The liquid fraction of digested material is rich in ammonium and may require nitrogen removal. The aim of this research was to evaluate the applicability of the anammox process for the biological N removal from a supernatant coming from the anaerobic digestion of a mixture of piggery manure, poultry manure, and of agro-wastes. The supernatant was pre-treated in a partial nitritation pilot-scale reactor located at the farm. A batch procedure for testing the short term effect of high-strength wastewaters on anammox activity is presented. The anammox process was successfully applied for the first time to undiluted digestate, and the average N removal efficiency achieved during 350 days of experimentation in a SBR lab-scale reactor was 91%

A. Teli

D. Scaglione

E. Ficara

F. Malpei

R. Canziani

V. Corbellini

SCAGLIONE, DAVIDE

FICARA, ELENA

TELI, ARONNE

CANZIANI, ROBERTO

MALPEI, FRANCESCA

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60(P8) Treatability of  digested  piggery/poultry manure  by anammox bacteriaD. Scaglione*, E. Ficara*, V. Corbellini*, A. Teli*,  R. Canziani* and  F. Malpei** Politecnico di Milano, DICA, Environmental Section, Piazza L.da Vinci 32, 20133, Milano, Italy(E-mail: davide.scaglione@polimi.it)AbstractThe liquid fraction of digested material is rich in ammonium and may require nitrogen removal. Theaim of this research was to evaluate the applicability of the anammox process for the biological Nremoval from a supernatant coming from the anaerobic digestion of a mixture of piggery manure,poultry manure, and of agro-wastes. The supernatant was pre-treated in a partial nitritation pilot-scale reactor located at the farm. A batch procedure for testing the short term effect of high-strengthwastewaters on anammox activity is presented. The anammox process was successfully applied forthe first time to undiluted digestate, and the average N removal efficiency achieved during 350 daysof experimentation in a SBR lab-scale reactor was 91%.KeywordsAnammox, activity test, digestate, manometry, piggery manure, poultry manureINTRODUCTIONAnaerobic digestion (AD) can effectively treat livestock wastewater and produce renewable energy asbiogas. The liquid fraction of the digested material is rich in ammonium and its disposal onagricultural soil is regulated by the European directive on nitrates (91/676/CEE). Therefore N removalmay be required in intensive breeding farms. This aspect has prompted attention toward advancedbiological processes such as the nitritation-denitritation process (Scaglione et al. 2013), and thecompletely autotrophic process (partial nitritation and anammox) that are cost effective thanks tolower aeration and external carbon source requirements (Van Hulle et al. 2010). Most studiesregarded digestates from piggery manure (Dosta et al. 2008) or diluted wastewater in lab-scalereactors (Yamamoto et al. 2008). Literature documented the treatment of neither digestates frommixed animal wastes nor undiluted agricultural digestate (Magrì et al., 2013). This work is part of atwo-years project (BRAIN) funded by the Italian Ministry for Agriculture, whose aim was to evaluateautotrophic N removal processes applied to digested liquid fraction coming from the anaerobicdigestion of a mixture of agro-wastes, mainly piggery and poultry manure. In this paper some lab-scale results are presented, namely: (i) activity batch tests, to assess anammox activity of digestatesupernatant after partial nitritation stage and the effect of different salinity concentrations; (ii) reportthe results of a 350-day operation of an SBR treating digested supernatant.MATERIALS and METHODSCharacteristics of the supernatantThe supernatant from a full-scale anaerobic digester (solid/liquid separation by centrifugation),located in a piggery farm in Northern Italy, was used. The digester was fed on thickened piggerymanure, poultry manure and agro-wastes. The supernatant showed a high variability in its chemicalcomposition during the course of the experimentation (pH =8.0±0.1, Conductivity =14.2 ± 2.1 mS cm-1; NH4+-N =1151 ± 251 mg L-1; COD = 2634 ± 1178 mg L-1; TSS = 430 ± 220 mg L-1; Alkalinity = 7128 ±2932 mgCaCO3 L-1). The supernatant was treated in a partial-nitritation 650-L SBR. The NO2/NH4molar ratio of the effluent from the partial nitritation pilot plant was slightly adjusted to remainwithin 1.25±0.25, thus suitable for feeding the subsequent anammox reactor.Soluble COD concentration in partial-nitrified supernatant was 853±257 mg/L while BOD5 and BOD20were 25±15 mg/L and 49±26 mg/L respectively.61Manometric batch testsThe assessment of the N2 production rate from the anammox reaction was performed by means ofan OxiTop Control system. This is a manometric device consisting of a pressure transducer and datalogger located inside a measuring head that is mounted on a glass bottle of 1140 ml volume, filledwith anammox biomass suspension. The bottle has got two lateral openings; one is sealed by arubber septum and is used for substrate injections, the second one is sealed by a teflon airtight valvefor gas discharge. During the course of the batch test, the overpressure due to N2 production by theanammox reaction is automatically registered by the measuring heads. Manometric determinationsof Anammox activity were performed according to the following procedure. Each bottle was filledwith 30 ml of granular anammox biomass and 700 ml of either mineral medium or a blend of real-wastewater and mineral medium, depending on the test type. Sodium bicarbonate (1g/L) was addedand the headspace was flushed with 95%N2 and 5% CO2 to ensure anaerobic conditions and to keepthe pH around 7.8. Bottles were thermostated at 350.5°C and stirred by a magnetic mixer.Substrates were added by spike injections through the rubber septum. Then, the gas volume ofcumulatively evolved N2 was calculated from overpressure data according to the ideal gas law. At theend of the test, concentrations of ammonium, nitrite and nitrate were measured. For each test, themaximum N2 production rate, the maximum specific Anammox activity, SAAmax (mgN2/gVSS/h) andthe nitrogen mass balance was verified.Lab-scale anammox reactorTo test the stability of the anammox process, a thermostated (34-36°C) 3-L lab-scale reactor wasused. It was equipped with time-controlled feeding/discharging pumps, mechanical mixing, and witha pH control unit. The reactor was operated in a SBR mode with a 8 h cycle (5 h of FILL, 2.5 h ofREACT, 0.5 h of SETTLE/DRAW).RESULTS AND DISCUSSIONManometric batch testsA first set of manometric tests were performed to evaluate the effect of exposing granular  anammoxbiomass sample to the effluent of a partial nitritation reactor. However, the high concentration ofnitrite in this wastewater (around 500 mgN/L) would have caused anammox inhibition. Therefore,the wastewater was first pretreated by adding activated sludge and a stoichiometric amount ofsodium acetate to remove nitrite by heterotrophic denitrification. After 24h of reaction, thesupernatant was centrifuged and the clarified fraction used, either as such or after dilution with amineral medium, for testing its effect on anammox biomass. The ammonium concentration was yethigh (around 190 mgN/L) so that only nitrite was spiked to achieve an initial concentration of 50mgN/L. Soluble COD concentration was 1018 mg/L while BOD20 and BOD5 were 80 and 30 mg/Lrespectively. The first set of activity tests aimed at evaluating the response of a non-acclimatedanammox biomass to the pre-denitrified digestate. To this purpose, a granular biomass sample wasused, having a specific activity of 0.11 gN2/gVSS/d. Results are reported in Figure 1. No inhibition wasobserved when the wastewater was blended with 50% mineral medium, while activity showed an85% decrease after 24h exposure to a blend containing 75% wastewater. For the last two samples,anammox activity recovered with time, more significantly for the less concentrated blend. The resultsconfirmed that the inhibition increases with the fraction of wastewater in the blend and that a longterm adaption may take place.62A second set of tests were used to evaluate whether the previously observed anammox inhibitioncould be due to the high salinity of the digested supernatant. In these tests, salinity of the mineralmedium was modified by NaCl addition. The NaCl concentrations tested were 7 and 13.5 g/L, whilesalinity of the digested supernatant corresponded to around 5 g/L as NaCl. As inoculum, two type ofbiomass were used: (1) the unacclimated granular sludge, and (2) an acclimatised sample drawn fromthe anammox. Results are reported in Table 1. For the unacclimated sludge the activity decreasedduring time with a final inhibition of 86%. At the same salinity concentration, the acclimatized sludgeshowed a faster adaptation with an activity recovery trend after 3 days of incubation. By doubling thesalinity level a slightly higher inhibition was observed.The inhibition level obtained are slightly higher than the one reported by Dapena Mora et al. (2007)who found a IC50 value of 13.5 g/L of NaCl in similar manometric batch tests on granular anammoxbiomass.Lab scale anammox SBRThe SBR reactor was inoculated with granular anammox sludge coming from a full-scale anammoxreactor located in Rotterdam (NL). The SBR was operated at an HRT of 2 d. A synthetic feed was usedinitially, at an average nitrogen loading rate (NLR) of 0.5-0.6 gN L-1 d-1, kept constant during theexperimentation. Then a blend with increased fractions of real wastewater were used (10%, 25%,33%, 50%, 70%, 100%), adjusting the NLR and the NH4/NO2 molar ratio by NaNO2 and NH4Cladditions. The reactor was operated for 350 days; during a 40 days stop in summer, the biomass waskept at 4°C. Batch tests were periodically performed to assess the maximum anammox activity(NRRmax). In figure 2-right influent and effluent nitrogen compounds are reported. The average Nremoval efficiency was 91±10% with an average nitrite removal efficiency higher than 97%.NO2-N/NH4-Nremoved ratio was 1.28±14% while NO3-N/NH4-Nremoved ratio was 0.10±72%. In Figure 2-left, the NRRmax is plotted together with the percentage of supernatant in the feed. During the initialFigure 1: Results of the first set of inhibitiontestsTable 1: Results of the second set of inhibitiontestsActivity lossDaysUnacclimated biomass Acclimated biomass7 g/L NaCl 7 g/L NaCl 13.5 g/LNaCl1 0% 0% 0%2 62% 57% 60%3 57% 80% 87%4 86% 66% 77%0,000,020,040,060,080,100,120,140 1 2 3g N2 / g VSS dTime [days]Synthetic medium 50% real WW 75% real WW 100% real WWSyntheyticmediumSupernatant replacement63start-up and restart after the first break, the activity increased from values between 1 and 2kgN/m3/d up to values between 3 and 4 kgN/m3/d in about 30 days, suggesting an increase of theactive biomass. In contrast, after each increase in blending real wastewater in the influent, theactivity decreased, suggesting that real wastewater had an inhibitory effect on the anammoxbacteria, similarly to what had been observed in the manometric batch tests. While working at 75%of wastewater in the feed and also during the first days of operation with undiluted wastewater, aminimum value of 0.14 kgN/m3/d was achieved. In these low-activity phase, temporary butsignificant increase in nitrite concentration in the effluent was observed. After 30 days of operationwith undiluted wastewater, the anammox activity began to recover and continued to rise until theend of the experimentation, achieving values greater than 1 kgN/m3/d. The trend observed in the lasttwo months of operation at 100% real wastewater suggests the ability of anammox bacteria to adaptto inhibitory substances that may be present in the agricultural digestate. Three particle-size testswere carried out at day 0, 260 and 350 of operation, and showed that the average diameter ofgranules increased from 775µm of day 0 to 1003µm and 1033µm of day 260 and 350 respectively.Figure 2: Maximum anammox activity in the SBR reactor during the experimentation and fractions ofreal wastewater blended in the feeding (left) and influent and effluent nitrogen compound (right).CONCLUSIONSThe following main conclusions can be drawn: a procedure to perform short-term batch tests to assess inhibition of anammox activity inpresence of high-strength wastewaters was developed and tested successfully; inhibition in presence of undiluted digestate supernatant from piggery/poultry manureprocessing with not acclimatized biomass; was as high as 85%; salinity concentration of 7 gNaCl/L caused 68% activity reduction in the test with acclimatedbiomass and 86% in the test with non- acclimated biomass; in the first days of operation with undiluted supernatant, the maximum anammox activity inthe SBR decreased sharply, but recovered afterwards, suggesting that biomass acclimated to thewastewater; in the long term, the anammox SBR proved successful in treating undiluted supernatant, withN removal efficiencies higher than 90%.AcknowledgementsThis research was funded by the Italian Ministry of Agriculture and Forestry (BRAIN Project, 2010-2012).REFERENCESDapena-Mora A., Fernandez I., Campos J.L., Mosquera-Corral A., Mendez R., Jetten M.S.M. (2007).Evaluation of activity and inhibition effects on Anammox process by batch tests based on thenitrogen gas production. Enzyme Microbiol Technol., 40, 859-865.0204060801000123450 50 100 150 200 250 300 350% real waste waterNRRmax (gN/L/d)time (days)NRRmax %real WW0,000,100,200,300,400,500,600,700,800,901,000 50 100 150 200 250 300 350gN/Ltime (days)N-NH4 in N-NO2 in NH4 out NO2out NO3out64Magrì A., Béline F. and Dabert P. (2013) Feasibility and interest of the anammox process as treatmentalternative for anaerobic digester supernatants in manure processing - An overview. Journal ofEnvironment Management 131 (2013) 170-184Yamamoto T., Takaki K., Koyama T., Furukawa K.(2008) Long-term stability of partial nitritation ofswine wastewater digester liquor and its subsequent treatment by Anammox. BioresourceTechnology 9;99(14):6419-6425.Scaglione D., Tornotti G., Teli A., Lorenzoni L., Ficara E., Canziani R. and Malpei F. (2013) Nitrificationdenitrification via nitrite in a pilot-scale SBR treating the liquid fraction of co-digestedpiggery/poultry manure and agro-wastes. Chemical Engineering Journal , 228, 935-943.Van Hulle S.W.H., Vandeweyer H.J.P., Meesschaert B.D., Vanrolleghem P.A., Dejans P., Dumoulin A.(2010) Engineering aspects and practical application of autotrophic nitrogen removal fromnitrogen rich streams. Chem .Eng J. 8/1;162(1):1-20.

Treatability of digested piggery/poultry manure by anammox bacteria

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Treatability of digested piggery/poultry manure by anammox bacteria

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