Treating high-strength saline piggery wastewater using the heterotrophic cultivation of Acutodesmus obliquus

Biochemical Engineering Journal 110 (2016) 51–58

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Regular article

Treating high-strength saline piggery wastewater using the heterotrophic cultivation of Acutodesmus obliquus Hyun-Chul Kim a , Wook Jin Choi b,c , A. Na Chae b , Joonhong Park c , Hyung Joo Kim d , Kyung Guen Song b,∗ a

Water Resources Research Institute, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul 143-747, South Korea Center for Water Resource Cycle Research, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea c Department of Civil and Environmental Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, South Korea d Department of Microbial Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, South Korea b

a r t i c l e

i n f o

Article history: Received 7 September 2015 Received in revised form 23 January 2016 Accepted 12 February 2016 Available online 15 February 2016 Keywords: Piggery effluent Waste-water treatment Microalgae Acutodesmus obliquus Heterotrophes Bioremediation

a b s t r a c t This study combined an algae treatment with post-ozonation for the robust treatment of piggery effluent in which high levels of organic constituents, inorganic nutrients, color, and salts remained. Due to a nearly complete light limitation resulting from a high level of color, the algae treatment was conducted with continuous O2 supplementation instead of using the combination of high lighting and CO2 injection. The microalga Acutodesmus obliquus KGE-17 showed tolerance to high salinity (up to 5.2% as chloride) and was capable of utilizing dissolved organics (1923 mg-COD/(g-cell)(day)) under the heterotrophic growth conditions. The use of A. obliquus for remediation of piggery effluent also resulted in an operational simplicity in the simultaneous removal of nitrogen and phosphorus in a single treatment process, in contrast to conventional biological nutrient removal processes. Subsequent ozonation was successful in decolorizing the algae-treated wastewater and in improving the biodegradability of residual organics in post-treatment processes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Biological processing is the most efficient way of removing organic matter and inorganic nutrients from a wide range of wastewaters. These living systems rely on mixed microbial cultures to decompose and remove targeted components from a solution. Salinity is considered to be one of the major constraints on microbial viability and species diversity in wastewater treatment systems. Piggery effluents commonly contain high concentrations of carbon, nitrogen, and phosphorous; along with a high salinity, these effluents can adversely affect biological growth and thus deteriorate wastewater treatability. It has been reported that saline wastewater with more than 2% salt results in low removals of organic and nutrients and poor settleability of microbial cells in conventional biological processes [1]. Thus, the application of salt-tolerant microorganisms in the biological treatment of saline wastewater has been attempted using a wide variety of bio-agents [2]. In particular, algae differ in their adaptability to salinity based on their tolerance. Use of algal strains for the remediation of piggery effluent can also result in operational simplicity in the simulta-

∗ Corresponding author. E-mail address: [email protected] (K.G. Song). http://dx.doi.org/10.1016/j.bej.2016.02.011 1369-703X/© 2016 Elsevier B.V. All rights reserved.

neous removal of nitrogen and phosphorus in a single treatment process, in contrast to conventional biological nutrient removal (BNR) processes that are comprised of nitrification/denitrification and luxury P uptake under strictly controlled operating conditions. BNR processes also typically require an amount of external carbon source for denitrification under anoxic condition. It has been reported that dark-colored wastewater usually derived from humus-rich habitats causes light limitation in the photosynthesis of microalgae and consequently results in a decrease in the rate of nutrient recovery from wastewater [3,4]. Some investigators decreased the chromaticity from piggery wastewater through oxidation with hydrogen peroxide [5], sodium hypochlorite [6], and ozone [7] to improve the performance of algae treatment. However, utilizing mixotrophic (i.e., heterotrophically growable) algal species as a bio-agent for the remediation of piggery wastewater seems to be more applicable than using oxidation processes for the removal of color. Along with the removal of inorganic nutrients using algae treatment, the removal of carbonaceous organics can also be achieved using specific algal strains, unless the organic carbon source for ‘heterotrophically-growable’ bio-agents is a limiting factor. We previously noticed that Acutodesmus obliquus can perform heterotrophic growth, besides the common autotrophic growth when using CO2 as the sole carbon source. The heterotrophic growth of microalga can proceed

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more rapidly by directly incorporating an organic substrate in the oxidative assimilation process for storage material production [8]. Biomass productivity is strongly associated with assimilating substrates and thus heterotrophic growth is more desirable for the better removal of contaminants compared to autotrophic growth. In contrast, an external CO2 supplement under continuous illumination is typically required for the photoautotrophic growth of microalgae, resulting in high operating costs for algae treatment. Local facilities for treating piggery effluent are currently facing stricter limits on their discharges of process wastewaters. In many cases, these limits are based on acceptable color levels, biodegradability, and biotoxicity, not simply on organic concentrations. In particular, acceptable color levels are generally location-dependent and are affected by the treatment capacity of the receiving posttreatment facilities and the volume of effluent discharged. The effect of light scattering causing apparent color can be eliminated by filtering of membranes with micro-pores, while chemical treatment processes are more applicable for removing true color, depending exclusively on the dissolved species in the wastewater. This paper discusses the feasibility of using ozone to comply with discharge permits that limit color in the effluent, especially by replacing conventional bleaching processes to reduce the use of chlorine. Chlorine-based oxidation can lead to satisfactory color abatement, but ozone remains the most efficient oxidizer [9]. Ozonation is the treatment most often mentioned in the literature for oxidative color removal [10,11]. Furthermore, many researchers have evaluated the applicability of ozonation in the context of advanced treatment for wastewaters, such as removal of antibioticresistant bacteria from piggery effluent [12] and degradation of pharmaceutical contaminants from urban wastewater [13]. Ozonation tends to increase the polarity and hydrophilicity of organic matter in the aqueous phase and only small portions of the organics can be completely mineralized, indicating a high feasibility of ozone use for the production of low-molecular and thus easilyassimilative organic fractions [7]. Algae treatment and post-ozonation were combined in this study for the robust treatment of pretreated piggery effluent in which high levels of organic constituents, inorganic nutrients, and salts still remained. Few studies have dealt with the concurrent removal of organic and inorganic components from highly-saline piggery effluent by means of mixotrophs; in particular, a lack of information prevails on the effect of light limitation on the growth of mixotrophic microalgae and the associated removal of residual contaminants. In this study, an evaluation was thus conducted with an emphasis on the simultaneous removal of inorganic nutrients and carbonaceous organics using saline tolerant microalgae species under heterotrophic growth conditions resulting from the light limitation in the photosynthesis of microalgae. Therefore, the goal of this study was to achieve the following objectives during the treatment of piggery effluent at high salinity: (1) a better understanding of how a microalga A. obliquus works on wastewater remediation under heterotrophic growth conditions; (2) operational simplicity in the simultaneous removal of TN, TP, and COD in a single biological process; and (3) substantial decrease in refractory organic fractions in the treated wastewater to alleviate the burden of post-treatment facilities (usually domestic wastewater treatment plants) and further receiving water bodies.

2. Materials and methods 2.1. Piggery wastewater pretreatment Piggery effluent was collected from commercial treatment facilities (ENR Solution Co., Ltd., South Korea). All facilities operate at 23 m3 /day and use the up-flow sludge blanket reactor process for

the typical mesophilic anaerobic digestion of organic substrates. The effluent was then subjected to sequential solid/liquid separations with flocculants that were added to facilitate the removal of suspended solids from the mixed liquor. The collected supernatant was filtered manually using a conventional filtration apparatus with 0.2 ␮m cellulose acetate membranes. The filtered piggery wastewater was then stripped with air using an acrylic column (ID 5 cm × H 100 cm) in which polyethylene carriers (AnoxKaldnes, Sweden) were filled up to 80 cm (see Supplementary data, SD-1). The wastewater was circulated at pH 10 from the top to the bottom of the column at a flow rate of 40 mL min−1 , while injecting air (60 L min−1 ) to create a counter flow. The wastewater was recirculated three times and the final effluent was neutralized (pH 7.5) for further algae treatment. Table 1 shows the average characteristics of the pretreated piggery effluent. 2.2. Biological treatment with a microalga obliquus A. obliquus KGE-17 was isolated from a livestock wastewater treatment facility in Gangneung, South Korea. The specie was identified using the same method reported in our prior work [14]. The algal strain was inoculated into 1000 mL tubular photo-bioreactors containing 800 mL BG-11 medium [15]. The photo-bioreactors were incubated under white fluorescent light illumination at 75 ␮mol photon m−2 s−1 at 25 ◦ C for two weeks while continuously supplementing with 5% CO2 (v/v) which was placed into the photo-bioreactors. Light intensity was determined using LI-250A (LI-COR, USA). The pH was measured using a pH meter (Orion 3 STAR, Thermo Scientific, USA). Microalga A. obliquus was employed to remediate piggery effluent because of its capability to grow in wastewaters with low to high salinity, as shown in our previous report [16]. Batch experiments were conducted to determine the growth rate of A. obliquus using a tubular borosilicate reactor (ID 3.4 cm × H 52 cm). Algal biomass from a two-week-old culture was harvested by centrifugation (3000 rpm for 10 min at room temperature) and was inoculated into the bioreactor containing 400 mL pretreated piggery wastewater. The bioreactor was incubated at 25 ◦ C for 138 h while aerating through an inlet port at the conical shaped bottom of the reactor. The piggery wastewater absorbed 85% of the light illumination within a path length of 1 cm due to high values of the chromaticity (6175 ± 26 mg Pt-Co L−1 ) and thus resulted in a nearly complete inhibition of photosynthesis. Instead of using the combination of high lighting and CO2 injection that is typically used in autoand mixo-trophic algal growth, algae treatment was conducted with continuous O2 supplementation to maximize the removal of carbonaceous organics from the piggery wastewater. During the incubation, a 10 mL mixed liquor was collected several times from the bioreactor for measurement of dry cell weight. Chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN), and ammonium-nitrogen (NH4 -N) were also measured after filtration with 0.2 ␮m membranes. Each measurement was carried out in triplicate and average values were reported. The Salicylate, Acid Table 1 Characteristics of pretreated piggery effluent. Parameter

Value (mean ± SD)

pH Chloride (mg L−1 ) Color (mg Pt-Co L−1 ) COD (mg L−1 ) TOC (mg L−1 ) UV254 (cm−1 ) TN (mg L−1 ) NH4 -N (mg L−1 ) TP (mg L−1 )

7.5 52524 6175 11100 3935 47 981 644 81

± ± ± ± ± ± ± ± ±

0.1 282 26 141 5 0 1 11 4

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7000 6000

COD (mg L-1)

8000

6000

4000

6000

5000 4000

4000 3000 2000

2000

(a)

1000

2000

0

(b)

DOC (mg L-1)

1600

12000

COD (mg L-1)

0 3

DOC SUVA

2000

0 14000

10000 8000 6000

2 1200 800 1 400

(b)

4000 0 700

0 1200

600

Nitrogen (mg L-1)

2000

TN NH4-N

(c)

Nitrogen (mg L-1)

1000 800

0 TN NH4-N

500 400 300 200

600

100

400

0

(c) 0

10

20

30

40

50

60

70

Ozonation duration (min)

200

Fig. 2. Impact of ozonation on the characteristics of algae-treated piggery wastewater. The ozonation of piggery wastewater continued up to 60 min at a dose of 87 mg O3 L−1 .

0 100

(d)

80

TP (mg L-1)

Color (mg Pt-Co L-1)

(a)

8000 COD Color

SUVA (L mg-C-1 m-1)

10000

Dry cell weight (mg L-1)

53

60 40 20 0 0

20

40

60

80

100

120

140

160

Time (h) Fig. 1. Growth of A. obliquus and the associated removal of contaminants. The residual concentrations of COD, TN, NH4 -N, and TP were determined after filtration (0.2 ␮m).

Persulfate Digestion, and Dichromate Methods were employed to measure NH4 -N, TP, and COD in the water samples. These methods are equivalent to Standard Methods 4500-NH3 G, 4500 P. B. 5, and 5220C, respectively for water and wastewater [17]. The Shimadzu TNM-1 coupled with the TOC-V CPN was used to measure TN.

The cell growth was determined by measuring the dry cell weight concentration using an equivalent procedure to that documented in the Standard Methods for the Examination of Water and Wastewater [17]. The growth coefficient (, per day) and the specific nutrient consumption rate (q, mg-N or −P/(g-cell)(day)) for a specific period were calculated as previously reported [7]. The maximum value of the growth coefficient (max ) and the maximum specific nutrient consumption rate (qmax ) were also obtained from the maximum slope on the dry cell weight concentrations and the assimilated nutrient concentrations plotted against time, respectively. 2.3. Ozone oxidation of bioeffluent Algae-treated piggery wastewater was ozonated at a dose of 87 mg L−1 for 60 min to identify the minimum O3 dose for compliance with color goals. The ozonation apparatus consists of a glass ozone contactor (ID 8 cm × H 33 cm), an ozone generator (Lab2B, Ozonia, Korea), and an oxygen tank. Ozonation was performed in the batch contactor on which a water jacket was installed to maintain an aqueous temperature at 15 ◦ C while injecting ozone at a flow rate of 1 L min−1 . During the ozonation, a 5 mL liquid sample was collected from the contactor at intervals and filtered with 0.2 ␮m

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Fig. 3. (a) Effects of sequential treatments on the distributions of five different organic fractions in piggery effluent. (b) LC-UVD spectra for organic materials in the piggery wastewater samples. Ozonation was 10 min at a dose of 87 mg O3 L−1 .

membranes to determine COD, the dissolved organic carbon (DOC), and the UV absorbance at 254 nm (UV254). The Shimadzu TOC-V CPN was used to measure DOC in the water samples. A DR/5000 spectrophotometer (Hach, USA) was used to measure UV254 and chromaticity. The specific UV absorbance (SUVA) value is calculated from the UV254 divided by the DOC of the water sample. Gaseous ozone exhausted from the contactor was detected and quantified using an ozone analyzer (Model H1, IN USA Inc., USA).

2.4. Analytical methods Advanced characterization techniques were employed to investigate the effects of algae cultivation and post-ozonation on the compositional and functional properties of organic components and to discriminate organics originating from algal cell growth. The molecular weight (MW) distribution of dissolved organic matter was determined using liquid chromatography-organic carbon detection (LC-OCD) (DOC-Labor, Germany), in which size-exclusion

Table 2 Effects of sequential treatments on fluorescent properties of dissolved organic matter in piggery effluent. Fluorescence intensities collected from four different regions were normalized based on the highest value (3.2 × 105 ) among the peaks observed for pretreated piggery effluent. Description

Peak A (Tryptophan protein-like)

Peak B (Aromatic protein-like)

Peak C (Humic acid-like)

Peak D (Fulvic acid-like)

Pretreated piggery effluent Algae treatment Ozonation (10 min) Ozonation (60 min)

0.42 0.03 0.05 0.05

0.87 0.08 0.08 0.10

0.61 0.45 0.20 0.01

1.00 0.54 0.16 0.04

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Fig. 4. Fluorescence EEM spectra for organic materials in piggery wastewater samples. Spectra (a) through (d) show the impacts of different treatment strategies on the fluorescent natures of dissolved organic matter in piggery effluent: (a) Pretreated piggery effluent, (b) algae treatment, (c) ozonation (10 min), and (d) ozonation (60 min). Ozonation was conducted for up to 60 min at a dose of 87 mg O3 L−1 .

chromatography was coupled with on-line equipment for real-time measurement of both UV254 and organic carbon concentration of effluent passing through the exclusion column. More detailed information of the LC-OCD system can be found in an earlier study [18]. Fluorescence spectra were collected using a Perkin-Elmer LS50B luminescence spectrometer, which uses a 450W xenon lamp source. All liquid samples were diluted with carbon-free electrolyte solution under ambient pH conditions. Spectroscopic analysis was carried out at a concentration of 1 mg C L−1 . The acquisition interval and integration time were maintained at 1 nm and 0.1 s, respectively. Right-angle geometry was used for liquid samples in a 10 mm fused-quartz cuvette. Three-dimensional spectra were obtained by repeatedly measuring the emission spectra within the range from 200 to 600 nm, with excitation wavelengths from 200 to 400 nm, spaced at 5 nm intervals in the excitation domain. Spectra were then concatenated into an excitation-emission matrix (EEM).

3. Results and discussion Prior to algae treatment, we employed air stripping to minimize the concentration of non-ionized ammonia (i.e., free ammonia) that could adversely affect the growth of microalgae in the pretreated piggery wastewater. Stripping was conducted using a standard stripping protocol which was identified through preliminary tests (see Supplementary data, SD-1). The result indicated that the NH4 -N concentration decreased to 644 ± 11 mg L−1 , corresponding to 14.1 mg L−1 free ammonia. This paper mainly focuses on the growth of mixotrophic microalga in high saline wastewater and the associated removal of residual contaminants under heterotrophic conditions; thus, we did not expand our research scope to consider the effects of pretreatment conditions (e.g., solid/liquid separations and air stripping) on the performance of downstream processes. 3.1. Algae treatment of pretreated piggery effluent The results of the batch experiment with pretreated piggery effluent using a microalga A. obliquus are shown in Fig. 1a, in which

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the concentration of dry cell weight is plotted against time. The stagnant growth of the algal strain was found to be a result of being acclimated to the significantly different growth conditions compared to when grown in a synthetic medium under autotrophic conditions. During the transition from the lag phase to the stationary phase, substantial removals of COD and inorganic nutrients were accomplished by algal cells. Interestingly, the growth of microalgae continued even up to the 90 h cultivation, despite there being insignificant change in the COD concentration after 40 h of cultivation. This observation demonstrates that microalgae produce storage materials (e.g., polysaccharides and polyphosphates) as reserve materials when resources are abundant and draw upon them in periods of starvation. Typically, the biological stabilization of carbonaceous organic compounds results in the accumulation of highly-condensed organics in the residual COD due to a substantial decrease in low-molecular and thus easily-assimilative organic fractions. This issue is discussed comprehensively in a later section of this paper. The TN removal was governed by the extent of the reduction in NH4 -N, which was the most dominant fraction among inorganic nitrogen species. The NH4 -N concentration can also be decreased via the stripping of free ammonia fraction during aeration, but such ammonia gasification during oxygen transfer in the algae bioreactor was negligible (data not shown). TP was decreased by less than 70% of the initial concentration by algal cells for the first 90 h of cultivation, after which it increased to some extent. It is well known that adenosine triphosphate (ATP) is synthesized by actively growing cells as a means of short-term energy storage and transfer. Under some heterotrophic cultures, ATP generated from the supplied energy is higher than under autotrophic and mixotrophic cultures [19]. ATP is hydrolyzed to adenosine-diphosphate and monophosphate to transfer energy to a metabolic process, resulting in increased ortho-phosphate in the aqueous phase [20]. The algal strain employed as bio-agents was capable of assimilating both nitrogen (175 mg-N/(g-cell)(day)) and phosphorus (1.5 mg-P/(g-cell)(day)) in a single treatment process (Fig. 1). The bio-agents also decreased COD (1923 mg-O2 /(g-cell)(day)) by utilizing dissolved organics as a carbon source under the heterotrophic growth conditions, and the removal of COD was associated with high productivities of algal biomass, accounting for 1850 mgcell/L/day. This was much higher than that reported in earlier work, in which a wide variety of auto- and mixo-trophic algal strains were grown on pretreated piggery wastewater supplemented with CO2 [21]. In general, the growth rate of microalgae can be decreased by the self-shading effect, causing poor light penetration due to the high population density of algal cells. Some investigators reported that the algal growth leveled out at a high population density of algal cells, accounting for >3000 mg L−1 due to a mutual interference in the light availability [21]. However, such phenomenon was not applicable to the heterotrophic growth. The maximum algal growth coefficient (max ) for A. obliquus under the heterotrophic conditions was 0.94/day, which was much higher than that observed in previous reports conducted under photoautotrophic growth conditions [22]. This result indicates that the heterotrophic growth of A. obliquus can proceed more rapidly by directly incorporating organic substrate in the oxidative assimilation process for storage material production. 3.2. Ozonation for compliance with the permit effluent limits Since biological treatment processes in domestic wastewater treatment plants are largely ineffective for removing color from the wastewaters, highly-colored wastewater should be properly treated for a full-scale treatment system before it is conveyed to the post-treatment facilities. In this study, ozonation was employed to achieve lower discharge concentrations and greater reliability for

compliance with color goals, especially by replacing conventional bleaching processes which are associated with the formation of harmful by-products. As ozonation continued for 20 min a significant decrease was found in the chromaticity which was further reduced at higher ozone doses (which were equivalent to longer reaction times), but only to a small extent. Similarly, a substantial decrease in SUVA was observed upon ozonation for 10 min, but an insignificant decrease was achieved with further increase of ozonation of up to 60 min. COD was stepwise decreased from 6.4 ± 0.1 to 1.2 ± 0.1 g L−1 when the concentration of ozone injected into algaetreated piggery wastewater was increased. A gradual decrease of DOC was found as the ozone dose was increased up to 2.9 mgO3 mg-C−1 , corresponding to 60 min of ozonation. Fig. 2 shows the much slower DOC reduction than COD reduction. The relative oxidation state of the byproducts with more oxygenated functionalities (e.g., hydroxyl, carboxyl, etc.) was much higher than for the parent organic molecules, as evidenced by a decrease in the COD/DOC mass ratio upon ozonation. The result also suggests that pretreatment with proper levels of ozone substantially improves the bioavailability of organic matter for heterotrophs in the bioremediation of piggery wastewater, which concurred with previously reported work [7]. The level of residual NH4 -N was gradually decreased from 299 ± 7 to 190 ± 10 mg L−1 with increasing ozonation duration. No significant change was observed in the level of residual TN upon ozonation for up to 60 min, and thus the ammonia gasification that could occur when bubbling the ozone through wastewater was negligible. This result indicates that ammonium was oxidized to nitrite/nitrate ions by ozone molecules. Amines constitute a highly reactive family of compounds with respect to ozone because the electrophilic attack is promoted by the presence of a negatively charged nitrogen atom [9,23–25]. In the context of mixotrophic wastewater treatment, ozonation can be alternatively used to increase light transmittance in the photosynthesis of microalgae. The ozonation pretreatment could result in the robust growth of microalgae and improved removal of inorganic nutrients during the subsequent cultivation of microalgae in the decolorized wastewater. However, the amount of newly produced algal biomass should be properly eliminated from the bioreactor due to the self-shading effect occurring in the photobioreactors. Moreover, repetition of the algal harvesting results in an accumulation of non- or less-biodegradable organic constituents in the culture, which may also require post-treatment. Therefore, the combination of pre-ozonation and algae treatment might only be applicable if the organic carbon source for heterotrophs is a limiting factor. 3.3. Characterization of piggery effluent organic matter The algae treatment combined with post-ozonation substantially changed the compositional and functional properties of organic compounds in pretreated piggery effluent. Bulk organics can be classified as illustrated in Fig. 3a based on the fact that the retention time of organic components in the exclusion column of LC-OCD depends on their MW. In this study, LC-OCD chromatograms were divided into five different fractions: biopolymers (>20,000 Da), humic-like substances (1200–500 Da), building blocks (500–350 Da), low MW acids (<350 Da), and low MW neutrals (<350 Da) [18]. The organic matter in pretreated piggery effluent were dominantly low MW acids (40% of DOC) and neutrals (24% of DOC). Cultivation of microalgae on the wastewater resulted in a significant increase of SUVA, which was attributed to the removal of hydrophilic fraction in bulk organics. SUVA has been used as an indicator of the humic content in water environmental systems [26–28]. A relative increase in UV254 upon algal culti-

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vation indicates that the hydrophilic organic components in the wastewater were more preferably assimilated by algal cells during the heterotrophic growth of microalgae. Algae treatment removed nearly all of the low MW acids and >42% of the low MW neutrals, while it could not effectively remove other fractions and rather doubled the level of biopolymers that were associated with extracellular polymeric substances formed during microbial metabolism and biomass decay. A 10 min ozonation achieved almost complete removal of large organic molecules designated as biopolymers in the piggery wastewater (Fig. 3a). The level of humic-like substances was slightly increased by ozonation for 10 min, after which it gradually decreased with increasing ozonation duration due to both mineralization and low-molecularization (see Supplementary data, SD-2). For low MW acids, the amount of organic carbon converted from larger organic components had an increasing tendency with the increasing ozonation duration. Unlike low MW acids, some fractions in low MW neutrals were removed by ozonation for 10 min, probably due to their electrophilic or nucleophilic characteristics that can very quickly react with molecular ozone. Fig. 4 shows that fulvic acid-like substances (peak region D) that dominated among the fluorescent organic constituents in pretreated piggery effluent. Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with some ␲ bonds [29]. The fluorescence EEM spectra of organic materials in wastewater samples were normalized for further interpretation based on the fluorescence intensities at the highest peak observed for the pretreated piggery effluent (Table 2). The results indicated that algae treatment was believed to remove protein-like substances (peak regions A and B) to a much greater extent than humus-like substances (peak regions C and D). On the other hand, the residual humus-like substances were dramatically removed by ozonation at a minimum of 10 min. These trends indicated that organic fractions with a higher degree of aromaticity were accumulated as a result of microalgae cultivation and that post-ozonation can be adopted to remove the fluorescent moieties in the residual hydrophobic organics. 4. Conclusions Algae treatment followed by ozonation were successful in treating piggery wastewater and resulted in the following benefits: (1) effective biological treatment with A. obliquus KGE-17 even at the high salinity typical of piggery wastewaters and (2) lower discharge concentrations and greater reliability for compliance with color goals. The simultaneous removal of inorganic nutrients and carbonaceous organics using A. obliquus was also achieved in a single treatment process under the heterotrophic growth conditions. Prior to conveying the effluent to neighboring domestic wastewater treatment facilities, highly-condensed organic fractions accumulated in algae-treated wastewater were effectively low-molecularized and/or mineralized by ozonation. Acknowledgment This work was supported by the Korea Institute of Science and Technology (KIST) Institutional Program (2E26300), the Eco-Innovation Technology Development Project (Grant no. 405112-037) funded by the Ministry of Environment, and the R&D Program for Society of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (2014M3C8A4030496), South Korea. This research was also partially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015R1A2A2A03005744).

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2016.02.011.

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