Journal of Environmental Management 275 (2020) 111233 Contents lists available nt ScienceDirect Journal of Environmental Management ELSEVIER journal homepage: hiip:/wwelsevier-comilocateljenvman Research artiele ® Saline fish wastewater in biogas plants - Biomethanation toxicity and ee safe use Carlos O. Letelier-Gordo", Enrico Mancini”, Per Bovbjerg Pedersen, Irini Angelidaki”, loannis A. Fotidis Satna nas of Ac Ren, Sco fr Aine, So Rech oe, Taal tay of Doma DK 85, Hr Dem = Dipemt of Enamel Eagening Tcl Unter of Dea, pga Bang 15, DK 2800, Kp. yg, Denk Seto! of Col gern Sota Unrey Nang, 21006, China ARTICLE INFO ApsTRACT wert Increasing marine land based recitulting aquaculture sjstens (RAS) and suites environmental eguatons Mocha methane potntiat ose eve cllenges to te aquacultie industry on how to teat and dispose saline fish wastewaters The sh Co-igenion trnsewrer cond be incorporated into biogas tesco, bit cent, the effets of salinity on the bio — ‘uethanatlon proces are poorly known, TAs study ened to asses the tox off wastewater with different == salutes on the bomethasntion proces and to proponeoptinusn eo-dgeston sectatos for nainal mete Potential and se ase in biogas plants. Ress showed tha, depending on saliity nnd organic conten, ii Possible to efficiently co igs from 3.220 61.85% sh wasterater (V/¥ wastewater /santte) and improve the ‘maximum methape production at from 272 to 61859, respecvely compared to cow manure mono digestion, Additional, salinity was idetified ar the min inhibitor of biometianation process with a half maxinal intibory concetetion (Cz) of 437 gL, while spate reduction was identified a 4 seconduy inhibitor 1. Introduction Aquaculture production isthe key provider of healthy protein sour ‘es, for the constantly growing human population, and has shown, ‘globally an exponential growth during the past en years (PAO, 2018), However, the strict enviconmental regulations are curently constrain lng the expansion ofthe aquaculture sector, especially in the Nordic and Baltic countries (Dalsgaard et al, 2013), Recirculating aquaculture systems (RAS) represent a promising technology for intensifying fish production with reduced environmental impacts, In recirculating ‘aquaculture systems, the water consumption is substantially reduced {minimization of make-up water), leading to water souree indepen dence and a low discharge of effluents with a constant pollutant con centration. This allows a higher degree of control at the end-of pipe treatment than in traditional aquaculture systems (Cripps snd Berghe, 20005 Martins eal, 20105 Sul etal, 2013). During the lst ve yeas, large-scale marine, land-based RAS, producing berween 1000 and 90, (000 tons of mainly Adantic salmon (Salmo salar) and yellowtail ‘amberjack (Seriola lalandi) per year are constructed worldwide (Dalsgaard, 2017). With these production levels, i has been estimated {har 0.9-77 tons COD/day’of fish organie waste will be discharged, requiring further handling and treatment (Dalsgeard and Pederson, 2016). With an increasing construction of marine land based RAS, the treatment of saline waste brings addtional challenges. Europe, saline waste from aquaculture production can neither be disposed in the ‘common municipal wastewater ueatment plants nor landfilled (VD, 2000), Therefore, the aquaculture industry will require the incorpore tion of end of pipe technologies for handling saline organie matter, phosphorus and nitrogen effents Biomethanntion isa anaerobic digestion process where organic ma terials are degraded producing a biogas, whicl is @ mixture if manly methane (50-70% v/v) and carbon dioxide (30-80% v/¥) (Angelidakt ral, 2011). The digestion process begins with extracellular bacterial hydrolysis, where insoluble organie polymers (i.e. enrbohyrates, pro teins lipids), are broken down fo soluble derivatives (i.e. sugars, amino acids, long chain fatty acids), that are further converted into carbon dioxide, hydrogen and volatile ftey acids (VFAS). The VFAS are subse quendly fermented into acctate, Finally, methanogens convert these Conresponding author: Deputment of Environmental Enginesing, Technical Univesity of Denna, Bygningorvet Byging 115, DK-2800, Kgs. Lyngby Denmark, ‘Email addrese osaforenv Ad (LA. Folds ps /do.org/10.1016/),Jeuvman.2020.111285, Received 72 Api! 2020; Received i evsed form 29 June 2020; Accepted 12 August 2020 Available online 19 August 2020 (0301-4797/© 2020 Eee Lid Al ight servedproducts to methane, carbon dioxide and HS (Angeli et al, 2011). When property managed, biomethanation i # cost eicent method for ‘organic waste reduction with concomitant production of renevable ‘energy as bioges. Lately, the use of biomethanstion process in treating ‘aquaculture organie waste has increased, partly because biogas plants ‘eager receive fish organic waste, with an optimal dry matter content (59%) for biogas production. For example, fish organic waste is frequently collected and disposed into biogas plants in Denmark and Norway (personal communication of Thomas Wagner Sandel, Sashimi Royal, Denmark, ‘The major source of waste produced in intensive aquaculture systems Is letary nutrients provided inthe feed that has uot been assimilated by the cultured species (Cho ets, 1994; Timmons eal, 2009). Conse ‘quently, the feed composition and digestibility of the fish species to ‘words the feed ingredients will dictate the waste properties (So 1994; Cho and Bureau, 2001; Amirkolaie, 2011), Therefore, solids originating in aquaculture systems are majorly composed of faeces and nal percentage of uneaten feed (Pinions et, 20095 van jn, 1996). ‘The collected solids exhibit normally alow total sold content (15-390) (Mirzoyan et al, 2008; Piedrahia, 2003), but during the last years, technologies that concentrate the organic waste has become more frequent especially in marine land-RAS. This hes allowed to obtain & waste containing 5-7% dry matter, resembling more industrial waste water streams rather than municipal wastewater plants (Lefebvre and Moletta, 2006; Xiao and Roberts, 2010). Fish solid waste is mostly ‘composed of proteins, lipids, nitrogen free extracts, ash and phospho: rous (Dalsgaard and Pedersen, 20114.) showing CIN ratios between 23 ‘nd 27 and TEN valies ranging berween 5 and 10 gL (Letelier Gordo eal, 2017; Geer, 2004 Zhang etal, 2016; Mirzoyan eta, 2008), ‘The organic matter reduction yields and potential production rates of biogas from aquaculture waste wsing biomtethanation have beet well ‘established. Using freshwater aquaculture organic waste, total solids (TS) reduction of more than 80% has been reached, while the methan production yields have varied from 460 mil CH, @-? VS Clanars and Franci, 1998) to 318 4 29 mL CH, g-! VS (Suhr ot al, 2015). Using brackish nd saline organic waste (salinity 0.5-89 gL), organic matter reduction (2.65-80%), as well as biogas production (0.02-0.18 L CH, 4 COD), has found to vary substantially (Zhang et sl, 2013). Drackish as well as in saltwater, total ammonia nitrogen (TAN), long hain fatty acs (LCFAS) and sodium (Na) have been suggested to be the main inhibitors of biogas production (Gebauer, 2004 Gebauer and Ekebrokk, 2006; Lo et al, 2013). Indeed, the main challenge in applying biomethanation process to treat saline wastewaters isthe in hibition eaused by Naas concentrations as low as 3.5 g Na" L-* have ‘been found (0 inhibit the biomethanation process (Che etal, 20145 Rinzema and Lettings, 1988; Zhang eta, 2013). However, the inhibi tory effect of salinity may depend on the adaptation of the microbial ccommiity to the salinity levels halophilic merobes) and/or on the presence of other cations and nutrients in the wastewater (Kars! and Dinger, 1996), For example, secondary factors, suelt as the levels of sulphate ($04?) and/or anumonia (NH) inthe aquaculture wastewaters ‘ean potentially interrupt biomerhansation process and lower the biogas yield, According to Cot and Rin (1991), a COD/SO%? ratio < 1.7 fa ‘yours sulpiate reduction rather chan methaniogenesis, which requires a cOD/SO;? ratio >2.7. Furthermore, it Is generally accepted that ‘ammonia levels >3.0 g NHy-NL~ can inhibit the methanogenic activity Inthe biomethanation process (Chen et al, 2014. In addition, Dy-produets from the biomethanation process, such ws hydrogen sl: phide (HS), can inhibit methane production, as ICsp (half-maximal inhibitory concentration) has found to be 160 mg HS L* for aeeto trophic and 220 mg HS L-! for hydrogenotphic methanogens (anaguchi etal, 1999), ‘The major aim ofthis study was to assess the toxlity of Fish exlture wastewater (Le. rainbow trout, Oncorhynchus mykiss) on the bio methanation process by estimating the inhibitory concentration (IC) values ofits salinity and frame te potential and safety of using RAS Jarl of monet! Monge 275 (2020) 131288 B ig 1. Reating tank and fc wast coletion syste A) Cultwe tak B) Sludge collection syste, ‘Tablet {Chemical cntactesiaton of inocula wastewaters from sh organic waste obtained at diferent salinities WO(O gL), WIS gL. *),W225gL "and W3G5 gL) (aan 0,0 ~ 3), Weld Mss wae “aar wan) es 296 copisu ty = 2590 sao TGR BoE Lone toa oeme aie tae cy aa 8 ty a Se ar * Toinl Chemical Oxygen Demands Toral Kjeldahl Niuogen, «Volatile Faty Aci. wastewaters as co substrates in biogas plants, In order to define the optimal methane potential of fish wastewater, «supplementary aim of the study was to determine the biochemical methane potential (BMP) of the rainbow trout exlture wastewaters at fur different reared salinity levels (0,15, 25 and 35 gL, 2, Materials and method 2.1, Fish wostewoter and blomethanation inocton ‘The wastewater from rainbow trout (Oncorhynchus mykiss) was reared at fur diferent salinities (0, 15, 25 and 35 g L~*) in 1m ret lartanks under a flow-through system configuration (Martins ea, 2010; Leteier Gordo ets, 2020) (Fig. LA) where salinity was regularly monitored using @ Seawater Refractometer (Hi 96822, Hanna‘Table 2 Experimental setup of the biochemical mcthane potential (BMP) for the diferent wastewaters and teste salinity concentrations (ean SD, n = 3). wen TRG L0H mae som asl3iaM aLseLGIO sainig Gi) _ 0 1s a = ‘Table 3 Levels of salinity assessed in thetic test. Sainig GI) Working vol (WA) Sly @L7) Woking wa AD. “ 090 to o90 3 2080 35 1090 as 1090 0 190 sts, US, Th fish were reared nt a density of 20 ke ih m0 the ‘amount of daily feed being 19% of the biomass (200 g/d with a 12h ‘automatic feeder), using Biomar Enviro 920 (Biomat A/S, Denn). ‘Once the fish condition was good and uo considerable feed waste was found, the pool samples of fish organic waste were collected and Immediately stored at 4°C during three consecutive days at the bottom fof n 20 L.swiel separator with the use of a 2 L collector forthe bio methanation experiments (Vg. 1B). The three day pooled semples were ‘kept at 4°C until the beginning of the experiment (10 days) ‘The methanogente inoculum used in the experiments was derived from a 2L (working volume) labseale continuous stired tank reactor (CSTR) fed with cow manure hydraulic retention time: 15 days, organie loading rate 230.1 gVSL*4-). Prior the experiment, the inoculum ‘was incubated for 7 days at 98 4 1 °C co degasify it, The chemical characterization of wastewaters and inoculum are shown it Table 1. 22. Biochemical methane potetal (BMP) test ‘The BMD test of the fish organic waste was performed according (0 (Holliger et el, 2016) using glass serum bores with 118 and 40 mL of total and working volume, respectively. Two inoculum/ wastewater ta ios (20/80 and 10/90 ¥/¥) were used for each salinity level; deionized water was added to Keep constant the working volnmie an the overall BMP experimental set-up is depicted in Tle 2. The test lasted 60 days ‘and esch treatment as performed in ripicates. Subsequently, the re. actors were lished with ges mintute of N/CO3(80/20% W/V) to create ‘anoxic conditions, and NayS+9H,0 (62.5 mg L) was added as a reducing agent. all she reactors were closed with butyl rubber stoppers, sealed with aluminium eaps and incubated at 53 1 °C. 2.3, Toxic test To assess the toxicity ofthe salinity onthe biomethanation process, Date reactors with methanogenic inoculum, fish organie waste from salinity 25 gL, and increasing levels of synthetic marine salt (Blue ‘Treasure SPS sea salt, MiniZoo, Denmark) were established (Table 3). ‘The rest lasted maxinn 60 days and each treatment was performed in triplione 24, Analytical meds TS, VS, TAN and TKN were measured ising APHA’s Standard Methods (APIA, 2013) and pH values with PHM99 LAB pH meter. Sulphate concentration in wastewaters was mieesured using. Spec troquant® Sulphate Cell Test (Merck, Denmark). The wastewater sam ples were previously filtered at 0.45 yan with Millex HV; Filter Unit Jarl of monet! Monge 275 (2020) 131288 (Merck, Denmark). The total VEA concentrations of the inoculum and the wastewaters were the stm theisindividal VRA conceatrations (se. acetate, propionate, iso butyrate, butyrate iso-valerate, valerate and hhexanoate), which were determined with ® gas-chromatograph (HP ‘5890 series Hl, Hewlett Packard, US) equipped with lame ionization detector and a FFAP fused silica capillary column, (30m x 0.53 mm i, film thickness 1.9 pm), and nitrogen as a carrier gas, The biogas composition was measured with @ gas-chromatograph (Trace 1310 GCTCD, Thermo Fisher, Denmark) equipped with Trace PLOT ‘TG-BOND Q 26004-6030 column (30 m > 0.32 mm LD., lm thickness 10.0 jms; Therm Fisher, Denmark) and helium. was used as easier gas for both methane, tbon dioxide, and hydrogen sulphide. 25. Caleularons, data tearment and sarsical analysis ‘The toxicity experiment values for TAN, sulphate and Na’, and the relationship between biomedanation inhibition () and the Salnity/ COD ratio was modelled using dhe dose-response equation (Eq. (1). Furthermore, 1¢ Guhibicory concentrations) values were determine the equation, Ay and Ag are the asymprotes of the sigmoidal curve As Ay peat @ To assess the combined effect of salinity and solids (inside the reaetor) on methane production, the Salinity/COD ratio was caeutated, and an average density of1028 gL for marine water was used, For the HS in the liquid, the Henry's law (Eq, (2)) was used: Ca xp @ with Cj as dhe molar concenteation inthe ligula phase (M), HP as the Henry's law constant (2100 mol m™* Pa") (Sue etal, 2000) and pas the partial pressure of HS inside the reaetor (in Pascal). In order to faleilate the dissolved HS in the liquid phase of the reactors twa ‘assumption have been made: 1, HS was considered as flly unionized, slehough some sin the form oF HS", the assumption is fel correct considering te pH inside the reactors (Lens et al, 1998). 2, The total pressure inside the reactors was considered as the sum of| the paral pressures of CO + CH + HS, from their respective concentrations inside the biogas. For the estimation of the potentially safe co-digeston scenarios of fish organic waste and manure at different typical reared salinity levels (Ge. 0, 5, 10,15, 25,35 gL”, the sigmoidal curve methane production Inhibition eaused by salinity) produced in the cutrent study by the experimental data was used, To calculate the potentially safe co: digestion scenarios i was assumed that: 1. The microbial communities of the CSTR reactors were aot acclims tized to any salinity levels 2. A vypical uninhibited methane potential of eow manne digested in (CSTR reactors, is 250 ml. CH, g"! VS, based on previous experiments, conducted by Fan etl. (2071). This methane potential corresponds toa manure with an organie matter content of 39.6 g VS L~. 8. The BMP value ofthe fish organic waste (no slinity effect) was 433 fk CH g-* VS; as it 89s found to be in tis study. 4. The minimum (50 g VS L~") and the maximum (70 g VSL! typical organic content of fish wastewater (personal communication, ‘Thomas Wagner Sauda, Sashint Royal, Denmark) were used i the diferent eo-digestion scenarios. 5, The salinity effect was calculated for hoth mesophilic (37 °C) and ‘hermophile (55 °C) conditions and their corresponding hydraulic retention times (HRT) of 23 and 15 days, respectively.300 250 200 150 +100 NmL.cH, 9 Cop" 50 ° > > goo oR 0 20 aa ol Fig. 2. Biochemical Methane Potential of sh organic waste performed at ferent salinities (WO: 0 L-1, WI: 15 Le, W2: 25g Le and WS! 38 gL) ‘and ino ato. [As methane rate change (MRC, Eq, (3)) was defined as the change (96) of methane produetion rate (MCR, in mL Cy L-reactor €~4 ex pected from the codigestion ofthe mantse and the different fish organic ‘waste, compared to the methane production rate (MMR, in mL CH, L “reactor €*) derived from the miono-digestion of manure (ck MMR, ame All statistical analyses were conducted using the OriginLab program (OriginLab Corporation, Northampton, Massachusetts). Student's test was used to test differences in the BMP values between diferent sain Ines, All vales are presented asthe averages over triplicates (enn sb,n=3) ee: @ 3. Results and discussion, i, Bochemical methane potential of trout wastewater ‘The BMP values of the four tout wastewaters tested, decreased significantly (p< 0.05) with the inereasing of the salinity levels ig 2). ‘The BMD values were statistically equal (P > 0.05) berween the two fish ‘organic wastes/inoculum ratios (20/80 and 10/90) For each salinity level. specifically, the BMP value for WO (0 mg salinity Lis in ‘agreement with tose obtained in the previous studies from freshwater ‘aquaculture systems (430 +30 mL GH g-TVS (Lana ané Fran’, 1998) ‘and 318 29 mil CH, g"! VS (Suhr et 9), 2015). Only few studios have measured biogas production in brackish and marine sh organic waste t previous studies from brackish systems with salinities between 0.5, 1415 g 1, methane production has varied from <12 up 10 450 mL (CH g- VS Clo eta, 2013; Mirzoyan et sl., 2008; Tal el, 2009), being generally lower (<1S0 mL. CHy g-? COD) than in this study (ig. 2). Only in two previous studies, the biogas production has been measured in full seawater stengrt (35 gL! saliniy): with values rnging between 114 and 184 ml. CHy g? GOD (Gebates, 200% ‘obser an Fike rok, 2006), being similar as reported in this study (ie. 2) Altogether, the results of the current study suggest chat, ‘although the methane production at incrensedseline fish organic waste is been reduced, sine aquaculture waste has a significant methane potential and could be suitable substrate for biogas production S.2, The effect of satiny on biomedhanation process The results of the toxicity experiment showed that inhibition of methane production in bomethanation process has # clear, rapidly Jarl of monet! Monge 275 (2020) 131288 100 & 8 Inhibition (%) Salinity (g L") Fig. 3. Methane production inhibition cass by salinity 100 _ © & Inhibition (%) os 04 (OS linity | COD Fig. 4. The effet of Salinty/COD rca an methane prodtion inti, Increasing correlation with salinity (Ad R® = 0.998; Fig. 8). Based on this, the calelated IC values for 208, 50% and 80% inhibition were 3.03, 4.97 and 5.94 g L”, respectively. This indicates that rather low salinities could be tox tothe biomethanstion process, since only 6 & Lis enough to lower mediane production by more dan 80%. Another ‘ateresting finding was that the Na concentration in the wastewaters tested, was ranging between O and 28 g L (see online Supplementary ‘la). However, & complete inhibition of the biomethsanation process ceurred at only 2.82 g Na* I, tn the literature, inhibitory concen. trations of Na by shock exposure and non adapted inocula are higher, nnging from 6 {0 13.¢ Na L-4, and only moderate inhibition has been found with concentrations betwee 3.5 and 5.5 g Na! L (Chen etal, 2008). Is generally accepted that methanogens ae the most sensitive tuieroorganisms in che biomethanation process (Clon et al, 2008; Haaghighatatshar, 2012; Kayhonien, 199. Thus, these results indicate thatthe biomtethanation inoculum used in the euerent experiment was tot acclimatized at even moderate Na levels. Interestingly, the inhibition of methane, production increased longside with the salinity/COD ratio (Adj. R® = 0,999; Fig. 0. The highest methane yield (277 19 ml CHy g™ COD) was found when salinity/COD ratio was zero, while the complete inhibition occured with sainity/COD ratio > 0.83. Therefore, it seems thatthe toxicity ofsolinity on anaerobic digestion does not depend only on the salinity ‘concentration, but also on che COD levels inthe anaerobie reactor. This ‘effect has been described before as “compatible solute strategy”, where many microorganisms, including. methanogente consortia, accumulate ‘organic solutes to balance the asmatie pressure between the environ rent and the cytoplasm at high osmolarity (Vysies and Stuckey, 2017) Nevertheless che aim of the ewrent study was not to investigate the effect of salinity and organic matter on the anaerobic microbiome development. Altogether, the rests indicate that microbiological and physicochemical interactions llow miethanogeas to be more robust at "he same salinity levels, which however, remains to be filly elvcidated, {us further investigation Is require. “13. Other factors inhibiting Dlomethanation process under saline ‘conditions In adaition to salinity and COD, other factors, seh as sulphide) ‘concentrations, total amionia nitrogen (TAN) and pH are known t0 affect the biomethanation process (nen tal, 2008). From these three factors, only sulphide seemed to contribute tothe biomethanation in hibition in the eurrene study Except methanogens, anserobie digestion reactors harbour sulphate rediucing baetcra, which convert sulphate, sulphite,orthiosulphate into sulphide, and compete with methane producing archaea for the same substrates (Haghighatasher, 2012). Therefore, COD/SO? rato is used to define ifthe methanogenie or the sulphate-redueing pathueay is fav ‘onred, 88 COD/SO;? ratios below 2.7 are known 10 favour the sulphate-reducing pathyeay (Chot end fm, 1991). In the current study, ‘COD/S0;? ratio was between 4 and 100 (W/W, see online Supplemien ‘ary data), indicating that methanogenic pathway was favoured over the sulphate reducing pathvtay in all the reaetors. In addition, complete inhibition occurred under » COD/SO;* ratio of 41, adit ta sl phate was not the major reason for the biomethanation process inhibi ton, However, the levels of HS in the gas phase were rather high (>15 gL" of HS) fr salinities above 15 g Lat the end of the experiment, Which means that sulphate reducing pathway was active. Thus, the ‘overall inhibition of the biomedhanation process was triggered by the combined inhibitory effect of salinity (min inhibitor), and sulphate reduction (secondary inhibitor. It seems that salinity strongly inhibited methanogenesis, which subsequently rendered the sulphate reducing pathway thermodynamieally favourable, further enlancing the overall Inhibition. Ar the moment chere are many fullseale biogas plants that ‘are operating efficiently under similar HS levels, with only consequence the higher operational cost for removing H,S from the biogas tomeet the legal and operational requirements (usually < 50 ppm) (Allesie and Hinge, 2014), suggesting that the inhibitory effet of HS on methane production is still only minor. in conteast ro sulphide, concentration of organic-N and TAN (low ‘TKN) and TAN inthe wastewaters were way below the threshold of 3 g NHEN L* Clable 1) that Is considered Inhibitory for the bio: tethasation process (Tiss e201). Ths iin eontras 0 eaeier studies conducted with aquaculture fish waste (Gebsier, 2004; Gebacr ‘and Eikebrokk, 20065 Luo etal, 2013) under high TAN (NEL + NH) ‘concentrations, which are knova to inhibit methane production (1st cr sl, 20180). This could be explained by the differences in fish diet, formulation and operation conditions between the studies. The sh ‘waste composition and related nutrient solubilization depends on the let formulation (Cho and Bureau, 2001; Letelier Gordo etal, 20155 Nijhot, 19949, as well as on the proper operation of the aquaculture system. Nowadays, formulated fish commercial diets have high a gesubilty towards protein and dry matter (>90% and >859%, respec tively (Dalsgaaré! and Pedersen, 201 a,b). Therefore, I is generally ‘expected that the proportion of protein content in respect co the dry natter in the fish waste should range between 20 and 25% Uneaten pellets, with approximately 40-50% protein content, will have a sub stantial impact on the waste protein concent. Thus, unesten pellet waste Jarl of monet! Monge 275 (2020) 131288 2 eee Methane Yield (mL CH, g" VS) 8 0 10 20 30 40 50 60 70 80 90 100 %WW in the feedstock (viv) 3 a3 -504 100. Methane Rate Change (%) %WW in the feedstock (viv) c) 250 200. 150: 3 ‘Methane Rate Change. (%) Oar = r 50] 127-2020 40°s0_ 60 70 60 90 100 100: wns sea %WW in the feedstock (viv) — wwo wws =~ wwto WHS W253 Fig. 5. The predicted a) methane yield b) methane rate change (MRC) for vwascewster (WW) Wh 50 g VS LI and e) methane ate change (MRC) for WHE With 70g VS Le, fC the WW coigestion wih cow manne under Five ‘ypical salinities. ‘and poor operating conditions can most probably explain the previously found high protein levels (29-60%: Gebiauer, 20045 Gober and Fike brokk, 2006), enusing biomethanation process inhibition by ammonia Finally, the pH of the reactors was within the optinal range for the biomethanation process, varying between 6.85 aud 7.21 (Vian et al, 2018), which did no ha any ree effect on the process inhibition. 2.4, Potentially safe codigestion scenarios The diferent co digestion scenarios of fish wastewater (WW) and ‘cow manure, for wastewater with different salinity levels, shossed that‘Table 4 ‘The predicted maximum co-digestion production (highest methane production rate) of WW (asteweter) and cow manure (9 wastewater/manute in ¥/¥), der five typical salinies and two WW organi contents, under msopile (27 ‘C) ane thesmnophilc (5 °) cous, Gngtie Saliy—G WMehane Matias Waar Meter (GES) gesion yim ate (7 "€) ate (5°C) Swi Cag eres, > 9 Ehren ‘an improvement between 3.22% (WW35, 50 g VSL") and 61.8596 (NWS, 70 g VSIL~" ofthe meximum methane production rate could be realised, compaced to the methane produetion rate of the mono digestion of cow manure (Fig. 5, Table 4). This means thar itis possible to optimally co digest from 2.7296 (v/v, WW35, 50 g VSL") up 48.0396 (v/v, WW5, 70 g VSL”) of fish wastewater with cow manure ‘and, at the same time, signfieantly improve the reactors’ methane produetion. Overall, it becomes elear that fish wastewater with higher ‘organic content and lower salinity are more desirable co-digestion substrates, since they contribute more on the methane production rate Increase and less on the sanity toxicity, respectively. Furthermore itis [possible thet the proposed yields could vary due to the competition Derwicen methanogens and SRB, especially as the later are normally found in the environments with high salinities, such es sea floor sedi ments and aquaculture systems (Hao esl, 201% Sorensen et al, 19815 Letelier Gordo etal, 2020), ‘These are very promising results, especially sine they were based on. the assumption of using non-acclimatized eo salinity methanogenic Inocula. Thus, ir ean be argued that biogas reactors, where @ stepwise ‘acclimatization of the microbiome to sanity oceus (¥an etal, 2019, they wil be able to optimally perform at even higher salinities an with higher methane production rates, However, any use of saline fish ‘wastewater as biomethanation feedstock should take place with great ‘caution (stepwise) since ts biogas reactors’ microbiome is distinct, and tus could be affected diferenly by salinity 4. Conclusions ‘The eurvent study shows that even highly saline sh wastewaters have an exploitable biomethane potential. Depending ow the salinity levels, It Is possible to efficiently co digest 3.22-61.85% (v/¥) fish Wastewater and improve the maximum merthase produetion rate oF & biogas reactor by sip to G1.85%, compared to cow miantre mono: digestion, However, toxicity tests revealed chat even Iow levels of salinity (ce. 4.97 g1.) are enough o inbibit non acclimatized methane production by 50%. Additionally, the suiphete reduction is contributing to the overall inhibition of the biomethanation process as a secondary inhibitor, alongside salinity (wei inubiton, ‘cRediT authorship contribution statement Carlos 0. LeteliersGordos Data curation, Formal analysis, Vista: zation, Funding acquisition, Investigation, Writing - original draft, Writing - review & editing. Enrico Mancini: Data curation, Visualiza tion, Fornial analysis, Methodology, Investigation, Writing - original Jarl of monet! Monge 275 (2020) 131288 draft, Writing -review & editing. Per Bovbjerg Pedersen: Supervision, Punding acquisition, Writing - review & editing. lini Angelidaki: Sw pervision, Writing review & editing. Ioannis A. Fotidis: Project ‘administration, Supervision, Methodology, Data curation, Forms ana ysis, Visualization, Writing“ original draft, Writing - review & eting Declaration of competing interest ‘The authors declare that they have no known competing financial Interest or personal relationships that could have appeared c influence the work reported in this paper. Acknowledgments This research was funded by The Ministry of Food, Agriculture and Fisheries of Denmark through the GUDP project WASTE-TREAT (GUDP 89190) “Cos effective solutions for Ené.of pipe treatment in sale water BRAS". The technical skills and invaluable assistance of Brinn Moller and Ulla Sproegel (DTU Aqua) are highly appreciated. We would Ike to \dink Thomas Wagner Sandal (Ses! Royal, Denmark forthe val ble information sbout the typical fish waste characterization. We Would like to thank Sanni Aalto for coreibuting in the revision of the Appendix A. 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