Bioremediation of aquaculture wastewater from Mugil cephalus (Linnaeus, 1758) with different microalgae species

1 Bioremediation of aquaculture wastewater from Mugil cephalus 1 (Linnaeus, 1758) with different microalgae species 2 3 1Valeria Andreottia,b*, Anuta Chindrisb, Gianni Brundub,c, Dario Vallaincb,c, 4 Matteo Francavillad, Joan Garcíaa 5 6 7 a GEMMA – Group of Environmental Engineering and Microbiology, 8 Department of Civil and Environmental Engineering, Universitat 9 Politècnica de Catalunya-BarcelonaTech, c/Jordi Girona, 1-3, Building D1, 10 E-08034, Barcelona, Spain. 11 b International Marine Centre IMC, Loc. Sa Mardini 09170, Torregrande 12 (OR) Italy. 13 c Department of Ecological and Biological Sciences (DEB) “Tuscia 14 University”, Viale dell' Università, snc 01100, Viterbo, Italy. 15 d Star*AgroEnergy Research Unit, Department of Agriculture, Food and 16 Environment, University of Foggia, Via Napoli, 25, 71122 Foggia, Italy. 17 18 19 1 Correspondence to: Valeria Andreotti, International Marine Centre IMC, Loc. Sa Mardini 09170, Torregrande (OR) Italy. E-mail: [email protected] 2 Abstract 20 Current aquaculture practices have a detrimental impact on the environment, 21 in particular due to the release of high concentration of nitrogen and 22 phosphorus that can induce eutrophication. This study investigates and 23 compares the capacity of three microalgae species Tetraselmis suecica, 24 Isochrysis galbana and Dunaliella tertiolecta, in the bioremediation of grey 25 mullet Mugil cephalus wastewater. 26 The experiment was conducted in batch conditions for 7 days using 27 completely mixed bubble column photobioreactors. After two days, T. 28 suecica and D. tertiolecta were able to remove more than 90% of Dissolved 29 Inorganic Nitrogen (DIN) and Dissolved Inorganic Phosphorous (DIP), 30 whereas I. galbana removed only 32% and 79% of DIN and DIP, 31 respectively. A higher biomass yield resulted for T. suecica (0.60 ± 0.03 32 g/L, mean ± SE). 33 This study confirms the potential to employ T. suecica in an Integrated 34 Multi Trophic Aquaculture system for bioremediation of wastewater and 35 identifies D. tertiolecta as another valid candidate species. Moreover, these 36 species can growth in unsterilized culture media, and this reduces energy 37 consumption, costs and efforts. 38 39 Keywords: phytoremediation, biotreatment, bioreactors, wastewater, algae. 40 41 3 1.1 Introduction 42 Aquaculture is one of the fastest-growing food producing sectors in the 43 world, providing almost about 50% of all fish for human consumption; 44 within 2030, this share is projected to rise to 62% (FAO, 2014). On the 45 other hand, aquaculture represents one of the major contributors to the 46 increasing levels of dissolved and particulate nutrients in the aquatic 47 ecosystems (Lamprianidou et al., 2015). A high nutrient loading into the 48 aquatic environment, in particular nitrogen and phosphorus may cause 49 eutrophication, oxygen depletion and siltation (Burford et al., 2003). 50 With the aim to reduce the impacts of traditional aquaculture, several 51 Countries around the world are developing Integrated Multi-Trophic 52 Aquaculture (IMTA) systems, which re-uses the wastewaters for the growth 53 of micro and macroalgae. Indeed, aquaculture wastewater provides nutrients 54 (ammonia, nitrite, nitrate, dissolved organic nitrogen and phosphate) 55 (Converti et al., 2006; Soletto et al., 2005; Abe et al., 2002) which can be 56 used for the production of microalgae. The uptake of dissolved nutrients by 57 microalgae is considered as the main way to remove nitrogen in aquaculture 58 wastewaters (Attasat et al., 2013; Sirakov et al., 2013). 59 Previous studies showed that it is possible to remove nutrients from 60 wastewater (fishes and shrimp production plants) employing microalgae and 61 macroalgae as key elements in biological treatments (Gao et al., 2016; 62 Michels et al., 2014; Sirakov and Velichkova, 2014; Bartoli et al., 2005; 63 Borges et al., 2005; Lefebvre et al., 2004; Hussenot et al., 1998; Lefebvre et 64 al., 1996; Hammouda et al., 1995; Shpigel et al., 1993). 65 4 This phycoremediation is an eco-friendly method that offers the advantage 66 to be a low-cost way to nutrient removal (Mulbry et al., 2008). In addition, 67 the biomass produced through bioremediation could have multi-purpose 68 uses including fuels, fertilizers, fine chemicals production and feed in 69 aquaculture (Mulbry et al., 2006; Vilchez et al., 1997). 70 One of the most common microalgae species employed in aquaculture 71 bioremediation wastewater is Tetraselmis spp. (Michels et al., 2014; Sirakov 72 and Velichkova, 2014; Borges et al., 2005). A recent study Michels et al., 73 (2014) showed for the first time that it is possible to use Tetraselmis suecica 74 for the nutrient assimilation of fishfarm wastewater throughout its 75 cultivation in controlled photobioreactors. 76 The aim of this study is to evaluate and compare the capability of T. 77 suecica, Isochrysis galbana and Dunaliella tertiolecta, widely used in 78 aquaculture as feed for rotifers (Mason 1963), echinoderms (Brundu et al., 79 2016a, 2016b; Paredes et al., 2015; De La Uz et al., 2013; Azad et al., 2011; 80 Miller and Emlet 1999; Zamora and Stotz 1994;), filter feeders (Nevejan et 81 al., 2003; Carboni et al., 2016) and fin fishes (Fabregas et al., 1986), for the 82 removal of dissolved inorganic nutrients (nitrogen and phosphorous) of 83 wastewater aquaculture. We evaluate the biomass yield of these species in 84 controlled bubble column annular photobioreactors, by using untreated 85 mullet wastewater as culture medium. Contrarily to previous studies that 86 sterilized the wastewater before its use for bioremediation to eliminate 87 zooplankton, bacteria and suspended solids (Michels et al., 2014), we 88 avoided the use of expensive pre-treatment procedures as filtration and 89 5 sterilization, aiming to reduce the costs of seawater treatment and simulate 90 more real operation conditions of a wastewater treatment system. 91 92 2.1 Materials and methods 93 2.1.1 Aquaculture wastewater 94 Aquaculture wastewater was provided by an experimental fish hatchery 95 located in the International Marine Centre - IMC Foundation (Oristano, 96 Sardinia, Italy). Juveniles of grey mullet Mugil cephalus (Linnaeus, 1758) 97 were obtained in laboratory and reared in a recirculating aquaculture system 98 (RAS) consisting of 4 tanks of 2000 L volume. In this system, the tanks 99 were linked in a single biological (trickling filter) and cartridge mechanical 100 filter (10 µm) and supplied with UV lamp (UVPE5, 80 W) and protein 101 skimmer (Panaque). Temperature was maintained at 23 ± 2 °C (mean ± SE) 102 with a chiller (TECO TR60, 0.91 Kw) and natural photoperiod (14/10 L/D) 103 was adopted (Figure 1). 104 Natural seawater (NSW) at 37.0 ± 1.0 ppt salinity was previously micro-105 filtered (0.5 µm) and UV lamp sterilized. Juveniles of 0.35 ± 0.43 g body 106 weight (BW) were fed at 3% BW per day with the commercial formulated 107 feed for sea fish supplied by Skretting SpA (PERLA LARVA) composed of 108 62% crude protein, 11% crude oils and fats, 9% crude ash, 0.8% crude fiber 109 and 1.2% crude phosphorus. Fishes were stocked at an average density of 110 0.5 g body weight/L. 111 Tanks were monitored daily for checking mortality; the uneaten food and 112 faeces were siphoned out twice a week for maintaining good water quality. 113 A 30% water exchange was weekly performed, and a part of this 30% was 114 6 employed as wastewater in our experiment. Wastewater was taken at the 115 inlet of the tank, after UV lamp. 116 117 2.1.2 Microalgae culture 118 The microalgae species were provided by the Agency for Agricultural 119 Research in Sardinia (AGRIS) and sourced from the Culture Collection for 120 Algae and Protozoa (CCAP: Oban, Scotland). Pre-culture inocula were 121 permanently kept in Erlenmeyer flasks in Pyrex glass with total capacity of 122 2 L, closed with cotton and covered with gauze and aluminum foil. NSW 123 was autoclaved at 121 °C for 30 min and enriched with Guillard F/2 124 medium (Guillard 1975; Guillard and Ryther 1962). Cultures were exposed 125 to a constant illumination (155 μmol/s/m2) provided by 4 fluorescent lamps 126 (OSRAM type Natura). Continuous aeration 3 L/min was supplied by 127 peristaltic pump (ECOH Air Pump) and temperature was maintained at 23 128 °C by air conditioning. 129 130 2.1.3 Experimental design 131 Nutrient uptake and biomass production of T. suecica, I. galbana and D. 132 tertiolecta were evaluated during seven days in batch conditions using two 133 completely mixed bubble column photobioreactors of 6 L; five runs were 134 done for a total of three replicates per treatment. 135 Lighting system was composed by four neon daylight lamp (four fluorescent 136 lamps type cool daylight, OSRAM Lumilux FQ 24W/865), with light 137 7 intensity of 100 μmol/s/m2. This system was monitored with a 138 Programmable Logic Controller (PLC) that it is a device that performs 139 discrete or continuous control logic in process plant or factory environments 140 (Figure 2). These controllers are hardware and software engineered 141 microcomputers, used to provide industrial control operations (Netto et al., 142 2013). Reactors were equipped with temperature and aeration regulation 143 control system; temperature was maintained at 23 °C, aeration was ensured 144 by a blower at flow rate of 3 L/min. On the contrary, pH was not controlled 145 and resulted at 7.7 ± 0.2. Phytoplankton laboratory-culture methods and 146 photobiorectors operation were adopted according to Saiu et al., (2016). 147 Microalgae growth was measured as dry weight biomass (DW) (Clasceri et 148 al. 1999). DW was measured once a day in 40 mL of water sample 149 previously filtered through 0.45 μm Whatman fiber-glass. After filtration, 150 filters were washed with 20 mL of deionized water to remove salts and dried 151 in an oven at 105 °C until constant weight, following Saiu et al., (2016). The 152 supernatant liquid fraction obtained after filtration was used for nitrate, 153 nitrite, ammonia and phosphorous analysis. In order to monitor the 154 microalgae nutrient uptake, nutrients were daily analysed by an automatic 155 chemical analyzer µCHEM based on Loop Flow Analysis (Systea, Italy). 156 Microalgae removal efficiencies of Dissolved Inorganic Nitrogen (DIN) and 157 Dissolved Inorganic Phosphorous (DIP) were calculated according to the 158 method used by Michels et al., (2014), as follow: 159 N removal efficiency (%) = ((DIN influent - DIN effluent) / DIN influent) x 160 100 161 8 P removal efficiency (%) = ((DIP influent - DIP effluent / DIP influent) x 162 100 163 DIN values were calculated as the sum of nitrite (NO2-), nitrate (NO3-) and 164 ammonia (NH4+), while DIP corresponded to the total dissolved phosphate 165 (PO43-). 166 167 2.1.4 Statistical analysis 168 Data were analyzed by Statistica 6.1 StatSoft, Inc. (2004). Differences in the 169 removal efficiences among phytoplankton species were analysed using 170 analysis of variance (ANOVA). Shapiro Wilk’s W test was used to verify 171 the normality of the data distribution and Levene's test was used to verify 172 the homogeneity of variances. Biomass was analyzed using repeated-173 measures ANOVA, with species as independent factor and days as repeated 174 factor. Tukey's honestly-significant difference (HSD) test was used to 175 evaluate all pair-wise treatment comparisons (p < 0.05). 176 177 3.1 Results 178 The nutrient concentration of the wastewater was regularly measured before 179 each experiments (Table 1). It was possible to observe that the composition 180 of wastewater was very similar in each experiment, being nitrate the N 181 species with the higher concentration. 182 9 3.1.1 Nutrients removal efficiency 183 At the end of the experiment a clearly higher DIN removal efficiency (p < 184 0.001, two-way ANOVA) resulted for T. suecica (94.4 ± 1.0%, mean ± SE) 185 and D. tertiolecta (95.4 ± 0.3%) in comparison with I. galbana (66.0 ± 186 1.5%). There were not statistical differences between the three species in the 187 removal of DIP at the end of the experiments (Table 2). 188 T. suecica and D. tertiolecta showed a similar pattern of nutrient uptake 189 (Figure 3 A, 3 C). Both species removed more than 90% of DIN and DIP 190 after 2 and 1 day, respectively. On the contrary, I. galbana showed a slower 191 nutrient uptake, lower than 35% and 80% removal for DIN and DIP, 192 respectively, after 2 days (Figure 3 B). The nutrient uptake of DIN showed 193 significant differences (p < 0.001) between I. galbana and the other two 194 phytoplankton species (Repeated-measures ANOVA). 195 196 3.1.2 Biomass yield 197 Ciliate protozoan Paramecium spp. was observed in all cultures through the 198 duration of the experiement, but we did not evaluate the abundance of this 199 species. This was mainly due to lack of the wastewater pre-treatment 200 procedures (i.e. filtration and sterilization). We found a significant 201 difference in biomass yield among the three species (Repeated measures 202 ANOVA, p < 0.001). T. suecica resulted in a higher DW (0.57 ± 0.02 g/L, 203 mean ± SE) than I. galbana (0.12 ± 0.01 g/L) from 3 days up to the end of 204 the experiment, 0.60 ± 0.03 g/L for T. suecica and 0.16 ± 0.02 g/L for I. 205 16 Bulletin, 50, 1386-1397. 337 Borges, M.P., Silva, P., Moreira, L. and Soares, R. (2005). 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Nutrients dissolved in the Mugil cephalus wastewater. Values are expressed as mean ± 513 SE (n= 3). 514 Tetraselmis suecica Dunaliella tertiolecta Isochrysis galbana NO3- -N (mg/L) 4.1 ± 0.4 4.2 ± 0.1 4.2 ± 0.4 NO2- -N (mg/L) 0.2 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 NH4+ -N (mg/L) 0.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 PO43- -P (mg/L) 0.3 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 23 547 548 549 550 551 Table 2. Influent and effluent DIN and DIP values (mg/L) and removal efficiency (%) of 552 Tetraselmis suecica, Dunaliella tertiolecta and Isochrysis galbana. Values are expressed as mean 553 ± SE (n= 3). Superscripts indicate significant differences among species. 554 555 Tetraselmis suecica Dunaliella tertiolecta Isochrysis galbana DIN Influent (mg/L) 4.5 ± 0.5 4.6 ± 0.1 4.6 ± 0.5 DIN Effluent (mg/L) 0.3 ± 0.1 0.2 ± 0.1 1.6 ± 0.1 DIN % 94.4 ± 1.0 a 95.4 ± 0.3 a 66.0 ± 1.5 b DIP Influent (mg/L) 0.3 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 DIP Effluent (mg/L) 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 DIP % 96.0 ± 2.5 91.2 ± 2.3 91.9 ± 4.0 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 24 584 585 586 587 Figure 1: Recirculating aquaculture system (RAS) for rearing of juvenile grey 588 mullets Mugil cephalus, consisting of four circular fiberglass tanks with 2000 589 L volume (V1, V2, V3 and V4). The system was equipped with biological (BF) 590 and mechanical filter (MF), protein skimmer (PS), chiller (C) and UV lamp 591 (UV). Dotted arrow = seawater outlet; continuous arrow = seawater intake. 592 Figure 2: Bubble column annular photobioreactors of 6 L volume (R1 and 593 R2) used for the growth of phytoplankton, supplied with LIGHT, 594 Programmable Logic Controller (PLC), gentle aeration (AIR), probes for 595 temperature (T) and pH (pH). 596 Figure 3: Nutrient uptake (%) of Dissolved Inorganic Nitrogen (DIN) and 597 Dissolved Inorganic Phosphorous (DIP) for Tetraselmis suecica (A), Isochrysis 598 galbana (B) and Dunaliella tertiolecta (C), during 7 days. Values are expressed 599 as mean ± SE (n= 3). 600 Figure 4: Microalgal growth curves as DW (g/L) of Tetraselmis suecica, 601 Isochrysis galbana and Dunaliella tertiolecta, during 7 days. Values are 602 expressed as mean ± SE (n= 3). Superscripts indicate significant differences 603 among species. 604 605 606 607 608 Figure 2 609 610 611 25 612 613 614 Figure 2 615 616