A new wastewater treatment process that involves coagulation, ozonation, and microalgae cultivation has been developed. Here, two challenges are discussed. The first was minimizing phosphorus removal during coagulation in order to maximize algal production. The second was to optimize microalgae cultivation; algal species that grow rapidly and produce valuable products are ideal for selection. Haematococcus pluvialis, which produces the carotenoid astaxanthin, was used. Growth rate, nutrient removal ability, and astaxanthin production of H. pluvialis in coagulated wastewater were investigated. After coagulation with chitosan, the turbidity and suspended solids decreased by 89% ± 8.4 → 8% and 71 → 73% ± 16%, respectively. The nitrogen and phosphorus contents of the supernatant remained at 86% ± 6% and 69 → 67% ± 24%, respectively. These results indicate that coagulation with chitosan can remove turbidity and SS while preserving nutrients. H. pluvialis grew well in the supernatant of coagulated wastewater. The astaxanthin yield from coagulated wastewater in which microalgae were cultured was 3.26 mg/L, and total phosphorus and nitrogen contents decreased by 99.0% ± 1.4% → 99% + 1% and 90.3% ± 7.6% → 90% ± 8% (Days 31–35), respectively.

INTRODUCTION

Wastewater is treated for the improvement and preservation of the quality of receiving water by removing organic compounds and nutrients. From a different perspective, however, the removal and disposal of nutrients may be a waste of a source that has a great potential for microalgae culture.

Conventional wastewater treatment processes using activated sludge pose concerns related to antibiotics. These chemical substances continuously flow into the treatment process and produce selective pressure for antibiotic-resistant bacteria. This raises a concern about using the activated sludge process because it provides a diffusion source of antibiotic-resistant bacteria (Baquero et al. 2008; Davies & Davies 2010; Rizzo et al. 2013).

Therefore, a new wastewater treatment process involving coagulation, ozonation, and microalgae cultivation has been developed. Suspended solids (SS) and colloids are removed during the coagulation process. The supernatant is subjected to an ozonation process, in which organic compounds, including antibiotics, are degraded into smaller compounds, and pathogenic organisms are inactivated. Finally, microalgae are cultivated using the ozonated wastewater to remove nutrients such as nitrogen and phosphorus. Because of the chemical oxidation and displacement of activated sludge that contains prokaryotic bacteria, there is little or no concern regarding the growth of antibiotic-resistant bacteria in this new process. The microalgae remove nutrients. Moreover, they produce valuable substances, which can be recovered along with the nutrients.

There are several valuable candidate substances that can be produced by microalgae: unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); biofuels; and carotenoids such as β-carotene, lutein, and astaxanthin. Carotenoids are lipid-soluble compounds with various colors such as red, orange, and yellow (Del Campo et al. 2007). They act as antioxidants to scavenge free radicals and are enhancers of the immune response. Some are involved in cell-to-cell communication. Recently, epidemiological studies have indicated an association between high vegetable intake and a lower risk of carcinogenic and cardiovascular diseases as well as age-related macular degeneration (Rodríguez-Bernaldo & Helena 2006). Their most important uses are as natural food colorants and as additives for animal feed and cosmetics (Pauline et al. 2005).

Haematococcus pluvialis is a microalga that produces the carotenoid astaxanthin. Several papers have been published on astaxanthin production using Haematococcus; Lorenz & Cysewki (2000) reviewed the commercial production and applications of astaxanthin. They showed that Haematococcus is cultivated in a two-step process that consists of vegetative and induction stages. In the first vegetative stage, Haematococcus grows under near-optimal conditions, while in the second stage the cells are subjected to environmental and nutrient stress. In this process, Haematococcus produces 1.5–3.0% astaxanthin by dry weight. Kang et al. (2005) reported H. pluvialis cultivation in primary-treated wastewater and piggery wastewater. They showed that the cell growth rate in primary-treated wastewater was 0.24 day−1, which was comparable to 0.23 day−1 in artificial medium; the cells were composed of 5.1 and 5.9% astaxanthin content using the two-step process; and the cells yielded 43 mg/L nitrogen and 2.6 mg/L phosphorus.

Optimizing the newly proposed process involves various challenges. Two of these challenges are discussed in this paper. The first challenge is to minimize phosphorus removal during coagulation. Phosphorus is easily removed by typical coagulants that contain iron or aluminum. However, to maximize algal production in the new process, removal of phosphorus must be minimized. The second challenge is to optimize microalgae cultivation. The growth rate, nutrient removal ability, and astaxanthin production of H. pluvialis in wastewater in combination with chitosan mediated coagulation were investigated. In this process, ozonation is expected to destroy organic molecules, including antibiotics, into smaller compounds, and inactivate remaining microorganisms after coagulation. As autotrophic microalgae cultivation was performed in this process, the molecular structure of the organic substances does not critically affect the cultivation. Therefore, in this paper, results without ozonation were discussed to focus on the fate of nutrients in coagulation and microalgae cultivation.

MATERIALS AND METHODS

Wastewater sample

In a preliminary study, it was found that natural coagulants were effective at removing SS and reducing turbidity with less phosphorus removal. Thus, in this research, chitosan, a polysaccharide obtained from the exoskeleton of crustaceans, which acts as a coagulant (Ahmad et al. 2006; Renault et al. 2009), was chosen for the investigation.

Raw primary effluent was collected once a week from a wastewater treatment plant, which mostly receives domestic wastewater by a separate sewer system in Okayama City, Japan. The wastewater samples were subjected to jar tests (n =23). For comparison, the coagulant poly-aluminum chloride (PAC) was used along with the chitosan. Table 1 shows the parameters of the wastewater.

Table 1

Parameters of the primary effluent

Parameter Average Range 
pH 6.9 6.8–7.1 
Turbidity (ABS) 0.51 0.403–0.732 
SS (mg/L) 46.5 37.1–66.1 
TP (mg P/L) 2.2 1.2–2.9 
TN (mg N/L) 20.1 17.2–21.2 
TOC (mg C/L) 21.8 8.2–34.7 
Parameter Average Range 
pH 6.9 6.8–7.1 
Turbidity (ABS) 0.51 0.403–0.732 
SS (mg/L) 46.5 37.1–66.1 
TP (mg P/L) 2.2 1.2–2.9 
TN (mg N/L) 20.1 17.2–21.2 
TOC (mg C/L) 21.8 8.2–34.7 

Coagulation experiments

Chitosan stock solution (1 g/L) was prepared using commercial chitosan (Chitosan 500, 032-14412; Wako Pure Chemical Industries, Ltd, Wako, Japan). The chitosan powder was dissolved in 0.1 N HCl and then diluted to the desired concentration using distilled water. A PAC stock solution (1 g/L) was prepared using commercial PAC from Kishida Chemical Co., Ltd (Kishida, Japan) and then diluted to the desired concentration with distilled water.

Coagulation was performed in four-spindle multiple stirrer units (Miyamoto Riken Ind. Co., Ltd, Riken, Japan). Primary effluent samples were divided into four beakers, each containing 500 mL. The pH values were adjusted using 0.1 N HCl. Each beaker was subjected to a rapid mixing step at 150 rpm for 5 minutes, a slow mixing step at 50 rpm for 15 minutes, and then left to stand for 30 minutes to allow sedimentation. Different volumes of the chitosan and PAC solutions were added to the beakers at the beginning of the rapid mixing. Samples were then collected in the upper part of the beakers to measure various parameters of the treated water.

Haematococcus pluvialis cultivation and extraction of carotenoids

H. pluvialis NIES-144 was obtained from the National Institute for Environmental Studies, Tsukuba, Japan.

For the first round of testing, culturing was performed in 50-mL conical tubes to evaluate astaxanthin production of H. pluvialis. Test cultures for carotenoid production were divided into two stages according to the study by Kang et al. (2005). During the first stage, culturing was performed under optimal conditions (temperature: 25 °C, light intensity: 3,000 lux, CO2 supply: 5%) in NIES-C medium (nitrogen and phosphorus concentrations: 36.4 mg N/L and 5.1 mg P/L, respectively) to promote optimal growth of microalgae. Fluorescent light was used as the light source. The light/dark cycles were 12 h:12 h and controlled using Programmable Timer NT301 (NISSO, Tokyo, Japan). This culturing was performed for 8 days. During the second stage, the induction phase, the culture was performed under stressed conditions with strong light and less nitrogen (temperature: 25 °C, light intensity: 35,400 lux, CO2 supply : 5%), in N-limited medium (nitrogen and phosphorus concentrations: 5.7 mg N/L and 5.1 mg P/L, respectively). A white light emitting diode (LED) was used to produce light that had about 10-fold greater intensity than that in the first vegetation stage. The light/dark cycle was 12 h:12 h and controlled using Programmable Timer NT301. The compositions of NIES-C and N-limited medium are summarized in Table 2.

Table 2

Compositions of media

 NIES-C (Ichimura 1971N-limited (Kang et al. 2005
pH 7.5 7.5 
Ca(NO3)2⋅4H2O(g/L) 0.15 − 
CaCl2·2H2O (g/L) − 0.13 
KNO3 (g/L) 0.10 − 
KCl (g/L) − 0.07 
β–Na2 glycerophosphate⋅5H2O (g/L) 0.05 0.05 
MgSO4⋅7H2O (g/L) 0.04 0.04 
Tris(hydroxymethyl) aminomethane (g/L) 0.5 0.5 
Thiamine HCl (μg/L) 10 10 
PIV metal solution (mL/L) 3.0 3.0 
Biotin (μg/L) 0.1 0.1 
Vitamin B12 (μg/L) 0.1 0.1 
 NIES-C (Ichimura 1971N-limited (Kang et al. 2005
pH 7.5 7.5 
Ca(NO3)2⋅4H2O(g/L) 0.15 − 
CaCl2·2H2O (g/L) − 0.13 
KNO3 (g/L) 0.10 − 
KCl (g/L) − 0.07 
β–Na2 glycerophosphate⋅5H2O (g/L) 0.05 0.05 
MgSO4⋅7H2O (g/L) 0.04 0.04 
Tris(hydroxymethyl) aminomethane (g/L) 0.5 0.5 
Thiamine HCl (μg/L) 10 10 
PIV metal solution (mL/L) 3.0 3.0 
Biotin (μg/L) 0.1 0.1 
Vitamin B12 (μg/L) 0.1 0.1 

Then, culturing was performed in both filtered wastewater and the supernatant of coagulated wastewater, using conical tubes, to evaluate cell growth and astaxanthin production rates of H. pluvialis in wastewater in comparison with those obtained with C-medium culture. These wastewater samples were obtained by filtering raw primary effluent or supernatant of the primary effluent after coagulation with Advantec No. 5B (Advantec Toyo, Tokyo, Japan). The culture conditions, aside from the medium, were the same as in the culture in artificial medium mentioned above.

The effect of gas supply was also investigated, in which H. pluvialis was cultivated in three conical tubes with different gas conditions; 5% CO2, air or without gas supply. NIES-C and N-limited media were used for the first and second stages, respectively.

Finally, continuous culture was performed using the same conditions for tube culture to evaluate astaxanthin production and nitrogen and phosphorus removal by H. pluvialis in wastewater. Figure 1 shows the reactors used for the culture. There are two tanks: one is for vegetative growth of H. pluvialis, and the other is for inducing astaxanthin production of H. pluvialis. The first tank has a volume of 1.2 L, where pH was controlled at pH 7.5 using 1 N HCl and 1 N NaOH. The wastewater flowed into the first tank continuously at a flow rate of 300 mL/day, and the hydraulic retention time (HRT) was 4 days. The second tank has a volume of 3.0 L, and HRT was 10 days. Coagulated wastewater after filtration with Advantec No. 5B (Advantec Toyo, Tokyo, Japan) was used as the medium.

Figure 1

Apparatus for continuous culture of H. pluvialis. The supernatant of coagulated wastewater was stored in a refrigerator. It was supplied to the first cultivation tank at a flow rate of 300 mL/day. The overflow from the first tank went into the second tank, in which light of higher intensity (35,400 lux) was supplied to induce astaxanthin production. The tanks were kept at 25 °C in an incubator.

Figure 1

Apparatus for continuous culture of H. pluvialis. The supernatant of coagulated wastewater was stored in a refrigerator. It was supplied to the first cultivation tank at a flow rate of 300 mL/day. The overflow from the first tank went into the second tank, in which light of higher intensity (35,400 lux) was supplied to induce astaxanthin production. The tanks were kept at 25 °C in an incubator.

To start the process, an aliquot of H. pluvialis culture was inoculated into the first tank. After the inoculation, absorbance at 660 nm as an index of algae concentration and SS was measured in the first tank, whereas total phosphorus (TP) and total nitrogen (TN) concentrations were measured in both tanks to evaluate nutrient removal rate.

Culture broth was taken from the second tank at 11, 17, and 31 days after inoculation to determine the pigment contents in the algal cells. The pigments were extracted based on methods proposed by Kang et al. (2005). The broth was centrifuged at 10,000 × g for 10 minutes. The supernatant was discarded, and the cell pellet was rinsed with distilled water before being homogenized with 100% acetone. After homogenization, the samples were centrifuged at 10,000 × g for 10 minutes, and the supernatant was then collected. The extraction procedure was repeated at least three times until the cell debris was almost colorless. All pigment extracts were combined and centrifuged again at 10,000 × g for 10 minutes. All of the above processes were performed in semi-darkness under the flow of nitrogen. Finally, the extracts were stored in a freezer at −20 °C.

Measurement of various indices

Turbidity, measured as absorbance at 660 nm, was determined using a spectrophotometer (HACH DR/2400, Loveland, CO, USA) with a 5-cm glass cell. SS and TP of the culture broth concentrations were measured based on Standard Methods (APHA 2005). TN of the supernatant after coagulation by chitosan was measured with a QuAAtro2-HR autoanalyzer (BLTEC, Osaka, Japan). Total organic carbon (TOC) was measured using a Shimadzu TOC 5000 Analyzer (Kyoto, Japan).

Carotenoid analysis was performed by measuring the absorption spectrum from 400 to 700 nm of the acetone extract in a 1-cm glass cell. The maximum absorbance wavelengths of chlorophyll a and b are 663 nm and 645 nm (Porra et al. 1989), respectively, and the maximum absorbance wavelength of astaxanthin is 475 nm (Buchwald & Jencks 1968). Astaxanthin and β-carotene concentrations were measured by high performance liquid chromatography (HPLC) at Japan Food Research Laboratories (Tokyo, Japan).

RESULTS AND DISCUSSION

Coagulation of wastewater with chitosan and PAC

Figure 2 shows the turbidity and SS in the samples before and after coagulation with chitosan or PAC. To remove turbidity, the optimum dose of chitosan was only 3 mg/L, whereas a relatively large amount of PAC (30 mg/L) was required to obtain the highest coagulation efficiency. An overdose of chitosan caused an increase in the turbidity of the supernatant, resulting in reduced treatment efficiency. Moreover, a small volume of sludge was generated by chitosan mediated coagulation, while a relatively larger volume of sludge was formed by coagulation with PAC. The lowest residual SS in the supernatant was obtained with 2 mg/L of chitosan, whereas the lowest SS concentration in treated wastewater was observed with 30 mg/L of PAC.

Figure 2

Turbidity and SS of the wastewater after coagulation with different doses of chitosan and PAC at pH 6. Vertical I-lines indicate standard deviation from eight determinations. The solid lines show the results of chitosan mediated coagulation, and the dashed lines show the results of the coagulation with PAC.

Figure 2

Turbidity and SS of the wastewater after coagulation with different doses of chitosan and PAC at pH 6. Vertical I-lines indicate standard deviation from eight determinations. The solid lines show the results of chitosan mediated coagulation, and the dashed lines show the results of the coagulation with PAC.

Figure 3 shows the TP concentration and TOC in the samples after coagulation. After coagulation with chitosan, the phosphorus concentration in the supernatant remained almost identical and was within the range of doses investigated in this research. However, phosphorus removal increased as the PAC dose increased. At the optimal dose of chitosan for removal of SS (2 → 3 mg/L), only 32% ± 23% → 33% ± 24% of the TP was removed, whereas 88% ± 8.7% → 89% ± 9% was removed by the optimal dose of PAC (30 mg/L). This may be due to direct adsorption of phosphate ions in the hydrolysis products and removal through the formation of phosphate precipitates with alum species produced by PAC. Moreover, the TOC was effectively removed by a chitosan dose of 3 mg/L, and an overdose of chitosan caused an increase in the TOC concentration in the supernatant. The TOC removal rates of chitosan and PAC were 59% ± 9.1% → 60% ± 9% and 68% ± 15%, respectively, at the optimum doses to remove SS (2 → 3 mg/L for chitosan and 30 mg/L for PAC). Figure 4 shows the TN concentration in the samples after chitosan mediated coagulation. Because the supernatant also contained nitrogen (17.2 mg/L), the supernatant after the coagulation was thought to be suitable for microalgae cultivation.

Figure 3

TP and TOC in the supernatant after coagulation; different doses of chitosan and PAC at pH 6. The solid lines show the results of chitosan mediated coagulation and the dashed lines show the results of PAC coagulation.

Figure 3

TP and TOC in the supernatant after coagulation; different doses of chitosan and PAC at pH 6. The solid lines show the results of chitosan mediated coagulation and the dashed lines show the results of PAC coagulation.

Figure 4

TN in the supernatant after treatment with different doses of chitosan at pH 6.

Figure 4

TN in the supernatant after treatment with different doses of chitosan at pH 6.

H. pluvialis cultivation in test tubes

Figure 5(a) shows the growth of H. pluvialis in 50-mL tube cultures in three different media: C-medium, supernatant after coagulation with chitosan, and filtered wastewater. TP and TN concentrations were 4.8 mgP/L and 36 mgN/L in coagulation supernatant, while those were 4.7 mgP/L and 30 mgN/L in filtered wastewater, respectively.

Figure 5

Algal growth in 50-mL culture tubes in (a) three different media and (b) coagulated wastewater with different gas supplies. (a) Triangles indicate filtered primary effluent, rhomboids indicate coagulated wastewater, and squares indicate C-medium. (b) Algae were cultivated with 5% CO2 supply, air supply, or without gas supply.

Figure 5

Algal growth in 50-mL culture tubes in (a) three different media and (b) coagulated wastewater with different gas supplies. (a) Triangles indicate filtered primary effluent, rhomboids indicate coagulated wastewater, and squares indicate C-medium. (b) Algae were cultivated with 5% CO2 supply, air supply, or without gas supply.

The specific growth rate in both filtered wastewater and in the coagulated wastewater was 0.34 day−1, which was higher than the growth rate of 0.29 day−1 in C-medium. These growth rates were comparable to or higher than those observed by Kang et al. (2005). Although TP and TN concentrations in coagulated wastewater were lower than those in C-medium, the growth rate in coagulated wastewater culture was higher than that in C-medium. This may be due to the availability of phosphorus and nitrogen for microalgae, or the existence of micronutrients like vitamins or other substances that stimulated the algal growth. This result indicates that coagulated wastewater is a suitable medium for H. pluvialis culture.

After cultures reached the stationary phase, they were placed under high-intensity lights, resulting in a change in the color of the culture broth from green to red. This phenomenon indicated that H. pluvialis produced red pigment. We also used filtered supernatant after coagulation as a medium to investigate the effect of CO2 supply and continuous culture.

Figure 5(b) shows the growth of H. pluvialis in 50-mL tubes in coagulated wastewater with different gas supply conditions. The maximum growth rate was 0.34 day−1 with a 5% CO2 supply. The specific growth rate of the culture supplied with air was 0.30 day−1, which was slightly smaller than that with 5% CO2. This result indicates that the 5% CO2 supply was the most appropriate among the conditions we evaluated, but air was also sufficient for H. pluvialis cultivation.

Continuous culture of H. pluvialis

Figure 6 shows the growth of H. pluvialis, while Figure 7 shows the changes in TP and TN concentration observed in the second tank. Turbidity went up to 0.826 in the first tank until 35 days. The cell dry weight was 0.151 g/L on Day 35 in the first tank. TP and TN concentrations in the second tank decreased with cell growth. On the same day, the nitrogen concentration was reduced from 30 to 5.6 mg N/L in the first tank, and it was completely consumed in the second tank. Similarly, phosphorus concentration decreased from 4.7 to 0.91 mg P/L in the first tank, and further decreased to 0.49 mg P/L in the second tank. These results show that 99 ± 1.4% → 99% ± 1% of nitrogen (Days 31–35) and 90.3 ± 7.6% → 90% ± 8% of phosphorus (Days 31–35) were removed by H. pluvialis cultivation.

Figure 6

Culture and cell growth of H. pluvialis (absorbance at 660 nm).

Figure 6

Culture and cell growth of H. pluvialis (absorbance at 660 nm).

Figure 7

Removal of TP and TN by H. pluvialis. Triangles show the TP concentration and squares show the TN concentration in the second tank.

Figure 7

Removal of TP and TN by H. pluvialis. Triangles show the TP concentration and squares show the TN concentration in the second tank.

Figure 8 shows the changes in UV spectrum of pigment extracted from H. pluvialis grown in the second tank. Between Days 11 and 17, the absorbance near 660 nm increased, which indicates that the chlorophyll content in the culture broth increased. Between Days 17 and 31, absorbance near 480 nm greatly increased while absorbance near 660 nm changed slightly. This suggested that astaxanthin content increased more than chlorophyll content in the culture broth.

Figure 8

Results of UV spectrum analysis of pigment extracted from H. pluvialis from the second tank.

Figure 8

Results of UV spectrum analysis of pigment extracted from H. pluvialis from the second tank.

Astaxanthin is produced in H. pluvialis as follows. First, β-carotene is produced from phytoene by phytoene desaturase. Then, astaxanthin is produced by ketoraze from β-carotene (Han et al. 2013). On Day 31, TN and TP concentrations were 1.3 mg N/L and 0.87 mg P/L, respectively. Because of low nutrient concentration, it was thought that phytoene desaturase production became active, resulting in β-carotene production, followed by the production of astaxanthin from β-carotene by ketolase that was produced under stress conditions.

The astaxanthin concentration at Day 31 in culture broth was 3.26 mg/L. Kang et al. (2005) reported the maximum concentration of 175.7 mg/L in batch experiments. Therefore, the astaxanthin concentration reported here was substantially lower than reported by Kang et al. (2005),. Astaxanthin is synthesized from β-carotene in algal cells as mentioned above. That is to say, there are two steps for astaxanthin production; production of β-carotene and transformation of β-carotene to astaxanthin (Han et al. 2013). The concentration of β-carotene at Day 31 in culture broth was no more than 0.39 mg/L, which was lower than that of astaxanthin (3.26 mg/L). This means that the transformation of β-carotene to astaxanthin properly functioned. Therefore, the production of β-carotene has to be induced to increase astaxanthin yield.

We think there may be at least two reasons for the lower astaxanthin concentration. The first is the light intensity in this experiment. The intensity was the same that used by Kang et al. (2005), but the reactor volume was 12 times larger than that used by them. Consequently, in this study, the energy of light per unit volume might not be sufficient. The cells possibly did not experience enough light stress to induce astaxanthin production. Another possible reason is the contamination of other microorganisms in the reactor. We did not include ozonation in this experiment, but filtered to remove particles after coagulation. However, the removal did not function perfectly. We found some other microalgae from the reactor in the microscopic observation after this experiment. Such contamination might inhibit the astaxanthin accumulation.

CONCLUSIONS

After coagulation with chitosan, turbidity and SS decreased by 89% ± 8.4 → 8% and 71 → 73% ± 16%, respectively. The nitrogen and phosphorus contents of the supernatant remained at 84 → 86% ± 5.9 → 6% and 69 → 67% ± 24%, respectively. These results indicate that coagulation with chitosan can remove turbidity and SS while preserving nutrients. Because the supernatant included sufficient nutrients, the supernatant is considered suitable as a medium for microalgae cultivation.

Haematococcus pluvialis grew well in the supernatant of coagulated wastewater and had a higher growth rate than in the artificial medium. TP and TN were satisfactorily removed by H. pluvialis cultivation, with removal rates of 99.0 → 99% ± 1.4 → 1% and 90.3 → 90% ± 7.6 → 8% (Days 31–35), respectively. The astaxanthin yield from coagulated wastewater was 3.26 mg/L, which is less than the content in cells. Further optimization will be required to increase astaxanthin production efficiency.

ACKNOWLEDGEMENT

This research was supported by the Japan Sewage Works Association.

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