Journal of Chemical Technology and Biotechnology

J Chem Technol Biotechnol 76:11471153 (online: 2001) DOI: 10.1002/jctb.497

Optimisation of the biological treatment of hypersaline wastewater from Dunaliella salina carotenogenesis
Carla A Santos,1 Ana M Vieira,2 Helena L Fernandes,2 Jose A Empis1 and lio Ju M Novais1*
1 2

gica e Qumica, Av Rovisco Pais, 1049-001 Lisbon, Portugal cnico, Centro de Engenharia Biolo Instituto Superior Te veis, Edicio G, Estrada do Pac do Lumiar, Instituto Nacional de Engenharia e Tecnologia Industrial, Departamento de Energias Renova o 22, 1649-038 Lisbon, Portugal

Abstract: The reutilisation of Dunaliella salina carotenogenesis medium, after microalgal biomass separation by centrifugation, was assessed. The wastewater had an NaCl concentration between 174 g dm3 and 254 g dm3 and an average total organic matter concentration of 1540 mg dm3 ash-free dry weight, of which 41% (w/v) was glycerol. The biological treatment was established at laboratory scale and batch operations used halophilic bacteria from the wastewater itself. The wastewater was 3 supplemented with NH,PO4 , K and Mg2 ions to enhance growth. The effect of each ion added per 4 se was initially investigated and a response surface methodology (RSM) used to identify the optimal conditions for maximisation of glycerol removal from the wastewater, which was considered to be the main objective. Addition of NH ions alone achieved 79% glycerol removal compared with only 59% in 4 the absence of supplement, after 8 days incubation. The combined addition of ions ([NaCl] = 214 g dm3, 2 3 3 [Mg ] = 114 mg dm , [K ] = 131 mg dm3, [NH] = 113 mg dm3, [PO4 ] = 40 mg dm3) increased glycer4 ol removal from the wastewater such that, after 2 days incubation, no residual glycerol was apparent in cultures. These ion combinations enabled the halophilic bacteria to efciently remove glycerol from the wastewater and consequently reduce organic matter. This treated wastewater should be appropriate for reutilisation as a carotenogenesis medium for b-carotene production from D salina. # 2001 Society of Chemical Industry

Keywords: halophilic bacteria; hypersaline wastewater; Dunaliella salina; wastewater treatment microorganisms has not been developed due to a lack of available information about the nutritional requirements4 and the biodegradation potential of these organisms.8 Ben-Amotz9 stated that D salina growth medium can be recycled, provided that the reused medium is treated biologically by reddish bacteria that naturally appear in the medium, but gave no details. In natural environments, such as salt crystallisation ponds and some salty lakes, halophilic bacteria may be found seasonally, imparting a reddish hue. These halophilic bacteria are heterotrophs and can be classied as slight halophiles (13% NaCl), moderate halophiles (315%) and extreme halophiles (1530%) based upon the salt requirements for their optimal growth.10 Extreme halophiles do not grow below 100 g dm3 NaCl. In habitats containing more than 10% (w/v) salts, moderate and extreme halophilic bacteria constitute the greatest proportion of the total microbial population.11 The former exhibit great metabolic diversity

INTRODUCTION

The wastewater resulting from b-carotene production by the microalga Dunaliella salina has an ion concentration higher than seawater and is therefore classied as hypersaline.1 The cost of growth and carotenogenesis media represents about one-third of the total intensive production costs of D salina biomass.2 Recycling these culture media lowers production costs and attenuates the environmental impact of the industrial process. In order to reutilise the culture medium for the carotenogenesis of D salina, it is necessary to minimise turbidity, as this can diminish the yield of algal carotenoids.3 Turbidity mainly results from cellular lysis occurring during centrifugation. The limiting effects of salt on the biological treatment of wastewater have been described previously.47 These limitations may be overcome if conventional mixed sludge is replaced by microorganisms that are well adapted to hypersaline media. Biological treatment of wastewater with halophilic

cnico, Centro de Engenharia Biolo gica e Qu mica, Av Rovisco Pais, 1049-001 lio * Correspondence to: Ju M Novais, Instituto Superior Te Lisbon, Portugal E-mail: julio.novais@ist.utl.pt Contract/grant sponsor: Fundacao para a Ciencia e Tecnologia; contract/grant number: BIO-1065 (Received 23 October 2000; revised version received 25 June 2001; accepted 3 July 2001)

# 2001 Society of Chemical Industry. J Chem Technol Biotechnol 02682575/2001/$30.00

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and the second grow on simple carbon compounds such as amino acids, sugars, organic acids and glycerol.1214 The nutritional characteristics of halophilic bacteria are inherently difcult to assess because the salt content of the growth medium affects nutrient requirements. In general, nutrient requirements become more complex as the salt concentration of the medium is increased.11 Some of these bacteria require Mg2 for cellular stabilisation and both NH4 and K 15 may also show this effect. Phosphorus has been suggested to be a limiting factor for growth.16,17 The metabolic and physiological characteristics discussed above indicate that these bacteria are plausible agents for the treatment of high salinity wastewater. The biotechnological potential of halophilic bacteria is already well known.18,19 Interest in the red pigment of halophilic archaebacteria, namely bacterioruberin, is increasing because of its antioxidant properties.2022 This paper describes the optimisation of conditions for biological removal of organic matter from the spent carotenogenesis medium of the microalga D salina, using halophilic bacteria normally present in the hypersaline wastewater.

230 g dm3 NaCl. After 6 days growth the microorganisms were inoculated in plates with a solid complex medium. These Petri dishes were placed in plastic bags that were sealed and incubated at 35 C. These microorganisms were characterised by microscopic observation and grown in complex medium containing 200 g dm3 NaCl at 35 C. Growth in the presence of 100 g dm3 NaCl was tested. Two dm3 of complex medium with 200 g dm3 NaCl were inoculated with the halophilic culture from plates and incubated at 35 C, 150 rpm. After 3 days growth cells were harvested by centrifugation for 4 min, at 5500 g with refrigeration at 3 C, in aseptic conditions. The pellet was resuspended in 100 cm3 of 200 g dm3 NaCl sterile solution to give the inoculum suspension.
Biological wastewater treatment Inoculum preparation

MATERIALS AND METHODS Wastewater

The cultivation of D salina for b-carotene production was performed at pilot-scale (5 dm3) in two stages, growth and carotenogenesis. After carotenogenesis the microalgae were harvested by centrifugation and the resulting supernatant uid was used as the wastewater requiring treatment. The carotenogenesis medium consisted of the residual growth medium after microalgae growth diluted three-fold with local tap water with added NaCl to 230 g dm3. The wastewater was characterised in terms of total, soluble and suspended solids (volatile and ash), lipid, protein, sugar, glycerol, phosphate, nitrate, nitrite and ammonium contents (see `Analytical methods').
Culture medium

A complex medium for isolation, cultivation and maintenance of extreme halophilic bacteria was used, with the following composition (per dm3): 5 g KCl, 5 g NH4Cl, 5 g MgCl2.6H2O and 5 g MgSO4.7H2O, 5 g Difco Yeast Extract and 2.5 g Difco Tryptone. Solid NaCl was added to the mineral salt solution to obtain the desired salt concentration and pH was adjusted to 7.3 with 1 mol dm3 NaOH. Mineral salts and carbon sources were autoclaved separately for 20 min at 120 C.23 Agar plates were made by adding 23 g dm3 Difco agar to the mineral salt solution before autoclaving.
Source of microorganisms

The wastewater treatment was performed at batch laboratory scale (500 cm3) after inoculation with the halophilic culture, varying the NaCl concentration and one ion concentration at a time. The following concentrations were tested: 174, 214 and 254 g dm3 NaCl, 43 and 168 mg dm3 NH4 as NH4Cl, 13 and 3 3 26 mg dm PO4 as K2HPO4, 98 and 196 mg dm3 K as KCl, 30 and 59 mg dm3 Mg2 as MgSO4.7H2O. After these initial runs the halophilic culture growth was performed, varying the four ions added. A response surface methodology (RSM)24 was used to study the combined effect of Mg2, K, NH4 and 3 3 PO4 , with an NaCl concentration of 214 g dm . The ion sources were the same as given above except that 3 PO4 was now supplied as Na2HPO4.2H2O to avoid K addition. For each run, 2 cm3 cell suspension was used to inoculate 250 cm3 of non-sterilised wastewater with added ions in 500 cm3 Erlenmeyer asks, and incubated at 35 C and 150 rpm. Growth was monitored by optical density at 520 nm. Samples (10 cm3) were taken for glycerol, ammonium and phosphate analyses at the start of incubation, during the stationary phase and at the end of the assay (8 days). Samples were centrifuged for 15 min at 16000 g in a Sigma 3MK refrigerated centrifuge and the supernatant uid stored at 18 C.
Experimental design and response surface methodology

The microorganisms came from a pilot-scale pond of D salina culture with 230 g dm3 NaCl. Samples of 2.5 cm3 were inoculated at 35 C in 25 cm3 of the liquid complex medium described above, containing
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A central composite design (CCD) of ve levels for four variables was applied.24 The variables concentrations were: Mg2 at 0, 23, 57, 91 and 114 mg dm3, K at 0, 27, 66, 104 and 131 mg dm3, NH4 at 0, 34, 3 3 84, 134 and 168 mg dm and PO4 at levels 0, 11, 26, 42 and 52 mg dm3. All results from the factorial design were used to calculate the individual ion effects and interactions between ions using Yate's algorithm.25 The results of glycerol removal and biomass
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production from each design were tted to the following empirical model using a least square regression analysis:
2 2 Y b0 b1 :X1 b2 :X2 bi :Xi . . . b11 :X1 b22 :X2

bii :Xi2 . . . b12 :X1 :X2 b13 :X1 :X3 bij :Xi :Xj where Y is the value of glycerol removal (or other property being optimised), i is the number of ions that are being varied, bij are the coefcients resulting from the tting and Xi is concentration of ion i. The surfaces obtained for glycerol removal were represented in a three-dimensional graph by allowing variation of two variables whilst keeping the other one constant at its central point.
Analytical methods

The average composition of the wastewater is given in Table 1. Glycerol, which was the major component of organic matter (41%), is a carbon source for halophilic bacteria and was taken as the indicator for organic matter. Ammonium and phosphate concentrations were low but could limit bacterial growth. The NaCl concentration varied between 174 and 254 g dm3.
Effect of ion addition

Salinity was measured with a portable refractometer (Atago (Japan), model S-28). Total solids and total suspended solids dried at 105 C and ash and volatile solids ignited at 550 C were determined according to the Standard Methods for the Analysis of Water and Wastewater.26 Total lipids were determined according to the method of Bligh and Dyer.27 Protein was determined with the Lowry method.28 Total sugars were determined by the phenolsulfuric method29 using a standard solution of glucose with NaCl in a concentration equal to that found in the samples, because NaCl modies the standard curve. Ammonium ions were determined using the blue indophenol method30 with equal NaCl concentration in samples and standards to eliminate NaCl interference. Phosphate was determined by Murphy and Riley's method,30 a specially adapted method for saline samples. Glycerol was measured using the nonspecic acetylacetone assay for saline samples.31 Nitrate was determined using the cadmium reduction method26 which had no interference by NaCl. Nitrite was determined by a colorimetric method26 that was free of NaCl interference. COD could not be determined because of chloride interference in the dichromate reux method,26 even when a chloride correction method was used.32

The growth of halophilic bacteria in wastewater was limited at each salinity tested (Table 2): from an initial value of 0.24 the OD increased to a stationary phase value of only 0.38 after 21 h for the low salinity culture and maintained stationary to the eighth day. The doubling time increased with increasing NaCl concentration and for the lowest concentration (174 g dm3) was about 17 h, indicating that the stationary phase was reached after less than two doubling periods of the initial culture. It was observed that increased NaCl concentration reduced biomass production as well as glycerol and protein removal. The poor growth of the halophilic culture could be a consequence of medium nutritional limitations that increased with salinity. Therefore nutrients suitable for halophilic bacteria growth (N, P, K and Mg) were added with the purpose of increasing growth because, as seen before, biomass production (OD variation) and glycerol removal had a similar evolution. Stationary phase was also reached after 21 h, except for those cultures supplemented with ammonium. The culture with 43 mg dm3 NH4 reached stationary phase after 7 days and the one with 168 mg dm3 was still growing at the end of the experiment, after 8 days incubation. All ions added were shown (Table 2) to increase biomass production but did not signicantly affect the doubling time. The presence of ammonium and 3 phosphate (except for PO4 at 26 mg dm3) increased glycerol removal. The presence of potassium ions reduced glycerol removal while magnesium ions had no effect. Ammonium ions did not reduce the nal
Table 1. Average composition of wastewater from the harvest of carotenogenic microalga Dunaliella salina

RESULTS AND DISCUSSION

The mixed halophilic culture obtained and maintained in 200 g dm3 NaCl, was red, Gram negative, showed rod and spherical shaped cells, and was able to grow in the presence of 100 g dm3 NaCl. The red colour is due to the presence of bacterioruberin, which is only biosynthesised by the extreme halophile group of bacteria. These bacteria require at least 100 g dm3 NaCl for growth, so that moderate halophilic bacteria are also likely to be present. Further purication of each type of bacteria was not attempted because the mixed culture was satisfactory for wastewater treatment. The doubling time of the mixed culture in the complex medium described, containing 200 g dm3 NaCl at 35 C, was 3.9 h.
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Salinity Total solids Volatile solids (ash free dry weight) Total suspended solids Volatile suspended solids Glycerol Protein Sugars Lipids Ammonium Phosphate b-carotene Nitrate Nitrite
a

174 g dm3 NaCl 174.97 g dm3 1.54 g dm3 1.21 g dm3 0.071 g dm3 0.638 g dm3 0.060 g dm3 0.135 g dm3 glucose 0.130 g dm3 0.195 mg dm3 <0.475 mg dm3a 0.15 mg dm3 <1 mg dm3a <0.050 mg dm3a

Method detection limit.

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CA Santos et al Optical density variation (u OD 520 nm) 0.089 0.078 0.036 0.219 0.358 0.124 0.118 0.176 0.132 0.130 0.107 Protein removal (%) 25 19 13 1 1 26 37 41 45 37 34 Glycerol removal (%) 59 36 19 78 79 62 53 53 48 58 58 Doubling time (h) 17 20 29 19 17 14 16 18 18 18 18

NaCl (g dm3) 174 214 254 174 174 174 174 174 174 174 174

Added ions (mg dm3) 43 NH 4 168 NH 4

3 13 PO4 3 26 PO4

Table 2. Wastewater treatment after 8 days halophilic bacteria growth, varying NaCl concentration and one extra ion at a time

98 K 196 K 30 Mg2 59 Mg2

protein content, in contrast with phosphate, potassium and magnesium ions, that all contributed to reduce nal protein. Ammonium ions were not completely removed under the experimental conditions, which could explain why the protein content was unaltered. The halophilic bacteria preferentially used ammonium as nitrogen source; in the absence of ammonium protein was used as nitrogen source, this process was stimulated by added phosphate, potassium and magnesium. The major impact on glycerol removal (79%) was obtained with ammonium addition but a removal of 62% could also be obtained using phosphate ions (13 mg dm3), the latter also improving protein removal. The other added ions showed similar effects. Optimal conditions for organic matter removal may be achieved by adding a combination of ions.
Effect of combined addition of Mg2, K, NH4 and 3 PO4

Growth of the halophilic culture in wastewater (initial OD, 0.55) with 214 g dm3 NaCl and variable 3 concentrations of Mg2, K, NH4 , PO4 , commenced after a 5 h lag phase. The majority of the 27 cultures tested reached stationary phase after a further growth phase (39 h), with the OD increasing by 0.45. Culture density then decreased until the eighth day. During the growth phase the average doubling time was about 32 h, less than two doublings of the initial culture. In these experiments the observed OD increase was greater than in experiments involving addition of single ion species. The effect of the combined addition of Mg2, K, 3 NH4 and PO4 on glycerol removal, biomass production, ammonium and phosphate consumption and doubling time is shown in Table 3; the data were calculated from factorial design. The average glycerol removal reached 83% after 44 h. Ammonium and phosphate ions induced the largest increase in both glycerol removal and biomass production. The effect of ammonium ions was similar to that of phosphate ions. For example, an ammonium
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increase from 34 to 134 mg dm3 increased glycerol removal by 9% (2%). Magnesium and potassium also stimulated glycerol removal; potassium had a greater effect than magnesium in increasing biomass production. Generally, ion interaction effects on glycerol removal and biomass production were not observed (Table 3), except for the case of magnesium and phosphate on biomass production. Potassium and phosphate each or in combination inhibited ammonium removal, this latter being enhanced by the interaction between magnesium and potassium. Phosphate removal was enhanced by interaction between magnesium and phosphate. Doubling time was reduced by all ions tested, with magnesium showing the greatest effect, but was slightly increased by interaction between potassium and ammonium. The response surface depicting glycerol removal as a 3 function of Mg2, K, NH4 and PO4 is shown in Fig 1 for a relevant set of parameters. The marked effects 3 of NH4 and PO4 on increasing glycerol removal and the slight effect of K and Mg2, are apparent from the shape of the surfaces. The surfaces corresponding to the variations of NH4 and Mg2 concentrations and NH4 and K concentrations have a similar shape to the response surfaces of Fig 1(c) and 1(d) respectively, 3 where NH4 had the same behaviour as PO4 . Although these results are in accordance with those calculated from factorial design, the CCD data give greater insight since the graft relationship between glycerol removal and ions concentration was quadratic and not linear as predicted by factorial design. Optimal conditions for maximum glycerol removal after 2 days growth were [Mg2] = 114 mg dm3, [K] = 131 mg dm3, [NH4 ] = 113 mg dm3, 3 3 [PO4 ] = 40 mg dm at [NaCl] = 214 g dm3 (calculated by the numeric method) achieving 100% glycerol removal and an optical density increase of 0.88 OD. The residual nutrients with these initial conditions were, after 2 days, 66 mg NH4 dm3 and 22 mg 3 3 PO4 dm . From an engineering point of view, biomass
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3 Table 3. Effects of Mg2, K, NH and PO4 ions on glycerol removal, biomass production (OD variation), ammonium and phosphate removal and doubling time, 4 after 2 days growth, at 214 g dm3 NaCl

Estimated effect standard deviation Glycerol removal (%) Average: Main effects Mg2 K NH 4 3 PO4 Interaction effects Mg2 K Mg2 NH 4 3 Mg2 PO4 K NH 4 3 K PO4 3 NH PO4 4 83 1 4 2 4 2 9 2 10 2 1 2 0.04 2 1 2 0.2 2 1 2 1 2 Optical density variation (ODu520) 0.45 0.01 0.04 0.02 0.06 0.02 0.20 0.02 0.17 0.02 0.01 0.02 0.01 0.02 0.04 0.02 0.02 0.02 0.01 0.02 0.03 0.02 Ammonium removal (mg dm3) 37 1 2 2 9 2 10 2 5 2 7 2 1 2 0.04 2 8 2 1 2 8 2 Phosphate removal (mg dm3) 11.6 0.4 0.7 0.8 0.02 0.8 0.8 0.8 8 0.8 0.07 0.8 0.2 0.8 1 0.8 0.9 0.8 0.4 0.8 0.5 0.8 Doubling time (h) 32.4 0.2 3.8 0.4 2.1 0.4 1.3 0.4 1.9 0.4 0.6 0.4 0.5 0.4 0.2 0.4 1.2 0.4 1.1 0.4 0.4 0.4

production can be dened as a secondary maximisation target, as it is a source of high value chemicals such as the red pigments. Biomass production (RSM analysis not shown) varied in much the same way as glycerol removal, except that potassium had a greater effect than for glycerol removal. Under conditions ensuring essentially 100% glycerol removal, biomass production was also at its maximum value. This

similar variation of glycerol removal and biomass production suggests that glycerol may mainly have been used as a carbon source. The optimisation of glycerol removal was nonetheless achieved at high residual ammonium and phosphate ion concentrations of 66 mg dm3 and 22 mg dm3, respectively. The residual ammonium ion concentration needs to be controlled since the ion

3 Figure 1. Effect of ion concentration on glycerol removal after 2 days of culture growth with an NaCl concentration of 214g dm3: (a) effect of NH and PO4 4 3 3 concentration at 57mg dm3 Mg2 and 65mg dm3 K; (b) effect of Mg2 and K concentration at 84 mg dm3 NH and 26 mg dm3 PO4 ; (c) effect of PO4 and 4 3 Mg2 concentration at 65 mg dm3 K and 84mg dm3 NH; (d) effect of PO4 and K concentration at 57 mg dm3 Mg2 and 84mg dm3 NH. 4 4

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is toxic for algae at concentrations exceeding 90 mg NH3 dm3, particularly at pH values in excess of 8.33,34 Calculation of the percentage of non-dissociated ammonia demonstrated that it did not achieve toxic concentration under the optimal conditions identied in this study. Furthermore it would only be toxic if simultaneously the temperature was greater than 40 C, the salinity greater than 174 g dm3 NaCl, pH above 9 and NH4 in the carotenogenesis medium was greater than 168 mg dm3. Of these critical conditions, the latter is highly improbable and the others are unusual. For the purpose of medium reutilisation in D salina production, an alga having a 14 day production cycle, total medium reprocessing, including biological treatment and biomass separation, should occur during a similar period. Biological treatment of the medium required only about 2 days, allowing up to 12 days for sedimentation; on the basis of the laboratory observations reported here, this is adequate time to achieve medium recycling.

Ciencia e Tecnologia for the nancial support through PRAXIS XXI (Project BIO-1065) and for a grant to Carla A Santos.

REFERENCES
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CONCLUSIONS

Although halophilic culture growth in the hypersaline wastewater was limited, the primary objective of organic matter removal, measured as glycerol removal, was achieved. Wastewater supplementation with phosphate and ammonium ions separately, increased glycerol removal. The combined supplementation of wastewater with potassium, magnesium, phosphate and ammonium ions was even more efcient for glycerol removal. An increase in sodium chloride concentration (in the range of 174254 g dm3) decreased glycerol removal and biomass production, but this inuence can be overcome by adding other ions. Each ion species added (phosphate, ammonium, potassium and magnesium) improved glycerol removal, but the effects of ammonium and phosphate were twice those of the other two ions individually. The optimum concentrations to achieve total glycerol removal, after 2 days, were: [NaCl] = 214 g dm3, [Mg2] = 114 mg dm3, [K] = 131 mg dm3, 3 3 [NH4 ] = 113 mg dm , [PO4 ] = 40 mg dm3. Optimal conditions must always be a compromise between the best glycerol removal and the lower residual ammonium and phosphate concentrations and further studies on combined supplementation are required to achieve this. The results presented show the potential of using halophilic cultures for glycerol removal from hypersaline wastewater. Future work should involve reutilisation of this treated wastewater as a carotenogenesis medium for D salina and comparison of b-carotene productivity with that obtained with fresh medium.

ACKNOWLEDGEMENTS

The authors wish to acknowledge Fundacao para a

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scavenging ability of bacterioruberin. Radiation Physics and Chemistry 50(3):267269 (1997). Egli L and Pfander H, Composition of carotenoids in three isolates of halobacterium sp. grown in different media. Abstract of a Poster Presentation at the 11th International Symposium on Carotenoids, August, Leiden, NL (1996). Takao K, Production of C50 carotenoid, JP07132096. Micro Arujie Corp KK (1995). Torsvik T and Dundas ID, Halophilic phage specic for Halobacterium salinarium STR. 1, in Energetics and Structure of Halophilic Microorganisms, Ed by Caplan SR and Ginzburg M, Elsevier/North-Holland, Amsterdam. pp 609614 (1978). Box GEP and Wilson KB, On the experimental attainment of optimum conditions. Journal of Royal Statistical Society, serie B 13(1):138 (1951). Box GEP, Hunter WG and Hunter JS, Statistics for Experimenters, John Wiley & Sons, New York. Chapter 10 (1978). American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 18th edn, Ed by Greenberg AE, Clesceri LS and Eaton AD, APHA, New York (1992). Bligh EG and Dyer WJ, A rapid method of total lipid extraction and purication. Canadian Journal of Biochemistry and Physiology 37:911917 (1959). Lowry OH, Rosebrough NH, Farr AL and Randall RJ, Protein measurements with folinphenol reagent. Journal of Biology and Chemistry 193:265270 (1951). Dubois M, Gilles K, Hamilton K, Regas P and Smith E, Colorimetric method of determination of sugars and related substances. Analytical Chemistry 28:350356 (1956). Strickland JDH and Parsons TR, A Practical Handbook of Seawater Analysis, Bulletin 167, 2nd edn, Fisheries Research Board of Canada, Ottawa (1972). Ben-Amotz A and Avron M, On the mechanism of osmoregulation in Dunaliella, in Energetics and Structure of Halophilic Microorganisms, Ed by Caplan SR and Ginzburg M, Elsevier/ North-Holland, Amsterdam. pp 529541 (1978). Baumann FJ, Dichromate reux chemical oxygen demand. Analytical Chemistry 46(9):13361338 (1974). Abeliovich A and Azov Y, Toxicity of ammonia to algae in the sewage oxidation ponds. Applied Environmental Microbiology 31:801806 (1976). Azov Y and Goldman JC, Free ammonia inhibition of algal photosynthesis in intensive culture. Applied Environmental Microbiology 43:735739 (1982).

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