Microbial monitoring of ammonia removal in a UASB reactor treating pre-digested chicken manure with anaerobic granular inoculum

Accepted Manuscript Microbial monitoring of ammonia removal in a UASB reactor treating pre-digested chicken manure with anaerobic granular inoculum Cigdem Yangin-Gomec, Goksen Pekyavas, Tugba Sapmaz, Sevcan Aydin, Bahar Ince, Çağrı Akyol, Orhan Ince PII: DOI: Reference:

S0960-8524(17)30724-1 http://dx.doi.org/10.1016/j.biortech.2017.05.070 BITE 18096

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

29 March 2017 9 May 2017 12 May 2017

Please cite this article as: Yangin-Gomec, C., Pekyavas, G., Sapmaz, T., Aydin, S., Ince, B., Akyol, C., Ince, O., Microbial monitoring of ammonia removal in a UASB reactor treating pre-digested chicken manure with anaerobic granular inoculum, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.070

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Microbial monitoring of ammonia removal in a UASB reactor treating pre-digested chicken manure with anaerobic granular inoculum

Cigdem Yangin-Gomec1*, Goksen Pekyavas1, Tugba Sapmaz1, Sevcan Aydin2, Bahar Ince3, Çağrı Akyol3, Orhan Ince1

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Istanbul Technical University, Department of Environmental Engineering, Maslak, 34469 Istanbul,

Turkey. 2

BioCore Biotechnology Environmental and Energy Technologies R&D Ltd., Istanbul, 34217, Turkey.

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Boğaziçi University, Institute of Environmental Sciences, Bebek, 34342 Istanbul, Turkey.

*Corresponding author (C. Yangin-Gomec) E-mail: [email protected] Tel: +902122853787 Fax: +902122856545

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ABSTRACT Performance and microbial community dynamics in an upflow anaerobic sludge bed (UASB) reactor coupled with anaerobic ammonium oxidizing (Anammox) treating diluted chicken manure digestate (Total ammonia nitrogen; TAN=123±10 mg/L) were investigated for a 120-d operating period in the presence of anaerobic granular inoculum. Maximum TAN removal efficiency reached to above 80% with as low as 20 mg/L TAN concentrations in the effluent. Moreover, total COD (tCOD) with 807±215 mg/L in the influent was removed by 60-80%. High-throughput sequencing revealed that Proteobacteria, Actinobacteria, and Firmicutes were dominant phyla followed by Euryarchaeota and Bacteroidetes. The relative abundance of Planctomycetes significantly increased from 4% to 8-9% during the late days of the operation with decreased tCOD concentration, which indicated a more optimum condition to favor ammonia removal through anammox route. There was also significant association between the hzsA gene and ammonia removal in the UASB reactor.

Keywords: ammonia removal; anammox; chicken manure; next generation sequencing; Planctomycetes; UASB reactor

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1. Introduction Serious environmental problems can be faced through unmanaged poultry wastes and/or their inappropriate land applications. While ammonia and greenhouse gasses (GHG) emitting from waste storage facilities can pose a threat to air quality control mechanisms (Yetilmezsoy and Sakar, 2008; Abouelenien et al., 2010), nitrogen and phosphorus contamination can lead to eutrophication of surface waters and consequently pollute soil and ground water (Güngör-Demirci and Demirer, 2004). Both ecosystem and human health are adversely affected by high concentrations of nitrogen compounds (toxic >1 mg N/L); whereas free ammonia nitrogen (FAN) is toxic to aquatic organisms at lower concentrations to 0.25 mg/L as well as nitrites (Knobeloch et al., 2000). In this regard, removal of nitrogen compounds from poultry manure is crucial to reduce its adverse effects on the environment. Conversion of organic materials such as manure and other bio-based alternatives to energy (biogas) through anaerobic digestion (AD) is a well-known process that has many benefits including ceasing GHG emissions and reducing the consumption of fossil fuels (Nasir et al., 2012). However; nitrogen content of the materials are not affected during AD in fact it causes its mineralization; hence, further operations may be needed in case of nutrient surplus (Magri et al., 2013). In this context, anaerobic ammonium oxidation (anammox) has gained certain attention since its discovery, because it is an efficient biological alternative to conventional nitrogen removal by nitrification-denitrification process (Molinuevo et al., 2009). Anammox is reported as one of the promising new means of removing nitrogen from wastes with high loadings of greater than 900 g-N/m3/d (Isaka et al., 2006). In anammox process, ammonium is oxidized to nitrogen gas under anaerobic conditions with nitrite as the electron acceptor and carbon dioxide is used for the growth of bacteria. The most important advantage of this process is that it consumes less biodegradable organic carbon and less oxygen, which makes it a more cost-effective system. Moreover, less biosolids are produced due to the slow

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growth rate of anammox bacteria and higher rates of N removal are achieved as well as providing a coupling with biogas production (Fux and Siegrist, 2004; Magri et al., 2013). Anammox reaction is catalyzed by the bacteria namely anaerobic ammonium-oxidizing bacteria. The process is autotrophic and the bacteria use dissolved carbon dioxide or bicarbonate for cell biosynthesis (Isaka et al., 2006). The anammox bacteria described so far are encompassed within the phylum Planctomycetes. Since their discovery, anammox bacteria have been isolated from various sludge samples such as sewage sludge, nitrifying and denitrifying sludge, anaerobic granular sludge as well as marine environments (Tang et al., 2010; Magri et al., 2013). This culture grows very slowly with a doubling time of approximately 11 d and it is extremely sensitive to changes in environmental conditions. Generally, it is reported that the presence of organic carbon source would adversely affect anammox bacteria due to the possible competition for nitrite between anammox bacteria and heterotrophic denitrifiers (Isaka et al., 2006; Molinuevo et al., 2009; Chen et al., 2016a). Because the specific maximum growth rate of the anammox bacteria (0.08 d -1 at 20oC) is much lower than that of the heterotrophic denitrifiers (6.00 d-1 at 20oC) (Lackner et al., 2008). On the other hand, Kumar and Lin (2010) reported that the coexistence of anammox and heterotrophic denitrification might reduce nitrate concentrations in the reactor. Certainly, the coexistence of these two processes can be feasible based on the availability of biodegradable organic carbon (Magrí et al., 2013). Despite all the benefits stated, the main challenge for conducting the anammox process is long startup periods due to the slow growth rate of anammox bacteria. Hence, seeding sludge is of great importance in order to enrich and maintain these bacteria in bioreactors. It has been indicated that anaerobic granular sludge contains Planctomycetes genes and has been successfully applied during the start-up of anammox bioreactors (Schmidt et al., 2004; Tran et al., 2006; Tang et al., 2009; Molinuevo et al., 2009). Recent molecular methods such as pyrosequencing are widely used to identify thousands of operational taxonomic units (OTUs) in complex sludge samples in order to ensure its suitability for anammox systems (Chen et al., 2016a). Furthermore, pyrosequencing can be

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also used throughout the operation period to gain microbial insights into the evolution of microbial community structure, such as in membrane bioreactors (Hu et al., 2012), sequencing batch reactors (SBR) (Liang et al., 2014), expanded granular sludge bed (EGSB) reactors (Liao et al., 2013) and anaerobic baffled reactors (ABR) (Chen et al., 2016a). However, few studies have focused on the microbial community dynamics in upflow anaerobic sludge blanket (UASB) reactors containing anaerobic granular sludge in terms of ammonia removal via anammox process (Tang et al., 2009; Tang et al., 2011; Xiong et al., 2013; Chen et al., 2016b). The objective of this particular study is therefore to evaluate the performance and to monitor the microbial community dynamics of an UASB reactor operated for ammonia nitrogen removal (anammox-UASB reactor) from diluted chicken manure digestate. In this scope, UASB reactor was inoculated with anaerobic granular sludge and changes in the relative abundance of microbial populations in time were identified by Illumina Miseq next generation sequencing (NGS). It is considered that, microbial monitoring could provide a better understanding of the relationship between the changes in the influent characteristics (e.g., organic strength, ammonia nitrogen etc.) and the route by which ammonia removal is favored in such systems.

2. Materials and methods 2.1. Anammox-UASB reactor and operating conditions A lab-scale continuously stirred anaerobic reactor digesting chicken manure (with a TS content of ca. 5.5%) with an effective volume of 2.5 L was operated in semi-continuous mode at mesophilic condition (35°C) in order to provide fresh digestate (i.e., the solids retention time was 60 d). Simultaneously, a second bio-system namely the UASB reactor; was operated in semi-continuous mode in the same constant temperature room at mesophilic condition following the first bioreactor reached to steady-state. The effective volume of the UASB reactor was 6.45 L (Fig. 1) which comprised of a Plexiglas column with 1.0 m height and 90 mm diameter. A special gas-solids-liquid

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separator on the top of the reactor collected the produced biogas. The UASB reactor was fed from the bottom port with 500 mL/d pre-digested chicken manure (i.e., digestate of the above-mentioned anaerobic digester) at an appropriate dilution ratio with tap water with a hydraulic retention time (HRT) of 13 d during the operating period of about four months. 2.2. Chicken waste and the inoculum Raw chicken waste was taken fresh from a chicken farm with a daily capacity of about 20,000 eggs from 275,000 livestocks. The waste produced in this industry was the manure only from the layinghen having average TS of about 28% with the volatile content of ca. 56%. The inoculum was taken from the mesophilic anaerobic Internal Circulation (IC) reactor treating the wastewater produced at a paper/cardboard industry. The seed was the granular sludge with a TS concentration ca. 95 g/L (VS/TS ratio of ca. 50%). The continuously stirred anaerobic reactor was seeded in a 1:3 ratio (v/v) using this active methanogenic sludge. On the other hand, the anammox-UASB reactor was inoculated with 800 mL of the original seed and 500 mL of the adapted seed (i.e., the biomass from the first anaerobic digester that has been already treating chicken manure). Hence, at the start-up period; the anammox-UASB reactor consisted of the inoculum sludge in a 1:5 ratio of the working volume. 2.3. Characterization of initial substrates The digestate of the continuously stirred anaerobic digester treating the laying hen waste with a TS of ca. 5.5% was used as the feed of the UASB reactor after diluted with tap water. Average characterization of the digestate before dilution was as follows: pH, 7.95; TKN, 3750 mg/L; TAN, 2320 mg/L; FAN, 190 mg/L; TP, 3750 mg/L; BOD5, 8900 mg/L; sCOD, 7350 mg/L, and tCOD, 20175 mg/L. On the other hand, influent feed characterization of the UASB reactor is presented in Table 1. Since the digestate of the anaerobic digester comprised of high TAN; it was diluted using tap water that consisted of the nitrite source namely NaNO2. Nitrite was included externally because the bacteria needed sufficient nitrite for removing ammonia from wastewater through anammox

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process. At the start-up; NH4:NO2 ratio in the influent of the anammox-UASB reactor was provided to be as 1.0 as well as TAN concentration was about 125 mg/L as recommended (i.e., TAN≤ 250 mg/L) for an effective anammox reaction (Dong and Tollner, 2003).

2.4.Analytical methods The performance of the bioreactors was investigated by measuring the following parameters; alkalinity, tCOD, sCOD, TS, VS, TSS, VSS, and total ammonia [the ammonium ion (NH4+) and unionised ammonia (NH3)] nitrogen concentrations according to Standard Methods (APHA; 2005). FAN concentrations were calculated according to the formula, as described by Hansen et al. (1998). Besides, nitrate and nitrite in aqueous solution were measured at HACH DR/2010 Spectrophotometer using the NitraVer®5 (cadmium reduction method) and NitriVer®2 (ferrous sulfate method) powder pillows, respectively. HI 2211-02 HANNA Model pH meter was used for the pH measurements. Daily biogas production was measured using the Ritter MilliGas Counter 770991000 model gas meter (Ritter, Germany). The content of the produced biogas (i.e. nitrogen, methane and carbon dioxide) was measured by HP Agilent 6850 Gas Chromotography (GC) equipped with a thermal conductivity detector (HP Plot Q column 30 m x 0.53 mm). Helium was used as the carrier gas at a rate of 2 mL/min and the oven temperature was maintained at 70°C. 2.5. Microbial community analysis The total DNAs were isolated from the 1 mL sludge samples by using PureLink Genomic DNA extraction kits (Invitrogen, U.K.) according to the manufacturers’ instructions. NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to determine the concentration. Three primer sets (Amx 694-Amx960R, hzsB_396F-hzsB_742R, hzsA_1597F-hzsA_1859R) targeting 16SrRNA gene was used to quantify the anammox bacteria. Detailed information on quantitative real time polymerase chain reaction qPCR analysis has been reported in a previous study (Aydin et al., 2015). 16S rRNA genes were sequenced following the Illumina MiSeq method 7

(Illumina, Inc., San Diego, CA, U.S.A.). The V4-V5 hypervariable region of the 16S rRNA gene was reproduced with region-specific primers and sequence analysis and the identification of operational taxonomic units (OTUs) were obtained using the methods suggested by Cole et al. (2014) and DeSantis et al. (2006). 2.6. Statistical analysis R 3.1.1 analysis was used to conduct Statistical analyses (www.r-project.org). Histogram, q-q plots and the Shapiro-Wilk’s test were performed to examine data normality. Variance homogeneity was also investigated by using The Levene’s test. One-way analysis of variance (ANOVA) or independent-samples t-test was used to check against the variations in ammonia removal and microbial community dynamics. To provide multiple comparison, The Tukey’s test was used. Values of tests were pointed out as mean and standard deviation. The applicability of microbial community and reactor performance were determined by Pearson’s test as a correlation test. Important difference was detected at the p<0.05 level of importance. 3. Results and Discussion 3.1. Treatment performance and biogas production At the start-up; the anaerobic digester treating the chicken (laying hen) waste was operated with a TS of ca. 5.5% according to optimization results of a previous study (Yangin-Gomec, 2017). Results indicated that the chicken waste could be successfully treated indicating a cumulative biogas production of ca. 60 L during a total digestion period of about 4 months operated in semi-continuous mode at a SRT of ca. 60 days (data not shown). In this context, the biogas yield was observed as about 0,469 L/gr VSadded. When this digester reached to steady-state condition, its digestate was then fed to the UASB reactor with a HRT of 13 days. Since the digestate comprised of high TAN (i.e., 2320 mg/L on average); it was diluted with tap water in order to provide ammonia nitrogen in the feed solution lower than 250 mg/L. Although operational and capital costs increase; Magri et al. (2013) also recommended dilution in order to prevent risk of biomass inhibition due to high

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concentrations of N inside the reactor where anammox takes place. Moreover, it is reported that NH4 + and NO2- are converted to N2 and nitrate (NO3-) under stoichiometric molar ratios of 1.00/1.32/1.02/0.26 for NH4+ consumption, NO2 consumption, N2 production, and NO3- production, respectively. In this particular study, NaNO2 was included externally into the influent of UASB reactor prior to daily feeding in order to provide NH4 :NO2≅1.0 as recommended for a successful anammox process. On the other hand, since the pre-digested chicken waste also comprised of high tCOD (i.e., 20175 mg/L on average); it needed more dilution that resulted in an average TAN concentration of 123±10 mg/L in the influent at the start-up. In this context, TAN was removed by 56±18% in the bioreactor in this study. Average TAN concentration was 54±22 mg/L (Fig. 2) as well as FAN was below 20 mg/L in the effluent of the system dependent on the pH value. Average influent and effluent NO3-N concentrations were about 140 and 50 mg/L, respectively. On the other hand, NO2-N concentrations of the effluent were always below 5 mg/L during the study. Moreover, TSS and VSS concentrations were 998±208 and 353±49 mg/L in the influent whereas 126 and 20 mg/L on average in the effluent, respectively. Hence, respective average TSS and VSS removals were ca. 87% and 94% during the operating period. The results also indicated that sCOD and tCOD removals in the anammox-UASB reactor were 26±8% and %63±11% respectively. Profile of tCOD during the operating time of the bioreactor is presented in Fig. 3. Although Chamchoi et al. (2008) reported that tCOD concentrations should not exceed 300 mg/L for an effective process for anaerobic ammonia oxidation; the bioreactor could be operated at much higher initial tCOD values in this study. Accordingly, tCOD with 807±215 mg/L in the influent was removed by 60-80% and efficiency decreased as tCOD concentration in the influent was lowered. Although ammonia removal via anammox has been developed for the treatment of many different wastewaters with low organic matter content (below 1700 mg COD/L); only a few studies have investigated the possibility of using the anammox process for ammonia removal from animal waste

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treatment water with high organic matter and nitrogen content (Breisha and Winter, 2010; Waki et al., 2007). For example, up to ca. 99% of ammonia was removed from a diluted partially oxidized pig manure effluent (121 mg COD/L) using the anammox process under different organic loadings in a semi-continuous UASB reactor (Molinuevo et al., 2009). Alkalinity and pH parameters were also monitored throughout the study. No deliberate influent pH control was carried out, and sufficient buffering capacity was still achieved. The buffering capacity of the solution was an important factor since it contributes to the pH variations. Respective influent and effluent alkalinity ranges were 807-1007 and 583-700 mg CaCO3/L during the operational period. Hence, sufficient alkalinity in the system led to smaller pH variations (i.e., 8.18±0.13) in the effluent. Moreover, influent pH value was 7.99±0.07. The importance of pH as an important control parameter during the operation of bioreactors was highlighted in previous studies, especially when ammonia removal has been investigated in anaerobic systems via anammox process (Strous et al., 1999; Waki et al., 2007; Tang et al., 2009). For example, Strous et al. (1999) studied the effect of pH on anammox process and batch experimental results indicated that the specific anammox activity at pH of 9.0 was only 1/5 of that at of pH 8.0 and optimum pH range for anammox bacteria was reported as 6.7–8.3. Under high pH, operational conditions were not suitable for the growth and metabolism of anammox bacteria leading to performance failure for long operational times. Correspondingly, high pH was accompanied by a high FAN concentration, which is toxic to microorganisms’ anabolic and catabolic processes (Vadivelu et al., 2006; Tang et al., 2009). In another study by Waki et al. (2007), the FAN concentrations of 13–90 mg/L negatively affected the performance of the anammox process. It was reported that the nitrogen removal performance was lower than half of that in the control reactor with FAN < 21 mg/L. In the present study, the FAN was always below 20 mg/L in the effluent of the anammox-UASB reactor because of the stable pH range. The relatively low pH and FAN ensured that the UASB reactor achieved a better nitrogen removal performance as also reported by Tang et al. (2009).

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According to the daily biogas results (i.e., the average being 30 mL/day); about 1.2 L cumulative biogas production was observed at the end of the operational period (Day 120). Change in the cumulative biogas production was shown in the Supplementary file. On the other hand, 43% N2 and 15% CH4 compositions were measured in the biogas while the rest was composed of other gases.

3.2. Changes in microbial biodiversity in the UASB reactor Firstly, qPCR was applied to assess changes of anammox bacteria during the operation of UASB reactor as given in Fig. 4. Quantitative changes of the hzsA gene was found to significantly (p<0.05) correlate with ammonia removal in the UASB reactor. On the other hand, positive correlation was not determined between hzsB gene and ammonia removal according to the results of statistical analysis by Pearson’s correlation test. Due to the limited availability of primers to detect anammox bacteria, the use of a high-throughput Illumina Miseq to perform microbial community analysis is widely considered to represent a promising culture-independent method by which the diversity in bioreactors can be determined (Aydin, 2016). In this study, anammox-UASB reactor treating diluted chicken digestate was also investigated using Illumina Miseq next generation sequencing (NGS) analysis. Change in the bacterial community throughout the operational period was detected in accordance with organic material and ammonia removals as seen in Fig. 5. The biomass samples taken from the UASB reactor at the first (Day 0), 90th (Day 90), and 120th (Day 120) days of operation were dominated by Proteobacteria, Actinobacteria, and Firmicutes followed by Euryarchaeota and Bacteroidetes, respectively at the phylum level. Synergistetes, which is a recently recognized phylum of anaerobic bacteria showing Gram-negative staining and have rod/vibrioid cell shape, stayed relatively stable during the operation between the ratio of 5-7%. The results showed that Proteobacteria was at the highest level in the reactor as well as its relative abundance slightly increased as the operation

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continued (i.e., 20% on Day 0, 22% on Day 90, and 25% on Day 120). The second abundant bacteria namely Actinobacter was at a ratio between 17-19% from Day 0 to Day 90, and its relative abundance decreased to 14% in Day 120. Firmicutes, on the other hand, slightly increased towards the end of the reactor operation from 10% in the first sample to 12% on Day 90, and to 14% on Day 120. Compared to the sample taken at the start-up of the UASB reactor; no significant change in Euryarchaeota was observed throughout the operational period (i.e., ~10% on Day 0, 10% on Day 90, and ~12% on Day 120). Besides, Bacteroidetes was dominant in equal level (9%) in the biomass samples namely Day 0 and Day 120. The Illumina analysis also revealed that Chloroflexi (i.e., responsible for maintaining the granule structure as well as for triggering bulking) (Fernandez-Gomez et al., 2013), Cyanobacteria, Lentisphaerae, Thermotogae, and Verrucomicrobia were also present in the UASB reactor at phylum level. Similar results were reported by Chen et al (2016a) as the predominant phylum changed from Chloroflexi to Protecobacteria with elevating COD concentrations. In an another recent study, Cao and colleagues (2016) investigated microbial dynamics during anaerobic ammonium oxidization with partial denitrification process. They highlighted similar dominant bacterial phylum as Proteobacteria, Planctomycetes, Chloroflexi, Chlorobi, Bacteroidetes and Acidobacteria. The identification of the dominant microbial communities at class level is depicted in Fig. 6. The most abundant class in the UASB reactor was Betaproteobacteria as about 17%. Bae et al. (2007) also identified that the most predominant subphylum was Betaproteobacteria (45%) and Rhodocyclales in Betaproteobacteria subclass. The second most abundant class in the reactor was Bacteroidia (i.e., a class that is generally identified in anaerobic environments) and Clostridia (i.e., belongs to the phylum of Firmicutes). Although, relative abundance ratios of these two classes stayed constant from Day 0 to Day 90 (i.e., Bacteroidia from 15% to 16% and Clostridia from 15% to 17%, respectively); their ratios slightly decreased to ca. 12% and 11% on Day 120, respectively. The relative abundance order continued by Deltaproteobacteria and Gammaproteobacteria at

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approximately equal levels in all biomass samples (i.e., 5.8% on Day 0, 7.9% on Day 90, and 4.9% on Day 120) whereas Alphaproteobacteria decreased as operation continued (i.e., 5%, 4%, and 3%, respectively). A slight increase was also observed in the bacterial class namely, Planctomycetia (i.e., from 2% on Day 0 to about 5% on Day 120) under the phylum of Planctomycetes. Methanomicrobia was also found at a comparatively high abundance (i.e., 8.2% on Day 0, 9% on Day 90, and 10.2% on Day 120). In the taxonomy of microorganisms, Methanomicrobia are a class of the Euryarchaeota that are a phylum of the Archaea. The Illumina analysis also revealed that Erysipelotrichi, Thermotogae, Epsilonproteobacteria, Acidimicrobiia, Bacilli, and Methanobacteria (known to grow on H2/CO2 and formate as carbon source) were also present in the bioreactor at class level in this study. The identification of the dominant microbial communities at genus level was as follows: Archaea communities were represented by Methanospirillum, Methanoasaeta, Methanosarcina, and Methanobacterium in the anammox-UASB reactor. Among them, Methanospirillum and Methanosarcina genes were dominant in equal level in all samples whereas Methanoasaeta and Methanobacterium slightly increased at the end of the operation period. Methanospirillum as well as Methanoasaeta are known to be hydrogenotrophic as also reported by Demirel and Scherer (2008). Among the identified genes in the UASB reactor; the most abundant genus was Anaerovorax (i.e., defined as strictly anaerobic, non-spore-forming bacteria of fermentative metabolism, often metabolizing aminoacids) followed by Bacteroides (defined as a genus of Gram-negative, obligate anaerobic bacteria). Illumina analysis also revealed that Anaerovorax slightly decreased to 3.2% from 5.1% whereas Bacteroides genus was identified as equal measure as the operation continued. Moreover, Paludibacter Fluviicola and Streptococcus were identified in the samples with relative abundance ratio between 1.2% and 2.8% at genus level. 3.3. Assessment of microbial community, ammonia removal and biogas production in the UASB reactor

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In this study, qPCR and NGS techniques were used to monitor the microbial relationships that exist between bacteria and archaea so that they can be better understood, specifically in terms of how they impede the improvement of ammonia removal through biological treatment in anaerobic bioreactors. Furthermore, the use of qPCR for generating functional genes were essential for maintaining an efficient and stable operation of anaerobic processes treating nitrogen-rich substrates. The NGS data revealed that the bacterial phylum Planctomycetes (anammox bacteria) doubled its relative abundance in the biomass samples taken from the bioreactor as the operation continued. The relative abundance ratio of this bacterial phylum was 4% on Day 0 and increased to 9% and 8% on Day 90 and Day 120, respectively, where tCOD concentrations in the influent were comparatively lower. This result can be attributed to the fact that the Planctomycetes acted as the main phylum for efficient ammonia removal in the bioreactor and showed a possible sign of an adaptation to more stabilized anammox conditions with decreasing organic carbon content. Bae et al. (2007) also investigated NH4+-N removal by anammox bacteria using UASB reactor when the NH4 +-N to NO2−N ratio in the feed was 1:1. It was reported that the granular sludge turned brownish red due to the attachment of anammox bacteria on the surface of the seeding anaerobic granules as the process was stabilized. The overall microbial community structure included Proteobacteria (47%), Planctomycetes (25%), Chloroflexi (18%), Chlorobi (8%), and Acidobacteria (2%). Although similar results were obtained in the present study, the differences in the relative abundances were derived possibly due to varying environmental and operating conditions (e.g., inoculum source, substrate characteristics, feeding conditions). The molecular analysis also implied that Methanosarcinales (acetoclastic methanogens) acted as the main class, which contributed to methane production in the UASB reactor. Thus, methanogens especially acetoclastic methanogens should be also targeted as an indicator to develop a further and better understanding of microbial community. The analysis of the results pertaining the functional genes also found that an increase in the hydrazine synthase (hzsA) gene was found to be closely related to the increase in the number of anammox bacteria represented

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by the Planctomycetes in the UASB reactor. These results are consistent with those of previous studies and suggest that hzsA gene could also reflect the performance of UASB reactor treating nitrogen-rich substrates (Chen et al., 2016a; Wang et al., 2017). This present study has also revealed that evaluation of qPCR and NGS data are useful for suggesting the potential to monitor ultimate microbial community via bioaugmentation for ammonia removal in such systems. However, additional studies are still required to investigate alternative methods for improving ammonia removal rate in anaerobic processes like UASB reactors treating pre-digested chicken manure with high organic and nitrogen contents. 4. Conclusions In this study, it was demonstrated that simultaneous nitrate reduction and anaerobic ammonium oxidation in a single reactor could be feasible for nitrogen removal from pre-digested chicken manure. Average NO2-N, NO3-N, TAN, and tCOD removals were 98%, 65%, 56%, and 63%, respectively. Anammox adaptation results in the bioreactor towards the end of the operation were in accordance with lower organic matter in the influent. With consequent confirmation of sequencing analysis, the dominant route for ammonia removal was distinguished by increased relative abundance of Planctomycetes. Based on the qPCR analysis, hzsA gene could be useful for monitoring ammonia removal in anaerobic reactors.

Acknowledgments Authors gratefully acknowledge the Department of Scientific Research Projects of ITU (Project Numbers: 38822 and 39541).

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Figure Captions Fig. 1. Schematic view of the anammox-UASB reactor used in this study. Fig. 2. TAN profile in the anammox-UASB reactor during the study. Fig. 3. tCOD profile in the anammox-UASB reactor during the study. Fig. 4. Quantification of anammox bacteria. Fig. 5. Microbial communities at phylum level in the anammox-UASB reactor treating diluted chicken manure digestate. Fig. 6. Microbial communities at class level in the anammox-UASB reactor treating diluted chicken manure digestate. Fig. S1. Change in the cumulative biogas production during operation of the UASB reactor.

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Table 1 Influent feed characterization of the UASB reactor.

Parameter

Unit

Value

pH

-

7.99±0.07

Alkalinity

mg CaCO3/L

890±75

Total Suspended Solids (TSS)

mg/L

998±208

Volatile Suspended Solids (VSS)

mg/L

353±49

Soluble COD (sCOD)

mg/L

295±46

Total COD (tCOD)

mg/L

807±215

Total Ammonia Nitrogen (TAN)

mg/L

123±10

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HIGHLIGHTS • • • •

Maximum ammonia removal of 80% was achieved with 20 mg TAN/L in the effluent. qPCR and lllumina Miseq sequencing were used to assess microbial community dynamics. A significant association was found between hzsA gene and anaerobic ammonia removal. The dominant route for ammonia removal was distinguished by Planctomycetes.

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