Evaluation of the dynamics of microalgae population structure and process performance during piggery wastewater treatment in algalbacterial photobioreactors

1 E alua ion o he dynamics o mic oalgae popula ion s uc u e and 1 p ocess pe o mance du ing pigge y was ewa e ea men in algal-2 bac e ial pho obio eac o s 3 Dimas Ga cía1,2, Es he Posadas1, Saúl Blanco3, Gab iel Acién4, Ped o Ga cía-Encina1, 4 Sil ia Bolado1, Raúl Muñoz1* 5 1Depa men o Chemical Enginee ing and En i onmen al Technology, Valladolid 6 Uni e si y, D . Me gelina, s/n, 47011, Valladolid, Spain 7 2Cen o pa a la In es igación de los Recu sos Acuá icos de Nica agua, CIRA/UNAN-8 Managua, Apdo. Pos al 4598 9 3The Ins i u e o he En i onmen , La Se na 58 - 24007 León, Spain 10 4Depa men o Chemical Enginee ing, Uni e si y o Alme ia, Cañada San U bano, s/n, 11 04120 Alme ia, Spain 12 *co esponding au ho : mu o [email protected] a.es 13 14 ABSTRACT 15 The dynamics o mic oalgae popula ion du ing pigge y was ewa e (PWW) ea men in 16 ou open pho obio eac o s ope a ed a 27 days o hyd aulic e en ion ime, and 17 inocula ed wi h Chlo ella sp. (R1), Acu odesmus obliquus (R2), Oscilla o ia sp. (R3) 18 and in he absence o inoculum (R4), we e e alua ed o 6 mon hs. In addi ion, he 19 algal-bac e ial biomass concen a ion, emo al o o ganic ma e , nu ien s and hea y 20 me als we e also assessed. The esul s e ealed a high di e si y and apid a ia ions in 21 he s uc u e o mic oalgae popula ions, Chlo ella sp. being dominan in R4 h oughou 22 mos o he ope a ional pe iod. S eady s a e a e age biomass concen a ion anged om 23 2445-2610 mg/L in R1-R3 o 3265 mg/L in R4. No signi ican di e ences we e 24 eco ded in he emo al e iciencies (REs) o o al o ganic ca bon (86-87%), ino ganic 25 *Manusc ip Click he e o iew linked Re e ences 2 ca bon (62-71%), o al ni ogen (82-85%) and o al phospho ous (90-92%). Finally, Zn-26 REs accoun ed o 26% in R3, 37% in R2, and 49% in R1 and R4. 27 28 Keywo ds: Algal-bac e ial p ocesses; biomass p oduc ion; hea y me al emo al; 29 mic oalgae dynamics; pigge y was ewa e ea men . 30 31 1. In oduc ion 32 The cu en global ene gy and clima e change c isis has igge ed he ques o 33 al e na i e g een ene gy sou ces wi h a low ca bon dioxide (CO2) oo p in (González-34 Fe nández e al., 2012a). In his con ex , mic oalgae ha e eme ged as a p omising 35 enewable ene gy pla o m due o hei abili y o ans o m sunligh di ec ly in o gas 36 bio uels (i.e H2) o an o ganic biomass eeds ock ha can be u he biocon e ed in o 37 mul iple liquid and gas bio uels (Richmond, 2004). Thus, mic oalgal biomass can be 38 anae obically diges ed yielding biogas (CH4 + CO2) and a nu ien ich diges a e 39 (Ehimen e al., 2011; González-Fe nández e al., 2012b). In addi ion, while he lipid 40 ac ion o mic oalgae can be anses e i ied in o biodiesel (Vimala asan e al., 2011), 41 he ca bohyd a e ac ion can be e men ed in o bioe hanol (Naik e al., 2010) o 42 biohyd ogen (Chand asekha e al., 2015). Mic oalgae exhibi mul iple ad an ages o e 43 con en ional ene gy c ops such as high a eal p oduc i i ies (50-100 n/ha·y), cul i a ion 44 in non-a able land (p e en ing compe i ion wi h ood) and high lipid o ca bohyd a e 45 ac ions depending on he cul i a ion condi ions. Likewise, mic oalgae can be 46 cul i a ed in esh, ma ine o was ewa e s (Cheah e al., 2016). 47 48 In his con ex , nu ien - ich was ewa e s ep esen a aluable eeds ock o educe he 49 cos s o mic oalgae and cyanobac e ia ( om now on e e ed o as mic oalgae) 50 3 cul i a ion, which will ul ima ely inc ease he cos -compe i i eness o mic oalgae-based 51 bio uels (Acién e al., 2016). Algal-bac e ial symbiosis can combine a low-cos mass 52 p oduc ion o biomass wi h he ea men o was ewa e o le els equi ed o discha ge 53 in o na u al wa e bodies. Indeed, bo h domes ic, indus ial and li es ock was ewa e s 54 ha e success ully suppo ed mic oalgae cul i a ion (Muñoz e al., 2003; Muñoz and 55 Guieysse, 2006). Du ing mic oalgae-based was ewa e ea men , bo h he o ganic 56 ca bon, ni ogen and phospho ous p esen in he esidual e luen a e assimila ed in o 57 algal-bac e ial biomass. Hea y me als and pa hogens a e also e icien ly emo ed 58 du ing mic oalgae g ow h as a esul o adso p ion and pH-media ed mechanisms. 59 Despi e mic oalgae cul i a ion in was ewa e en ails signi ican economic and 60 en i onmen al ad an ages o e axenic mass p oduc ion o mic oalgae in mine al sal 61 media, con o e sy s ill exis s in li e a u e abou he possibili y o main aining 62 monoalgal cul u es wi h a cons an biomass composi ion du ing mic oalgae-based 63 was ewa e ea men . This is cen al o he de elopmen o mic oalgae-based 64 bio e ine ies o bio uel p oduc ion, whose iabili y depends on he supply o a biomass 65 wi h a consis en yea - ound composi ion and cha ac e is ics. Hence, while mos s udies 66 conduc ed unde labo a o y o ou doo s condi ions ocused on he emo al o key 67 pollu an s p esen in was ewa e , li le a en ion has been paid o he moni o ing o he 68 dynamics o mic oalgae popula ion. 69 70 Pig p oduc ion is a key economic sec o in many coun ies in Eu ope, accoun ing o 71 148.7 million pigs heads and 44.3% o he o al Eu opean li es ock (EU, 2015; 72 MAGRAMA, 2015) in 2015. Eu opean pig a ming gene a es 217- 434 million m3/y (4-73 8 L/day/pig) o pigge y was ewa e con aining high concen a ions o o ganic ma e 74 and nu ien s (De Godos e al., 2009). The es ima ed a e age o ganic ma e and nu ien 75 4 load p esen in EU pigge y was ewa e s in 2015 amoun ed o 8.923.000 n chemical 76 oxygen demand (COD)/y, 890.000 n ni ogen (N)/y and 223.000 n phospho ous (P)/y 77 (EU, 2016). In addi ion, pigge y was ewa e can con ain high concen a ions o hea y 78 me als such as Zinc and Coppe , ypically used as g ow h p omo e s in swine nu i ion 79 (Abe e al., 2012; De la To e e al., 2000). 80 81 The expe imen al wo k he ein conduc ed e alua ed he dynamics o mic oalgae 82 popula ion du ing pigge y was ewa e ea men in ou open con inuous 83 pho obio eac o s inocula ed wi h wo g een mic oalgae species, a cyanophy a, and 84 wi hou inoculum. In addi ion, he in luence o he mic oalgae inoculum on he s eady 85 s a e o ganic ma e , nu ien and hea y me al emo al was assessed. 86 87 2. Ma e ials and me hods 88 2.1. Mic oalgae 89 Chlo ella minu issima Fo and No áko á was ob ained om an indoo high a e algal 90 pond (HRAP) ea ing cen a e a he Dep . o Chemical Enginee ing and En i onmen al 91 Technology om Valladolid Uni e si y (Spain). Acu odesmus obliquus and Oscilla o ia 92 sp we e kindly p o ided by he Depa men o Chemical Enginee ing om Alme ia 93 Uni e si y (Spain). 94 95 2.2. Pigge y was ewa e 96 F esh cen i uged pigge y was ewa e (PWW) was collec ed a a nea by a m a 97 Can alejo (Spain) and s o ed a 4°C. The a e age composi ion o he pigge y was ewa e 98 dilu ed a 15% was: 1340±34 mg/L o o al suspended solids (TSS), 1375±121 mg/L o 99 5 o al o ganic ca bon (TOC), 314±55 mg/L o ino ganic ca bon (IC), 393±26 mg/L o 100 o al ni ogen (TN), 9.4±0.4 mg/L o o al phospho us (TP) and 0.7±0.2 mg/L o zinc 101 (Zn). Ni a e (NO3-), ni i e (NO2-), coppe (Cu) and a senic (As) concen a ions 102 emained below de ec ion limi (Table 1). 103 104 <Table 1> 105 106 2.3. Expe imen al se -up 107 The expe imen al se -up consis ed o ou 15.8 cm deep 3 L open pho obio eac o s 108 illumina ed a 2800 µmol/m2·s o 12 hou s a day (08h00 o 20h00) by LED lamps 109 a anged in a ho izon al con igu a ion 20 cm abo e he pho obio eac o su ace 110 (Figu e1). The pho obio eac o s we e imme sed in a wa e ba h o p e en he high 111 empe a u es imposed by he LEDs i adia ion. Imme sion wa e pumps we e used o 112 mix he algal-bac e ial cul i a ion b o h in he eac o s. The pho obio eac o s we e ed 113 wi h pigge y was ewa e dilu ed a 15% using an au o con ol 205U7CA mul i-channel 114 casse e pump (Wa son-Ma low, UK). The pH in he cul i a ion b o h was 115 au oma ically main ained a 8.0 ia CO2 addi ion (CARBUROS METALICOS- 116 Ba celona, Spain) using a C ison mul ime e M44 con ol uni (C ison Ins umen s, 117 Spain). 118 119 ˂ Figu e 1˃ 120 121 2.4. Expe imen al design 122 6 Pho obio eac o s 1, 2 and 3 (namely R1, R2 and R3, espec i ely) we e inocula ed wi h 123 Chlo ella minu issima Fo and No áko á, Acu odesmus obliquus and Oscilla o ia sp., 124 espec i ely, a an ini ial TSS concen a ion o 220 mg/L (co esponding o ini ial cell 125 concen a ions o 1.750, 0.295 and 0.332·109 cells/L, espec i ely). Pho obio eac o 4 126 (R4) was no inocula ed and se ed as con ol. The pho obio eac o s, which we e 127 ini ially illed wi h ap wa e , we e ope a ed a a hyd aulic e en ion ime (HRT) o ≈ 27 128 days (es ima ed based on he in luen PWW) o 176 days. Pho obio eac o s e luen s 129 o e lowed sepa a ely as a unc ion o he e apo a ion a es. Liquid samples o 30 mL 130 we e weekly d awn om he in luen PWW and e luen o R1, R2, R3 and R4 o 131 de e mine he concen a ions o TOC, IC, TN, NO2-, NO3-, TP and TSS. E luen 132 samples we e il e ed h ough 1 µm glass ibe il e s p io analysis. Likewise, he 133 mic oalgae popula ion s uc u e in R1, R2, R3 and R4 was weekly assessed om 134 biomass samples p ese ed wi h lugol acid a 5% and o maldehyde a 10%, and s o ed 135 a 4 ºC p io o analysis (only 8 samples om each pho obio eac o we e analyzed). The 136 dissol ed oxygen and empe a u e o he cul i a ion b o hs we e measu ed wice pe 137 day, while he in luen and e luen low a es we e daily eco ded in all 138 pho obio eac o s o moni o wa e e apo a ion losses. Finally, he C, N and P con en o 139 he algal bac e ial biomass was measu ed unde s eady s a e a he end o he 140 expe imen . 141 142 The C, N and P emo al e iciencies (RE) we e calcula ed acco ding o Eq. (1): 143 (1) 144 whe e C eed and Ce ep esen he dissol ed concen a ions o TOC, IC, TN, TP and Zn 145 in he PWW and pho obio eac o s e luen s, espec i ely, while Q eed and Qe ep esen 146 7 he PWW and e luen low a es. The p ocess was conside ed unde s eady s a e when 147 he TSS concen a ions in he pho obio eac o s emained s able o a leas ou 148 consecu i e samplings (~ 1 mon h). The esul s we e he e p o ided as he a e age ± 149 s anda d de ia ion om duplica e measu emen s along one mon h o s eady s a e (days 150 150-176). 151 152 2.5 Analy ical p ocedu es 153 A C ison M44 mul ime e and a C ison PH 28 me e we e used o he on-line 154 measu emen o he pH. Dissol ed oxygen (DO) and empe a u e (T) we e eco ded 155 using an OXI 330i oxime e (WTW, Ge many). A LI-250A ligh me e (LI-COR 156 Biosciences, Ge many) was used o measu e he ligh in ensi y as pho osyn he ically 157 ac i e adia ion (PAR). TOC, IC and TN concen a ions we e de e mined using a TOC-158 V CSH analyze equipped wi h a TNM-1 module (Shimadzu, Japan). Ni a e and ni i e 159 we e analyzed by high pe o mance liquid ch oma og aphy-ion conduc i i y (HPLC-IC) 160 in a Wa e s 515 HPLC pump coupled wi h a Wa e s 432 ionic conduc i i y de ec o and 161 equipped wi h an IC-Pak Anion HC (150 mm × 4.6 mm) column. TSS and TP 162 concen a ions we e de e mined acco ding o S anda d Me hods (APHA, 2005). The 163 analysis o he C, N and P con en in he algal-bac e ial biomass was ca ied ou using a 164 LECO CHNS-932 elemen al analyze wi h p e-d ied and g inded algal-bac e ial 165 biomass. The concen a ion o Zn, Cu and As was de e mined using a 725-ICP Op ical 166 Emission Spec opho ome e (Agilen , USA) a 213.62. The iden i ica ion and 167 quan i ica ion o mic oalgae we e conduc ed by mic oscopic examina ion (OLYMPUS 168 IX70, USA) acco ding o Phy oplank on Manual (Sou nia, 1978). 169 170 8 3. Resul s and Discussion 171 3.1. Dynamics o mic oalgae popula ion 172 Chlo ella sp., he inocula ed mic oalgae species in R1, was de ec ed h oughou mos o 173 he expe imen al pe iod in his pho obio eac o and dominan a days 37 and 86 (a 174 concen a ions o 0.5·109 and 0.9·109 cells/L, espec i ely). Acu odesmus obliquus was 175 also iden i ied in R1 and became he dominan species by day 58. Finally, Aphano hece 176 sp. was de ec ed o he i s ime by day 58 and was dominan om day 122 o he end 177 o he ope a ion o R1 (Figu e 2a). Simila ly, he inocula ed mic oalga species in R2 178 (Acu odesmus obliquus) was iden i ied along he en i e pho obio eac o ope a ion, wi h 179 a signi ican dominance by days 37, 58 and 122 a cell concen a ions o 1.3·109, 180 1.8·109 and 0.3·109 cells/L, espec i ely. Chlo ella sp. was iden i ied in R2 om he 181 i s ope a ional days and emained a simila cell concen a ions h oughou he en i e 182 expe imen ( om 0.3·109 o 0.7·109 cells/L). Finally, Aphano hece sp. became 183 dominan in R2 by he end o ope a ion, wi h inal cell concen a ions o 2.9·109 cells/L 184 (Figu e 2b). Oscilla a ia sp. was eplaced by Chlo ella sp. and Acu odesmus obliquus in 185 R3 om he i s ope a ional days (a e he inocula ion a change in colo om g een o 186 ed was no iced), Chlo ella sp. being he dominan species h oughou he en i e 187 ope a ion wi h a maximum concen a ion o 8.2·109 cells/L by day 58 (Figu e 2c). The 188 highe pollu ion- ole ance o Chlo ella sp. o PWW, combined wi h he high 189 empe a u e and i adia ions p e ailing in his s udy, could ha e caused his apid 190 eplacemen o Oscilla o ia sp (Talbo e al., 1991). Despi e R4 was no inocula ed, 191 Chlo ella sp. and Aphano hece sp. we e p esen in he pho obio eac o om he i s 192 days, Chlo ella sp. being he dominan species along he 6 mon hs o expe imen . The 193 9 g adual inc ease in numbe o cells o Aphano hece sp. in R1, R2 and R4 sugges he 194 in luence o he cha ac e is ics o he PWW on mic oalgae popula ion (Figu e 2). 195 196 The highe dominance o Chlo ella sp. in he ou pho obio eac o s con i med he high 197 ole ance o his g een mic oalgae o he pollu an s and concen a ions ypically p esen 198 in PWW (Kim e al., 2016; Kuo e al., 2015; Yuan e al., 2013). Indeed, he high 199 abundance o Acu odesmus obliquus and Chlo ella sp. (bo h belonging o he 200 Chlo ophy a phylum) along he expe imen al pe iod in R1, R2 and R3 ma ched he 201 mic oalgae pollu ion- ole ance classi ica ion epo ed by Palme e al. (1969), who 202 anked Scenedesmus and Chlo ella 4 h and 5 h, espec i ely. I can be hypo hesized ha 203 o ganic pollu ion exhibi ed a highe in luence on mic oalgae popula ion s uc u e han 204 o he en i onmen al pa ame e s such as wa e ha dness, ligh in ensi y, pH, DO o 205 empe a u e (Palme , 1969). On he o he hand, Aphano hece sp., which was no 206 p e iously classi ied as a pollu ion ole an mic oalga, was mainly iden i ied a he end 207 o expe imen in R1 and R2 (Palme , 1969). Howe e , Aphano hece mic oscopica 208 nägeli and Aphano hece Cla h a a success ully suppo ed he emo al o o ganic ma e 209 and ni ogen om pa boiled ice was ewa e (REs o 83.4 and 72.7% o COD and N-210 TKN, espec i ely) in a 4.5 L ubula pho obio eac o ope a ed ba chwise o 24 hou s 211 (Quei oz e al., 2007). Likewise, Bas os e al. (2014) epo ed COD and N-TKN REs o 212 97 and 78%, espec i ely, in a 4L ba ch ubula eac o ea ing pa boiled ice 213 was ewa e o 24 hou s. 214 215 The lack o monoalgal cul u es in he ou pho obio eac o s h oughou he 216 expe imen al pe iod and he apid a ia ions in mic oalgae popula ion s uc u e he e 217 eco ded (mainly in R1 and R2) e ealed he di icul y o main ain monoalgal cul u es 218 16 al., 2006). Highe Zn-REs by biosopo ion would be expec ed a highe pHs acco ding 369 o Muñoz e al. (2006), who obse ed an inc ease in Zn accumula ion in o he algal-370 bac e ial biomass om 5.0 o 11.7 mg Zn/g biomass when pHs was aised om 7 o 9, 371 espec i ely. The de e mina ion o coppe and a senic emo al e iciencies was no 372 possible based on he low concen a ions o hese me als in he PWW (below he 373 de ec ion limi o he ins umen = 0.6 mg/L). 374 375 4. Conclusions 376 This esea ch e ealed he di icul y o main ain monoalgal cul u es du ing PWW 377 ea men in open-pho obio eac o s ope a ed unde simila en i onmen al and 378 ope a ional condi ions. The high abundance o Chlo ella sp. in mos pho obio eac o s 379 con i med he high ole ance o his mic oalga o he pollu an s. The acclima ion o 380 na i e species o he cha ac e is ics o he PWW esul ed in highes biomass 381 concen a ions. An e icien PWW ea men occu ed ega dless o he mic oalgae 382 species inocula ed, which con i med he obus ness o algal-bac e ial p ocesses de o ed 383 o ca bon and nu ien emo als om li es ock was ewa e s. Finally, he hea y me als 384 can be emo ed by bioso p ion in o he algal-bac e ial biomass p oduced du ing PWW 385 bio emedia ion. 386 387 5. Acknowledgmen s 388 This esea ch was suppo ed by INIA, he Eu opean FEDER p og am (RTA2013-389 00056-C03-02), he Regional Go e nmen o Cas illa y León (P ojec VA024U14 and 390 UIC 71) and MINECO (Red No eda ). 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C ( ), N ( ), and P ( ) con en in he biomass p esen in he 539 pho obio eac o s unde s eady s a e. 540 Figu e 1. Schema ic diag am o he algal-bac e ial pho obio eac o se -up using ca bon dioxide supplemen a ion o pH con ol. Figu e Click he e o download Figu e: Figu e 1_Ga cia_BITE.docx Figu e 2. Time cou se o mic oalgae popula ion s uc u e in (a) R1, (b) R2, (c) R3 and (d) R4. Acu odesmus obliquus ( ), Aphano hece sp. ( ), Chlo ella sp. ( ), Oscilla o ia sp. ( ) and o al numbe o mic oalgae cells (●). Figu e Click he e o download Figu e: Figu e 2_Ga cia_BITE.docx