Wickman J. and Chalup M. S. (1983) A simple algal production system designed to utilize the flashing light effect. Biotechnol. Bioeng. 25, 2319-2336. Marshall S. M. and Orr A. P. (1927) The relation of the plankton to some chemical and physical factors in the Clyde Sea area. J. Mar. Biol. Assoc. 14, 837-868, Marsot P., Mouhri K. and Cembella A. D. (1992) Assimilation du nitrate en cycles diurnaux par Phaeodactylum tricornutum. Plant Physiol. Biochem. 30, 665-673. Materassi R., Tredici M. R., Milicia F., Sili C., Pelosi E., Vincenzini M., Torzillo G., Balloni W., Florenzano G. and Wagener K. (1984) Development of a production size system for the mass culture of marine microalgae. In Energy from Biomass (Edited by Palz W. and Pirrwitz D.), pp. 150-158. D. Reidel Publ. Co., Dordrecht. Megharaj M., Pearson H. W. and Venkateswarlu K. (1992) Removal of nitrogen and phosphorus by immobilized cells of Chlorella vulgaris and Scenedesmus bijugatus isolated from soil. Enzyme Microb. Technol. 14, 656-658. Mohn F. H. (1988) Harvesting of micro-algal biomass. In Micro-algal Biotechnology (Edited by Borowitzka M. A. and Borowitzka L. J.), pp. 395-414. Cambridge University Press, Cambridge. Nalewajko C. and Lee K. (1983) Light stimulation of phosphate uptake in marine phytoplankton. Mar. Biol. 74, 9-15. Oswald W. J. (1988) Large-scale culture systems (engineering aspects). In Micro-algal Biotechnology (Edited by Borowitzka M. A. and Borowitzka L. J.), pp. 357-394. Cambridge University Press, Cambridge. Parsons T. R., Yoshiaki M. and Lalli C. M. (1984) A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford. Richmond A. (1986) Outdoor mass cultures of microalgae. In CRC Handbook of Microalgal Mass Culture (Edited by Richmond A.), pp. 285-329. CRC Press Inc., Boca Raton, Florida. Richmond A., Boussiba S., Vonshak A. and Kopel R. (1993) A new tubular reactor for mass production of microalgae outdoors. J. Appl. Phycol. 5, 327-332. Robinson P. K., Reeve J. O. and Goulding K. H. (1988) Kinetics of phosphorus uptake by immobilized Chlorella. Biotechnol. Lett. 10, 17-20. Roubicek R. V., Patton J. T., McCorkle J. H. and Rakow A. L. (1985) Novel Pond Designs. In Algal Biomass Technologies: An Interdisciplinary Perspective (Edited by Barclay W. and Mclntosh R.), pp. 218-221. Ryther J. H. (1983) The Evolution of Integrated Aquaculture Systems. J. Worm Maricult. Soc. 14, 473-484. Snell, F. D. (1981). Photometric and Fluorometric Methods of Analysis: Non-metals, pp. 585-586. John Wiley & Sons, New York. Soeder C. J. (1980) Massive cultivation of microalgae: results and prospects. Hydrobiologica 72, 197-209. Soeder C. J. and Hegewald E. (1988) Scenedesmus. In Micro-algal Biotechnology (Edited by Borowitzka M. A. and Borowitzka L. J.), pp. 59-84. Cambridge University Press, Cambridge. Torzillo G., Pushparaj B., Bocci F., Balloni W., Materassi R. and Florenzano G. (1986) Production of Spirulina biomass in closed photobioreactors. Biomass 11, 61-74. Weissman J. C. Eisenberg D. M. and Benemann J. R. (1978) Nutrient removal from wastewater. Biotechnol. Bioeng. Symp. 8, 229-316.