Struvite Precipitation as a Means of Recovering Nutrients and Mitigating Ammonia Toxicity in a Two-Stage Anaerobic Digester Treating Protein-Rich Feedstocks

Accumulation of ammonia, measured as total ammonia nitrogen (TAN), a product of protein decomposition in slaughterhouse wastes, inhibits the anaerobic digestion process, reducing digester productivity and leading to failure. Struvite precipitation (SP) is an effective means to remove TAN and enhance the buffering of substrates. Different Mg and P sources were evaluated as reactants in SP in acidogenic digester effluents to reduce its TAN levels. In order to measure impact of TAN removal, a standard biochemical methane potential (BMP) test was conducted to measure methane yield from treatments that had the highest TAN reductions. SP results showed 6 of 9 reagent combinations resulted in greater than 70% TAN removal. The BMP results indicated that SP treatment by adding Mg(OH)2 and H3PO4 resulted in 57.6% nitrogen recovery and 41.7% increase in methane yield relative to the substrate without SP. SP is an effective technology to improve nutrient recovery and methane production from the anaerobic digestion of protein-rich feedstocks.


Introduction
Anaerobic digestion (AD) is an attractive technology to treat high-strength slaughterhouse wastes as it provides energy recovery (methane), nutrient recovery (nitrogen and phosphorus), and pathogen destruction [1,2]. Energy recovered in the form of methane by treating slaughterhouse wastes in AD was reported to be 1300 Megajoule (MJ) per bovine, 140 MJ per pig and 1.3 MJ per broiler [3][4][5]. As much as 23.0 g of nutrients (total of nitrogen, phosphorus and potassium) per bird are potentially recoverable from poultry slaughterhouse wastes [5]. With respect to pathogen destruction, both fecal coliform and salmonella were reportedly completely eradicated in a thermophilic digester (50˝C), while 99.9% and 90%-99% of oocysts of Eimeria tenella were inactivated in a thermophilic digester and a mesophilic digester, respectively [2].
In AD, proteins and lipids are hydrolyzed and acidified to intermediates including hydrogen, NH 3 (measured as total ammonia nitrogen or TAN) and volatile fatty acids (VFA) in the acidogenic stage of the process. Hydrogen and VFAs are then converted to methane and carbon dioxide by a different group of bacteria in the methanogenic stage. High concentrations of TAN are known to severely inhibit methanogenic microorganisms, reducing digester performance and leading to failure. Strategies of TAN management are needed when treating high protein wastes in AD.
One of the best management strategies is the use of a two-stage AD system where acidogenesis (and the production of TAN and VFA) are physically separated from methanogenesis to reduce TAN and VFA inhibition of methanogens. However, high TAN concentrations (over 2.0 g¨L´1) produced during acidogenesis of slaughterhouse waste proteins [6] are well above the known inhibitory level of 1.5 g¨L´1 [7]. Reduction of TAN in the substrate through external intervention can therefore enhance digester performance. Considering TAN is not biologically removed in AD, removing it via physical stripping or chemical precipitation from the acidogenic digester effluent could be a way to reduce concentrations and associated inhibition downstream in the methanogenic digester.
Magnesium ammonium phosphate (MAP) commonly known as struvite is a compound with low aqueous solubility under alkaline conditions [8]. When concentrations of TAN, magnesium and phosphorus reached critical values, struvite is naturally formed and is known to attach to and clog pipes in wastewater treatment. Struvite is also a slow release fertilizer with commercial value and its precipitation from different high-TAN wastewaters, including AD effluents, has been studied extensively [9,10]. Çelen and Türker [9] reported 50% to 90% TAN removal from AD effluents through struvite precipitation (SP) using H 3 PO 4 and MgO or MgCl 2 under short reaction times (~10 min), high pH (~9.0) and room temperature (25˝C). In order to improve AD performance by TAN reduction, in some cases magnesium (Mg) and phosphorus (P) were directly added to the digester [8,11]. Lee et al. [8] reported reduction of 67% TAN and 73% of P in the substrate after adding MgCl 2 to a single-stage food waste digester thus increasing methane yields from 180 to 290 mL¨g´1 COD added . Romero-Güiza et al. [11] added a low-grade MgO byproduct to a single-stage digester treating pig manure and found methane yields increased from 130 mL¨g´1 VS added (before MgO addition) to 190 mL¨g´1 VS added (after 30 kg MgO¨m´3 addition). SP directly in the digester has been shown to significantly improve methane yield, however, accumulation and deposit of struvite within the digester gradually reduce the effective volume of the digester. In addition, once settled inside digesters the precipitate is difficult to remove. Establishing a struvite precipitator external to the digester, between the 1st and 2nd stages of a two-stage digester, is a practical way to remove TAN from substrates before feeding the substrate to the 2nd stage methanogenic digester.
Integrating an external SP with a two-stage digester treating high-protein substrates can result in nitrogen (N) recovery, reduced TAN inhibition, and increased buffering in the methanogenic digester. In order to evaluate such a process and its effectiveness a study was conducted with the following goals: (1) identify the most suitable reagents that maximize N recovery while minimizing residual methanogenic toxins; (2) test Mg and P sources and quantify TAN removal in the effluent of an acidogenic digester; and (3) perform a biochemical methane potential (BMP) test to quantify methane yields from the treated substrates, compared to controls that did not receive SP.

Selection of Mg and P Sources for SP Test
The Mg and P sources that can be used in SP and residual toxins that could remain, along with their inhibition thresholds in AD, are summarized in Table 1. Since the substrates after SP are fed to the methanogenic digester, the criteria for selecting Mg and P sources included both highest TAN removal and least amount of residual toxins (e.g., ions and elements such as N, S, Na + , K + or Cl´) that could potentially inhibit methanogenic activity. Since these elements are highly soluble in the substrates and will remain in the treated substrates, the reagent dosages would have to be calculated to minimize inhibition of methanogenesis. Therefore, based on the minimal toxic residue criteria, MgO, Mg(OH) 2 , MgCO 3 , and MgHPO 4 were selected as Mg sources, and H 3 PO 4 and NaH 2 PO 4 were selected as P sources for further evaluation as reagents in SP of acidogenic effluents.

Experiment 1-Optimization of Struvite Precipitation
TAN of substrates was found to be 1.3˘0.1 g¨L´1, a value approaching inhibition levels for methanogenesis, potentially leading to lower process efficiency and TAN removal is expected to reduce these impacts ( Table 2). Concentrations of Ca, P and Mg in the substrate were too low to contribute to SP in any significant way as found in a preliminary test (Table S1). Treatments G1 and G5 using H 3 PO 4 had TAN removal of more than 70% (Table 3), while G2 and G6 using NaH 2 PO 4 were less than 60%. As H 3 PO 4 is a relatively strong acid, MgO and Mg(OH) 2 are more readily dissolved in it providing more active Mg 2+ for SP relative to their mixtures with NaH 2 PO 4 [17,18]. TAN removals in G1 and G5 treatments were higher than those reported in the literature treating different substrates under similar conditions. In particular, TAN removal in G1 was 77.5%, compared to 54.4% and 25.7% removal reported by Celen and Türker [9] and Yetilmezsoy and Sapci-Zengin [19], respectively. Such large differences could be caused by higher availability of Mg 2+ formed from the dissolution of MgO that is enhanced by the lower pH in this study (pH = 6.4) relative to that in the literature where pH were 7.9 [9] and 7.95 [19]. Substrates used in the above studies had a higher pH of 7.9 and after adding equimolar H 3 PO 4 , MgO dissolved in the substrate was relatively small. TAN removals using Mg(OH) 2 were comparable to those using MgO when combining different P sources. Treatments G2 and G6 using NaH 2 PO 4 had lower TAN removal of 50.5% to 58.8%. These results indicate higher removal of TAN compared to Li et al. [20] who treated pharmaceutical wastewater with pH of 12.2 and TAN of 1.12 g¨L´1 by adding MgO and NaH 2 PO 4 (Mg 2+ :NH 4 -N:PO 4 -P = 1:1:1) and maintaining pH of 9.0 for 15 min under well mixed conditions. The TAN removal in the above study was reported to be less than 40%, which was lower than the 50.5% TAN removal observed in this study. Based on the work of Celen and Turker [9], it is known that TAN removal via SP is complete within 5 min when operating in a pH between 7.5 and 9.0. Therefore, the 20-min reaction time used in this study relative to the 15-min used by Li et al [20] was not the reason for difference in performance. The higher TAN removal obtained, is a result of the lower pH in the used substrate (pH = 6.4) relative to pH in Li et al. (pH = 12.2). Treatment G6 had similar TAN removal to G2, because of the similar chemical reaction of MgO and Mg(OH) 2 in SP. Treatments of G3 and G4 containing MgCl 2 had higher TAN removals of 88.3%-89.0%. These values are in the same range as reported in other studies treating different wastewaters using SP [9,21,22]. MgCl 2 is a highly water soluble compound and provides the highest ion concentrations of Mg 2+ without the addition of acids, resulting in high TAN removal regardless of the strength of acids used (e.g., H 3 PO 4 ).
Treatments containing MgCO 3 also had similar high TAN removals in the range of 75.9% to 76.5%. In contrast to treatments using less soluble MgO and Mg(OH) 2 , with MgCO 3 , large amounts of CO 2 bubbles were observed when reagents were added to the substrates. This bubbling of CO 2 resulted in some mixing of the solution and potentially enhanced mass transfer and ionization of MgCO 3 to Mg 2+ .
It was surprising that treatment G9 containing MgHPO 4 resulted in very low TAN removal (5.2%). This result is not in agreement with previously reported results in the literature. For example, Sugiyama et al. [23] reported TAN removals of 49% and 77% for 1 and 3 h of SP reaction time, respectively, using MgHPO 4 at pH of 8 and temperature of 25˝C using an equimolar ratio of NH 4 Cl to MgHPO 4 . This difference in performance could be explained by the fact that the MgHPO 4 powder was dissolved in the HCl solution first and then added to the substrate. The Mg 2+ and PO 4 3´i ons were released in the acid solution and possibly precipitated in the form of compounds such as Mg 3 (PO 4 ) 2 instead of struvite [24]. A dark colored precipitate was seen with treatment G9, different from other treatments, which had a white precipitate characteristic of struvite, showing possibility of other compounds having precipitated in addition to struvite. A longer reaction time is probably necessary to form struvite when using MgHPO 4 as the sole Mg and P reagent. These possibilities need further validation. TAN was also potentially removed by free ammonia volatilization from the substrate, which was continuously stirred at high pH and temperatures [7,19]. In this study, which was conducted at 25˝C, 20 min, and pH 8.5, only 15.2% of TAN was in the form of free ammonia, calculated from the equation in Cuetos et al. [25]. Therefore, considering the high solubility of ammonia in water, ammonia volatilization is not expected to have a significant impact on TAN reduction relative to TAN removal by SP [19,20].

Experiment 2-Biochemical Methane Potential of TAN Reduced Substrates
In order to generate sufficient substrate for BMP tests, larger volumes of substrates were treated with SP reagents following the procedure of experiment 1. Characteristics of substrates to be used for BMP testing (before and after SP) are shown in Table 4. Untreated substrates had very low concentrations of Mg, Na and P to impact SP in any appreciable manner. There was also an absence of micronutrients at concentrations toxic to AD [15,26]. After SP, no significant COD change was observed between treated and untreated substrates. Previous researchers have reported COD reduction in wastewaters after SP ranging between 10% and 20% [20] and 22.4% to 53.3% [19]. These COD reductions are typically caused by co-precipitation of organics and struvite [19]. In this study, because of prior treatment through acidogenesis, organics were mostly converted to soluble VFA and did not participate in the co-precipitation. TAN removal of each treated substrate ranged between 57.4% and 93.8%. In treatment G1 (MgO treatment), it was observed TAN removal of 57.4%, which was lower than the 77.5% removal observed in experiment 1. For other treatments, results of TAN removals were comparable between experiments 1 and 2. The SP treatment substantially increased TS and VS of substrate because of salt residues such as Mg(OH) 2 , and NaH 2 PO 4 [27,28], however, it did not appreciably change feedstock rheology which allowed for the treated substrate to be easily pumped into the liquid-state anaerobic digester. The C/N of substrates increased from 5.0 to 22.7 as a result of TAN removal, providing the additional benefit of nutrient balance in AD. It is not surprising that residues of certain reagent elements such as Na, P and Mg remained in the substrates. After SP, Na concentrations were 3526 to 7432 mg¨L´1 depending on the amount of NaOH added for pH adjustment. High Na concentrations in substrates can potentially inhibit AD [15]. However, inhibition can be alleviated if microorganisms are properly acclimatized to the conditions [29]. Residual substrates had equimolar ratios of TAN and P, but less than equimolar contents of Mg. This suggests that TAN and P removal were directly in the form of struvite, however, in addition to contributing to struvite, Mg was also removed through the precipitation in the form of other compounds. Magnesium was reported as the limiting factor in SP in previous studies (e.g., [30]), confirming the observations in this study. Residual Mg in the treated substrates ranged from 23.96 to 90.44 mg¨L´1, far less than the moderately inhibitory threshold of 1000-1500 mg¨L´1 [31]. Residual total P concentrations were in the range of 195 to 1246 mg¨L´1. P is an important requirement for living microorganisms and plays a vital role in their growth and metabolism [32]. Based on the authors' best knowledge, few studies have looked at the impact of high P concentrations on AD. Wang et al. [32] and Lei et al. [33] reported preferred concentrations of 414 and 465 mg P¨L´1, respectively, in the anaerobic digestion of different substrates. The residual P in the substrates in this experiments (with the exception of G3) was relatively high and can be reduced by adding excess Mg in SP. Due to different final TAN concentrations in different treatments that may itself cause an impact, the TAN concentration was normalized to 0.6 g¨L´1 in all treatments prior to conducting the BMP test (Table 5). Table 5. Biochemical methane potential (BMP) assay of struvite precipitation (SP) treated substrates. Cumulative methane productions and yields are presented in Figure 1. As expected, the control with only inoculum had negligible methane production (3 mL, Figure S1) compared to other treatments, confirming the very low biodegradable organic fraction in the inoculum. All treatments showed long lag phases in the range of 30 to 140 days, which is longer than reported in other similar work [1,5]. The inoculum from the methanogenic digester operated at 26˝C may also have required some time to adapt to the higher test temperature of 38˝C. The pH in all treatments increased from 7.3 to around 7.6 and then stabilized for the rest of the BMP test, with a slight pH drop that occurred at the 122nd day ( Figure 2). The buffering effects to maintain an acceptable pH were from TAN and P compounds including solution of Na 3 PO 4 , Na 2 HPO 4 or NaH 2 PO 4 . It is known that these compounds have a high buffering capacity and are often used to make common buffering reagents. Table 6 shows performance parameters of each treatment. The BMP results of treated substrate BG5 showed improvement over the untreated control, while other treatments had no significant difference. Treatment BG0 that contained untreated substrate had a relatively longer lag phase than most other treatments. However, methane yield from BG0 was in the range of most other treatments with the exception of BG5. These results indicate that SP shortened the adaptation time. Among BMP of treated substrates, COD and TAN of feedstocks had negligible differences ( Table 7). The major differences impacting the BMP test were residual Cl´, Na (in form of sodium ion) and P (total phosphorus). Chloride toxicity was reported at a concentration of 5500 mg¨L´1 in the case of 0.6 g¨L´1 tannin input in AD [16]. Since measured levels in the treatments were lower (335 to 3026 mg¨L´1), it was concluded that chloride toxicity may not have been a factor in this experiment. Inhibition level of Na is known to be 3500 mg¨L´1 in AD [31] and since measured Na levels in all treatments were lower, no negative impacts were anticipated.       Treatment BG5 had the shortest lag phase (8.7 days) and highest methane yield (180.2 mL¨g´1 COD added ) in this experiment. BG1 with 897 mg¨L´1 of P had the longest lag phase of 125.7 days, suggesting that microorganisms required a long adaption period to adjust to high P substrate. The best performance of BG5 showed that 623 mg¨L´1 of P was an optimum level, compared to the 408 to 422 mg¨L´1 of P in treatments of BG7 and BG8. This result is slightly higher than results in a previous study [33] that reported the optimum P concentration was 465 mg¨L´1. COD removal of all treatments ranged from 42% to 65%.

Optimizatiom of SP Treatment
In SP treatments, TAN was significantly removed from substrates, which improved anaerobic process by shortening the lag phase time or increasing methane yields. However, residuals such as Na, Cl´, Mg (total magnesium) and P were introduced at different levels to the substrate. If not properly managed, the high concentrations of such elements can result in inhibition of AD, economic loss of P as a valuable nutrient, and potential eutrophication of water bodies if the P was discharged to the environment.
Actions should be taken to minimize the residual Na and P in the treated substrates lower than the inhibition levels. Adding extra Mg and reducing the SP reaction pH can facilitate P precipitation and reduce usage of NaOH for pH adjustment, respectively. Because of the low solubility of most Mg sources and high solubility of P sources, the addition of excess Mg source is required to provide more active Mg 2+ to improve SP. Celen and Türker [9] reported that when TAN and P are present in equimolar concentrations, SP was improved when Mg concentration was increased. Yetilmezsoy and Sapci-Zengin [19] also concluded that TAN removal efficiency increased when excess Mg was added using MgCl 2 . Few studies have looked at using MgCO 3 in SP in wastewaters, probably because of its higher price compared to MgO, MgCl 2 or Mg(OH) 2 . The Mg provided by MgO is cheaper than that of MgCl 2 [9]. To keep cost down, low-grade MgO can be used to remove the TAN (e.g., [11]). The residual Na from NaOH addition can be decreased if the reaction pH is controlled at 7.5 or 8.0 without seriously impact of SP effect [9]. Treatment BG5 had 41.7% more methane yield than treatment BG0 and obtained 57.6% nitrogen recovery in the form of struvite (calculated from Tables 4 and 5). SP in other treatments recovered more than 57% nitrogen but did not seem to have much impact on methane yields, though the longest lag phase occurred in treatment BG1. Finally, based on nitrogen recovery performance and BMP it is concluded that BG5 was the best candidate among those tested and is recommended.

Economic Analysis
Economic benefits were estimated to evaluate feasibility of SP in AD of poultry slaughterhouse blood wastes. Figure 3 and Table 8 show a chemical mass flow and the estimated economic benefits from SP for downstream methanogenesis, respectively. The net economic benefits from struvite and additional methane sale in the SP system using MgO and Mg(OH) 2 are $434.42 and $262.13-738.10 d´1, respectively. The cost of Mg accounts for 11.2%-39.6% of output benefit, however, additional methane sale only accounts for 0-2.2%. Hence, using low-cost Mg sources could significantly increase economic benefits of this process. Several researchers [11,34] suggest use of cheaper MgO/Mg(OH) 2 sources such as low-grade MgO and Mg(OH) 2 slurry to improve economic benefits and enhance process feasibility in large scale AD. The feasibility of using low-cost Mg can be evaluated using methods introduced in this study or by software simulation [35] to avoid residual toxins in the treated substrates that could inhibit downstream methanogenesis. In addition, it should be mentioned that the system using Mg(OH) 2 showed a shorter lag phase in the BMP test (Table 6), suggesting shorter HRT in continuous operation. This will allow for a smaller digester, thus reducing capital costs. Comprehensive evaluations, including the price of Mg, toxicity in methanogenesis and methanogenic digester capital costs, should be made before choosing the Mg source in this system.
Molecules 2016, 21, 1011 9 of 16 more than 57% nitrogen but did not seem to have much impact on methane yields, though the longest lag phase occurred in treatment BG1. Finally, based on nitrogen recovery performance and BMP it is concluded that BG5 was the best candidate among those tested and is recommended.

Economic Analysis
Economic benefits were estimated to evaluate feasibility of SP in AD of poultry slaughterhouse blood wastes. Figure 3 and Table 8 show a chemical mass flow and the estimated economic benefits from SP for downstream methanogenesis, respectively. The net economic benefits from struvite and additional methane sale in the SP system using MgO and Mg(OH)2 are $434.42 and $262.13-738.10 d −1 , respectively. The cost of Mg accounts for 11.2%-39.6% of output benefit, however, additional methane sale only accounts for 0-2.2%. Hence, using low-cost Mg sources could significantly increase economic benefits of this process. Several researchers [11,34] suggest use of cheaper MgO/Mg(OH)2 sources such as low-grade MgO and Mg(OH)2 slurry to improve economic benefits and enhance process feasibility in large scale AD. The feasibility of using low-cost Mg can be evaluated using methods introduced in this study or by software simulation [35] to avoid residual toxins in the treated substrates that could inhibit downstream methanogenesis. In addition, it should be mentioned that the system using Mg(OH)2 showed a shorter lag phase in the BMP test (Table 6), suggesting shorter HRT in continuous operation. This will allow for a smaller digester, thus reducing capital costs. Comprehensive evaluations, including the price of Mg, toxicity in methanogenesis and methanogenic digester capital costs, should be made before choosing the Mg source in this system.    [38]. Mg(OH) 2 is in slurry form ($0.28 kg´1) or as powder ($0.99 kg´1); 4 Average suggested market value of struvite in [39]; 5 Calculated from the average residential natural gas price of year 2010-2015 [40]. Unit price and flow rate are in unit of $¨m´3 and m 3¨d´1 , respectively. The natural gas containing 100% methane is assumed for easy calculation.

Substrates
Acidogenic digester effluents were collected from three identical 40-L pilot acidogenic digesters treating co-substrates of poultry slaughterhouse wastewaters (PPWW) and poultry blood (3:1, v/v) that was diluted 50% with de-chlorinated water to reduce its initial organic strength. Each digester was stirred by recirculating substrates from the bottom to the top using a diaphragm pump, at an approximate flow of 5.7 L¨min´1, that was operated by a programmable timer for 15-min on and 15-min off. The digesters had been actively operating at mesophilic conditions (26˘2˝C) for approximately six months at the point of sampling. Collected samples were stored in a refrigerator (ď4˝C) for further testing prior to use. These compounds were selected based on ease of market availability and the low concentration of residual toxins that remain after reactions (Table 9). A solution of 300 g NaOH¨L´1 was prepared and used for pH adjustment in the SP protocol. Nine combinations of reagents, namely, MgO + 85% H 3

Struvite Precipitation Protocol
Amount of reagents required for 500 mL of substrate was calculated so as to result in a molar ratio of 1:1:1 of Mg:NH 4 :PO 4 . The substrate TAN was measured to be 1.36 g¨L´1, while Mg nor P in the feedstock were measured because expected amounts in the substrate were known to be relatively low (based on values in the range of 0.006 to 0.0655 g¨L´1 measured during preliminary studies). Reagents and substrates were reacted in 500-mL Erlenmeyer flasks with aluminum covers and a magnetic stir bar placed in the flask to stir the substrates at 500 rpm on a stir plate. A volume equal to 510 mL substrates was placed in the Erlenmeyer flask and heated to room temperature in 1 to 3 min using a water bath at 50˝C. A 10-mL aliquot of substrates was sampled at the start of the experiment (T0). Subsequently, Mg and P reagents were added to the substrates in sequence (MgHPO 4 was dissolved using small amount of diluted HCl solution and then added to the substrate). After adjusting substrate pH to 8.5, 10-mL substrate was sampled after the 20 min (T20) time point. A 1-mL aliquot from the T0 samples and from the supernatants of the T20 samples (1 h after sample collection) was analyzed for TAN concentrations. The SP after 20 min of reaction time was considered complete based on previous studies [9,19]. Each treatment was replicated twice.

Treatments Used in Experiments
Due to high levels of TAN removal (>70%) observed with different reagent combinations, treatments G1, G5, G7 and G8 were selected for further BMP analyses. Treatment G3 was also selected because of highest TAN removal and the popular use of these reagents in previously reported SP studies. A volume equal to 1700 mL of substrates was placed in a 2000-mL Erlenmeyer flask and processed exactly as was done in the SP experiment described earlier. After 20 min, stirring was stopped and the substrate was allowed to stand in the flask for one hour to allow for gravitational settling of struvite precipitates. The supernatant was poured slowly into storage bottles and stored in a refrigerator (4˝C) for further use in chemical analyses and BMP testing.

Inoculum
Inoculum used in the BMP tests was collected from a 87-L methanogenic digester treating pH adjusted effluent (pH 7.2-7.4) from a 40-L acidogenic digester. This methanogenic digester was operated at an OLR of 0.4 g COD¨L´1¨d´1 (corresponding to HRT of 37.8 days) and had been actively operating at mesophilic conditions (26˘2˝C) for approximately four months when inoculum was collected. The inoculum was placed in a pre-incubated anaerobic digester at 38˝C for three days to deplete any un-degraded biological residues present in the inoculum before use in the BMP assay, as suggested by Angelidaki et al. [41]. The characteristics of the inoculum were 2.9˘0.1 g¨L´1 of TS, 1.3˘0.1 g¨L´1 of VS, 1.4˘0.0 g¨L´1 of TAN, and 1.6˘0.4 g¨L´1 of COD.

Biochemical Methane Potential (BMP) Assay
The BMP was tested using 500-mL batch anaerobic digesters with an effective substrate volume of 300 mL incubated at 38˝C. Substrate to inoculum ratio was 70/30 (v/v) which corresponded to 3 g-VS untreated substrate to 1 g-VS inoculum [42]. Because different treatments had different final TAN concentrations after SP, before use in the BMP test, they were mixed with untreated substrate (which had higher TAN concentration) to normalize all treatment TAN concentrations to 0.6 g¨L´1. The pH of the mixture of untreated/treated substrates and inoculum was then adjusted to 7.3 using HCl solution before placing in the BMP test digesters. The headspace of the digesters were purged using N 2 gas and sealed using butyl rubber stoppers and aluminum crimps. To obtain a complete profile, the BMP digesters were incubated for 278 days with treatment and controls replicated three times. Blank digesters of triplicate were run using the inoculum and DI water replacing substrates. Methane yields were calculated at normal temperature and pressure (NTP) and expressed as mL CH 4¨g´1 COD added . Biogas production and methane concentrations in the biogas were measured every 4 to 12 days depending on the level of activity. The pH was measured every 7 to 57 days by collecting 1-mL sample from serum bottles and measuring pH using a laboratory pH probe. Small amounts of substrates (12 mL) were taken from each digester for pH measurement in the whole experiment period. The pH was measured more frequently, every 7 days in the first month, and less frequently close to the end of experiment, 57 days.

Calculation of Cumulative Methane Production
The cumulative methane production was calculated using the following equation where M n is the cumulative methane production till the nth day (mL); m n is the biogas production on the nth day (mL); C n is the methane content in biogas on the nth day; H is the headspace in the bottle and measured as 239 mL.
In this equation, n ř 1 pm nˆCn q is the cumulative methane volume measured by discharging biogas and testing methane percentage in biogas on the nth day and HˆC n is the methane volume in the bottle headspace on the nth day.

Modeling the Kinetics of Methane Production
A modified Gompertz model was used to model cumulative methane production during the incubation period [5] as shown below: where M is the cumulative methane production (mL); e is 2.718282; R m is the maximum specific methane production rate (mL¨d´1); P m is methane production potential (mL); and λ is the lag phase time (days).

Analytical Methods
The pH was measured using an Accumet portable AP61 pH meter (Fisher Scientific, Hampton, NH, USA). COD was measured in samples that were diluted 100-fold using the HACH method 8000 (HACH, Loveland, CO, USA). Total solid (TS), volatile solid (VS) and total suspended solid (TSS) were analyzed following standard laboratory methods used in the wastewater industry [43]. TS and VS were measured by drying 30-mL samples at 105˝C for 24 h and then burning at 550˝C for 1 h. TSS was measured by filtering 10 mL samples through a 1.6-µm filter and drying the filter with residue at 105˝C for 24 h. Total Nitrogen (TN) and TAN were measured on a 100-fold diluted sample using HACH method 10072 and 10031, respectively (HACH, Loveland, CO, USA). Micronutrients and chloride concentration of samples were analyzed at the Soil, Plant, and Water Analysis Laboratory of University of Georgia. For micronutrients analysis, 0.5-g or 1-mL sample was added to 5 mL of concentrated HNO 3 and digested in a microwave oven following the USEPA method 3051A. The digested solutions were analyzed using an Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES) (Spectro Arcos FHS16 AMETEK ICP-OES, SPECTRO Analytical Instruments Inc., Mahwah, NJ, USA). For Clá nalysis, the samples were diluted 100 to 2000 fold using DI water and filtered through a 0.45-µm syringe filter. Further analyses were carried out in an ion chromatograph (Metrohm 861 Advanced Compact IC, Metrohm Ltd., Herisau, Switzerland) running at a flow rate of 0.7 mL¨min´1. The CHNS concentrations were measured using a FLASH 2000 CHNS-O analyzer (Thermo Fisher Scientific, Waltham, MA, USA) on freeze dried samples. Approximately 1 mg dried samples were weighed in tin capsules and placed in the instrument that quantified elements by combustion and detection of elements in the off gases. Volume of biogas produced was measured by volume-displacement in a Eudiometer water column (Selutec, Hechingen, Germany), while methane concentration was measured using a GC-FID (SRI310C, SRI Instruments, Torrance, CA, USA). The method used a stainless steel column (80/100 HayeSep D 6'ˆ1/8", Supelco, Bellefonte, PA, USA); oven and detector temperatures of 40˝C and 380˝C, respectively; Carrier gas, fuel gas and oxidizing gas were helium (10 mL¨min´1), hydrogen (25 mL¨min´1) and air (250 mL¨min´1), respectively. Total biogas volume generated was measured by puncturing the rubber lid of each digester with a needle and syringe, which was connected to a Eudiometer by airtight tubing. Biogas samples (0.1-mL) were taken from the headspace of each digester using a gastight syringe and tested in the GC.

Statistical and Regression Method
A one-way ANOVA and Tukey HSD test (JMP software, Pro 10, SAS Institute, Cary, NC, USA) were used to compare methane yields and COD and TAN removal from different substrates in the BMP study. Differences between treatments were considered significant at p ď 0.05. The nonlinear regression of the modified Gompertz model was performed using SigmaPlot 12.

Economic Analysis
A typical poultry processing plant processes 200,000 birds daily and discharges 17,200 kg blood when 50% of the total blood generated is collected [44]. Blood can be diluted using poultry slaughterhouse wastewaters and fresh water at a volumetric ratio of 1:3:4 (blood: wastewaters: fresh water) and fed into digesters as described in the experiment. Assuming the diluted blood has the same density as water, 137,600 L¨d´1 acidogenic digester effluent containing 1.4 g¨L´1 TAN enter the proposed SP system. In the system using MgO as Mg source, all the effluent is treated (BG1); in the system using Mg(OH) 2 , 84% (115,584 L¨d´1) is treated by SP and 16% (22,016 L¨d´1) is used to mix the SP treated effluent (BG5) as indicated in Table 5. MgO and Mg(OH) 2 are chosen in this calculation because of their wide use in environmental applications and the lower NaOH requirement [38] ( Table 3).

Conclusions
The SP treatment using different groups of Mg and P sources recovered nitrogen and improved methane yields, while increasing buffering capacity of acidogenic digester effluents. The treatment using Mg(OH) 2 and H 3 PO 4 was the best candidate for TAN removal and methane yield, compared to the control. Residual components after SP, including P and Na, could lead to inhibition of AD and the loss of valuable nutrients. This could be minimized by process control strategies such as addition of extra Mg and lowering the operational pH. SP treatment also contributes to positive economic benefits due to struvite recovery and/or additional methane production.