Availability of carbon sources on the ratio of nitrifying microbial biomass in an industrial activated sludge

International Biodeterioration & Biodegradation 129 (2018) 133–140

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Availability of carbon sources on the ratio of nitrifying microbial biomass in an industrial activated sludge

T

Leon Steuernagel∗, Erika Lizette de Léon Gallegos, Asma Azizan, Ann-Kathrin Dampmann, Mohammad Azari, Martin Denecke Institute of Urban Water and Waste Management, Department of Civil Engineering, University of Duisburg-Essen, 45141 Essen, Germany

A R T I C L E I N F O

A B S T R A C T

Keywords: Nitrifying bacteria Industrial wastewater treatment Respirometry Quantitative polymerase chain reaction Inorganic carbon source Nitrification without COD

Chemical industry wastewater is known to have disproportionate nutrient concentrations with varying C:N ratios, thereby individually affecting the growth of heterotrophs and autotrophs in activated sludge. The present study underlines the impact of the changing availability of organic and inorganic carbon on the ratio of nitrifying biomass in an industrial activated sludge. To quantify changes, oxygen uptake rates by nitrifiers were monitored using respirometry, while nitrifier abundance was determined by real-time PCR using 16S rRNA primers. The obtained results point to the prominent influence of the organic or inorganic carbon source, reducing or increasing the active nitrifying biomass ratio and AOB gene quantity respectively. When the active nitrifier ratio dropped below 10% of the total activity or when nitrifiers were limited for inorganic carbon, ammonia oxidation was compromised. Nitrifiers were able to recover sufficient activity after a carbon starvation period of 34 days through the supply of gaseous CO2 or bicarbonate. The results of this study suggest a superior role of gaseous CO2 over bicarbonate as carbon source for nitrifiers and the feasibility to operate a high performing nitrification without any COD.

1. Introduction One of the major tasks of industrial activated sludge processes is the biological transformation and removal of nitrogen compounds by nitrification and denitrification (Juretschko et al., 2002). Effective treatment of wastewaters with high ammonia loads depends on a highly active nitrifying sludge. Such systems are suggested to operate with separated biological steps for organic carbon removal, denitrification and nitrification (Wiesmann, 1994). Biological nitrification comprises the conversion of ammonia over nitrite to nitrate which is catalyzed by ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) respectively (Bothe et al., 2000). To achieve maximum nitrification rates, optimal levels of pH, alkalinity, temperature, salinity and growth substrate need to be met (Gerardi, 2002; Torà et al., 2010). The major fraction of nitrifying bacteria in activated sludge plants are autotrophs that require an inorganic carbon (IC) source for biosynthesis (Bitton, 2005). Nitrifiers assimilate one-carbon compounds such as CO2 via the Calvin-Benson-Bassham-Cycle, catalyzed by the enzyme Ribulose-1,5bisphosphate carboxylase/oxygenase or RubisCO (Schramm et al., 1998; Utåker et al., 2002). Hommes et al. (2003) showed that certain organic compounds are available as carbon source for N.europaea but inorganic carbon (IC) is the preferred substrate for biosynthesis. Jiang

∗

et al. (2015) stated that gaseous CO2 is a less preferable source of inorganic carbon compared to bicarbonate. Both active transport of bicarbonate and passive transport of CO2 through the cell membrane were assumed in previous works of Denecke and Liebig (2003) and Chain et al. (2003). The existence of carbonic anhydrase (CA) in N.europaea enables the bacteria to manage CO2 and bicarbonate levels inside the cell (Chain et al., 2003; Dworkin et al., 2006; Jahnke et al., 1984). Autotrophic carbon uptake for biosynthesis consumes more energy compared to heterotrophic carbon uptake yielding lower maximum growth rates for autotrophic bacteria (Dworkin et al., 2006; Ni and Yu, 2008; Ottow, 2011). Using a batch set-up, several studies revealed a severe reduction of nitrification rates by deficient inorganic carbon supply or by the suppression of thriving heterotrophic bacteria due to competition for oxygen (Okabe et al., 1996; Toor et al., 2015; Vendramel et al., 2011). Since municipal wastewater has a relatively constant substrate composition in terms of carbon and nitrogen with a ratio of 100:5 (Forster, 2003), a heterotrophically dominated biocenosis can be expected as the ratio of nitrifiers was found to be inversely correlated to the influent C:N ratio (Xia et al., 2008). In this environment, heterotrophic metabolism is expected to fulfil the requirements of nitrifiers for IC. However, industrial effluents can be subject to varying C:N ratios, thereby influencing heterotrophic and autotrophic growth

Corresponding author. E-mail address: [email protected] (L. Steuernagel).

https://doi.org/10.1016/j.ibiod.2018.02.001 Received 17 October 2017; Received in revised form 31 January 2018; Accepted 3 February 2018 Available online 17 February 2018 0964-8305/ © 2018 Elsevier Ltd. All rights reserved.

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dynamically and changing their respective biomass fractions. To ensure process and performance stability, further information and investigation on the biomass composition is vital (Wagner and Loy, 2002). Previous studies of high performing nitrification steps expressed maximum sludge loading rates as the mass flow rate of NH4-N d−1 per biomass, either measured as total (TSS) or volatile (VSS) suspended solids, without considering the ratio of active nitrifying biomass (Wiesmann, 1994; Yusof et al., 2010). With autotrophic AOB and NOB being the predominant nitrogen oxidizing organisms in activated sludge, considering their fraction will make the evaluation of maximum ammonia sludge loading rates more reliable. This study aims to contribute to the better understanding of autotrophic/heterotrophic biomass composition dynamics in industrial nitrification sludges under changing carbon availability. After the identification of nitrifiers via fluorescence in situ hybridization, respirometry with selective ammonia monooxygenase (AMO) inhibition and real-time polymerase chain reaction (real-time PCR) targeting AOB and NOB were applied to monitor changes in biomass composition of an industrial nitrification sludge. For 140 days, two laboratory scale plants with an ammonia influent concentration of 1060 mgN L−1 were operated under aerobic conditions. One of the reactors was aerated using CO2-enriched air (1–2.5 vol % CO2) to investigate the possibility of gaseous CO2 acting as the sole carbon source for nitrifiers and countering carbon starvation. Respiratory activity of nitrifiers and AOB/NOB abundance were expected to indicate changes of the sludge's active nitrifying fraction in the presence or absence of an organic carbon (OC) source in the influent.

Table 1 Substrate and trace element composition of growth medium. Substrate solution NH4Cl KH2PO4 Na2SO4 CaCl2 MgSO4·7H2O Nutrient solution

Nutrient solution 4050 408 1278 408 900 3

mg mg mg mg mg mL

L−1 L−1 L−1 L−1 L−1 L−1

Na2-EDTA FeSO4·7H2O ZnSO4·7H2O CoCl2·6H2O MnCl2·4H2O CuSO4·5H2O NaMoO4·H2O NiCl2·6H2O Na2SeO3 HBO4

150 5000 430 240 990 250 220 190 210 14

mg mg mg mg mg mg mg mg mg mg

L−1 L−1 L−1 L−1 L−1 L−1 L−1 L−1 L−1 L−1

Table 2 Description of trial phases. L1

Phase I Phase II Phase III

L2

trial-day

Regime

C-source

Regime

C-source

0–61 62–97 98–140

OC-supply C-starvation IC-supply

Acetate – Bicarbonate

CO2-supply C-starvation CO2-supply

CO2 – CO2

while gaseous CO2–supply was cut off in L2. On day 98, CO2 was resupplied to L2 and bicarbonate was added to the feed of L1, changing the carbon source from organic to inorganic. Temperature, dissolved oxygen (DO) and pH were controlled by a programmed logic controller with respective values of 35 ± 0.2 °C, 3 ± 0.5 mg L−1 and 7.3 ± 0.3 using a 0.1 M NaOH–solution to counter acidification during nitrification and to keep the CO2/bicarbonate equilibrium at a constant level throughout the whole experiment, whether CO2 was supplied or not.

2. Materials and methods 2.1. Reactor set-up Two continuously operated laboratory plants comprising an aerated reactor and a post-clarifier (Fig. 1) with working volumes of 4 L and 2.5 L respectively were inoculated with activated sludge from a fullscale nitrification basin treating chemical industry effluent. This nitrification step operates at average influent concentration of 120 mgC L−1 and > 300 mgNH4-N L−1 at nitrogen loading rates around 220 mgNH4-N L−1 d−1. A sample of 10 L was drawn from the return sludge line of the secondary clarifier of the nitrification step and kept at 4 °C during transport. 1 L of homogenized sample was added to each labplant. For both laboratory reactors (L1 and L2), ammonia volume loading rates were set to 360 mgNH4-N L−1 d−1. Synthetic wastewater fed to the reactors (Table 1) was a three-fold concentrate of the solution by Liang et al. (2010). One plant (L1) additionally received 21960 mg L−1 of C2H3NaO2 via the feed during the period of “OCsupply” which was later replaced with 4500 mg L−1 of NaHCO3 during the period of “IC-supply” (Table 2). Both reactors were aerated with atmospheric air. A scrubber bottle containing a 3 M NaOH-solution was attached to the atmospheric air stream of the second plant (L2), trapping atmospheric CO2. Bottled CO2 was initially injected separately into the second reactor (L2) at a rate of 10 ± 5 mL min−1, making it possible to supply or deprive inorganic carbon. Later during Phase I, the gaseous CO2 supply was increased to a rate of 20 ± 5 mL min−1. Throughout the trial period, both systems were subjected to carbon starvation from day 62–97. L1 was deprived of the influent acetate

2.2. Sample preparation & chemical analysis Samples drawn from laboratory reactors were filtered using a 0.45 μm syringe filter for subsequent chemical analysis. In order to minimize CO2 mass transfer into the gas phase, 15 mL sample tubes were fully filled and analyses were conducted immediately after preparation. Concentration levels of inorganic nitrogen species (NO2-N, NO3-N and NH4-N) were monitored using Hach Lange Kits (LCK303, LCK432, LCK340).Total inorganic carbon (TIC) was determined with a Dimatoc 2000 TOC-analyzer (DIMATEC). 2.3. Respirometry Respirometry samples were drawn from both lab reactors weekly. Prior to analysis, samples of 20 mL from each reactor were washed with a Phosphate-Buffer-Solution containing 7.04 gKH2PO4 L−1 and 11.58 gNa2HPO4·2H2O L-1 at pH 7.3 and aerated by shaking the bulk solution in a tube of 50 mL of volume. Liquid phase static gas-static liquid (LSS) respirometry was conducted according to Vanrolleghem (2002). The total induced oxygen uptake rate (OURt) was measured after addition of NH4Cl and C2H3NaO2, inducing a final concentration of 100 mg L−1 for N and C. Quantification of the AMO-inhibited oxygen uptake rate (OURi) was achieved by adding the AMO-inhibitor allylthiourea to Fig. 1. Plant set-up of treatment lines L1 (left) and L2 (right).

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reach a final concentration of 58 mg L−1 (Moir et al., 1996). During respirometry, sample temperature was kept at 35 °C with a Haake D3 bath circulator. Oxygen uptake by nitrifiers (OURn) was calculated by subtraction of OURi from OURt. According to Contreras et al. (2008), endogenous respiration by nitrifers contributes about 1–3% to the oxygen uptake during substrate utilization and is negligible. Therefore, endogenous respiration was not separately quantified in order to least affect IC levels in sludge samples through excessive washing or longterm aeration for residual substrate depletion. In order to identify whether the dominating fraction of the biomass is nitrifying, the oxidation rates of nitrogen and carbon were calculated from the respective OUR. Sludge samples with an N/Cox ratio > 1 were defined as “nitrifier dominated”.

N OURt 14 ⎛ ⎞ =⎛ − 1⎞· ⎝ C ⎠ox ⎝ OURi ⎠ 24 ⎜

2.5.2. Real-time PCR Absolute quantification of AOB and NOB by real-time PCR was carried out using 16S rRNA primers (Table 3) and SYBR Green as a fluorescent dye. The method evaluation for real-time PCR assays was conducted as stated in the MIQE guideline (Bustin et al., 2009). For the absolute quantification of those targets, customized DNAstandards (Life technologies, Darmstadt, Germany) were used. Additional information can be found in Supplementary materials A.1. The DNA-standards were prepared according to the supplier's manual and their corresponding genomic units (GU) were calculated using Equation (2) (Vanysacker et al., 2014). The plasmid length of the DNAstandards corresponding to the AOB group was 2899 bp and 2905 bp, while for NOB, it was 2585 bp. The starting concentration for all standards was estimated at 1 × 10−7 g μl−1.

DNA concentration standard [g μl−1] x (6.02x1023) = GU μl−1 (plasmid length [bp] x 660)

⎟

(1)

Mixed liquor suspended solids (MLSS) and volatile suspended solids (VSS) were measured according to DIN 38409–1 (1987) in respirometry samples to calculate the specific oxygen uptake by nitrifiers.

The protocol for sample measurement included the following steps: initial denaturation at 95 °C for 5 min, followed by 40 amplification cycles of denaturation (95 °C; 0.5 min), annealing (61.2 °C for AOB and 64.3 °C for NOB; 1.15 min) and extension (72 °C; 1 min), and a final amplification step after the last cycle at 72 °C for 10 min. All analyses were conducted on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, München, Germany) and evaluated with the BioRad CFX Manager IDE software. The reaction volume was set to 25 μl, including: 5 μl of sample specific DNA dilutions, 12.5 μl of MESA GREEN qPCR Master Mix Plus (Eurogentec, Seraing, Belgium), 5.5 μl of nuclease-free water and 1 μl of each forward and reverse primer (Biomers.Net, Ulm, Germany) with a concentration of 50 nM for AOB and 100 nM for NOB. The genomic units for AOB and NOB were calculated individually and against the obtained standard curve of each of the targets (Supplementary materials A.2). Both level of detection (LOD) and level quantification (LOQ) were determined at 50 GU μl−1 for both targets. Additionally, in every sample run, a plate calibrator consisting of a standard curve with three different dilutions was included to allow better comparison between different sample runs. Efficiency of the amplification was estimated using the plate calibration with acceptable values in the range of 90–110%. Technical controls were also included in every sample run, i.e. positive control (PC), negative control (NC) and non-template control (NTC), to prove the accurate working conditions of the run.

2.4. Fluorescence InSitu Hybridization Prior to fixation, following Daims et al. (2006), 50 mL of the initial sludge sample was treated mechanically in a glass bead mill for 3 min to break cell agglomerates. 30 mL of sample were mixed with 60 mL of cyclohexane and 50 mL Milli-Q water in a separation funnel and extracted after organic/inorganic phase separation. The protocol described in Nielsen et al. (2009) was followed using the oligo-fluorophores (5'end -Cy3) listed in Table 3. Sample slides were examined using an epifluorescence microscope (Carl Zeiss Microscope Axio Imager.M2; Carl Zeiss Microimaging, Jena, Germany) in combination with the AxioVision SE64 Rel. 4.9.1 Software. 2.5. Quantification of bacteria by real-time PCR 2.5.1. Sampling & DNA extraction The sampling period for real-time PCR analysis covered test Phase I and III. All samples for real-time PCR were stored at −20 °C directly after sampling. To characterize the community composition of AOB and NOB, genomic DNA was isolated from industrial activated sludge samples using FastDNA SPIN Kit for Soil (MP Biomedicals, Eschwege, Germany) as described in Dunkel et al. (2016). DNA concentration and purity of the samples were measured photometrically using the Nanodrop 2000 Spectrophotometer (Thermo Scientific, Dreieich, Germany). All extracted DNA samples were stored at −20 °C until use for PCR applications.

3. Results and discussion The presence of Nitrosomonas and Nitrospira in the initial sludge sample was detected using FISH (Supplementary materials A.4). Nitrobacter was not detected. After inoculation, biomass compositions in L1 and L2 were expected to change from their initial state due to the supply of different carbon sources. Fig. 2 shows the N/Cox ratio of L1 and L2 with the corresponding fraction of oxygen consumed by nitrifiers. Since carbon sources were switched in L1, a large range of N/ Cox values from 0.018 to 4.79 was detected. Only during the phase of IC supply, the oxygen uptake was classified as “nitrifier dominated”. 17 of 19 N/Cox ratios measured in L2 samples were identified as “nitrifier dominated”, ranging from 1.13 to 7.75. These results indicate a selection process due to the different phases of carbon availability which are described in the following sections.

Table 3 List of 16S rRNA primers and probes used for FISH and real-time PCR analysis. Target group

Oligoprobe

Sequence (5’ → 3′)

Reference

AOB

CTO189fa

GGA GRA AAG CAG GGG ATC G CTA GCY TTG TAG TTT CAA ACG C GTA GGC CST TAC CCY ACC

Junier et al., 2008

CCT GCT TTC AGT TGC TAC CG GTT TGC AGC GCT TTG TAC CG GGA ATT CCG CGC TCC TCT CCT GTG CTC CAT GCT CCG

Zeng et al., 2014

CTO654r Nmo254

NOB

NSR1113F NSR1264R Ntspa662 NIT3

(2)

Junier et al., 2008 Stephen et al., 1998

3.1. Phase I: OC-supply/CO2-supply

Zeng et al., 2014

During OC-supply, OURn/OURt continuously decreased in L1 until no measurable inhibition by allylthiourea on the OUR was observed after day 42 (Fig. 4). sOURn varied between 0.62 and 7.44 mgO2 (gVSS h)−1. Similar values (5–8 mgO2 (gVSS h)−1 were shown by Yang et al. (2009), where a mixed culture of heterotrophs and autotrophs in

Daims et al., 2001 Wagner et al., 1996

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Fig. 2. Calculated N/Cox ratio from measured active nitrifier fraction in L1 (a) and L2 (b) during the different C-supply and -starvation phases.

initiation phase which might be due to the proceeded selection of nitrifying bacteria from the beginning of the experiment. The maximum oxygen uptake by nitrifiers of the carbon starved systems was 13.78% for L1 and 25.85% for L2 compared to their overall maximum activity. These results are in accordance with previous studies (Denecke and Liebig, 2003; Guisasola et al., 2007) and will be discussed further in 3.5. TIC levels in L1 and L2 decreased from 2264 to 1.9 mg L−1 in L1 and from 67.7 to 0.01 mg L−1 in L2. Considering the reported values at which a nitrifying system is regarded as carbon limited, the C-Starvation phase was the only period of time where carbon limitation of nitrifiers appeared in L1. The absence of any carbon source is suspected to inhibit both ammonia oxidation by AOB and assimilation by heterotrophic bacteria due to stagnating growth. Both systems showed their poorest performance in ammonia degradation, reaching maximum NH4 concentrations of 955 mgN L−1 in L1 and 640 mgN L−1 in L2.

suspended growth was supplied with C:N-influent ratios between 9 and 20. The average OURn/OURt values in their study range from 0.18 to 0.25. While acetate was supplied in the present study, heterotrophic bacteria experienced a growth advantage, possibly outcompeting nitrifiers for oxygen (Okabe et al., 1996). Similar findings were presented by Rodriguez-Sanchez et al. (2016) where the addition of organic matter to a partial nitritation biofilter caused a decrease in abundance of Nitrosomonas while heterotrophs prospered. The calculated ratio of oxidized nitrogen to oxidized carbon (N/Cox) reached a minimum of 0.03 (Fig. 2). Since IC levels in L1 were constantly above 750 mg L−1, carbon limitation of nitrifiers was ruled out as a possible reason for the low activity (Supplementary materials A.6). In the first 50 days, L2 yielded low oxygen uptake rates with an average sOURn of 11 mgO2/(gVSS h)−1 and an N/Cox ratio of 2.53. During that time, the average TIC-concentration was 34.14 mgTIC L−1 so the supply of gaseous CO2 may have been insufficient. Guisasola et al. (2007) proposed that IC-limitation occurs at concentration levels below 36 mgC L−1 (expressed as 3 mmolC L−1) which is in good agreement with the presented results. Another possible reason is an adaption phase of nitrifiers to their main source of IC. Jiang et al. (2015) reported a reduction in nitrification performance when the sole carbon source for a pure culture of N.europaea was switched from bicarbonate to gaseous CO2 with a subsequent performance recovery due to adaption after 40 days. The sludge in the presented study was not subjected to external gaseous CO2 aeration before the beginning of the experiments and was therefore reliant on dissolved inorganic carbon from heterotrophic metabolism in the bulk liquid of the treatment plant. With an increase of the bulk liquids’ TIC-concentration by increasing the CO2 gas-flux, nitrifying activity increased to 45.2 mgO2/ (gVSS h)−1, yielding a N/Cox ratio of 7.75. The effect of a possible IClimitation on nitrifier dominated biocenoses will be discussed further in 3.5.

3.3. Phase III: IC-supply/CO2-resupply Nitrifying activity was recovered by substituting acetate with bicarbonate as the sole carbon source in L1 and resupplying gaseous CO2 to L2. Both lines yielded very similar N/Cox ratios up to 4.75 and 4.72 respectively (Fig. 2). Total sOUR in L1 increased up to 49.6 mgO2/ (gVSS h)−1, with 43.9 mgO2/(g VSS h)−1 being contributed by nitrifiers. This shows that in the absence of organic carbon, a directly supplied IC-source causes a shift towards a nitrifier dominated biomass with higher nitrifying capacity. Furthermore, it demonstrates that avoiding excessive organic carbon in the influent is paramount in order to keep a high performing nitrification biocenosis. With accumulating TIC in the L2 bulk liquid, specific oxygen uptake by nitrifiers increased rapidly over the next four weeks, peaking at 117 mgO2/(gVSS h)−1 (Fig. 3). At comparable ammonia concentrations to our respirometry set-up, specific oxygen uptake rates of 92 mgO2/(gVSS h)−1 and 184.6 mgO2/(gVSS h)−1 (expressed as 130 mgO2/(gCOD h)−1) were measured in nitrifier enriched cultures by Jih et al. (2008) and Contreras et al. (2008) respectively. Both studies supplied sodium bicarbonate as sole carbon source with either bulk-phase or influent concentrations of ≥2000 mg L−1, representing non-carbon-limited systems. Although Jiang et al. (2015) concluded that gaseous IC is the less preferable source of inorganic carbon for Nitrosomonas europaea, similar activities of bicarbonate and CO2 supplied systems suggested that the autotrophic

3.2. Phase II: C-Starvation During C-starvation, no carbon source was introduced to either experimental line, depriving the anabolism substrate for both heterotrophs and autotrophs. In this trial phase, nitrifiers yielded the lowest rates for mean and maximum specific oxygen uptake (Table 4). L2 shows a higher minimum sOURn during C-Starvation compared to the Table 4 Summary of specific oxygen uptake rates of nitrifiers during different trial phases. Phase

L1 mean

L2 min.

max.

mgO2/gVSS/h I II III Overall

OC-Supply C-Starvation IC-Supply

4.11 1.71 14.50 6.77

0.62 0.03 0.54 0.03

7.44 6.05 43.92 43.92

max./overall max.

mean

%

mgO2/gVSS/h CO2-Supply C-Starvation CO2-Resupply

16.94 13.78 100.00

136

20.80 14.92 74.40 35.41

min.

5.59 7.56 36.11 5.59

max.

max./overall max. %

65.07 30.25 117.00 117.00

55.62 25.85 100.00

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88 mg L−1 when CO2 was the sole carbon source which is in good agreement with Peng et al. (2015). In their study, a series of batch tests was conducted and reduced ammonium oxidation rates of AOB were found when TIC decreased below 72 mgC L−1 (expressed as 6 mmolC L−1). In our set-up, every substantial decrease in TIC concentration in L2 resulted in a delayed increase of NH4-N in the bulk liquid. The approximate duration of the delay was 5 d which might be connected to the hydraulic retention time of 4.64 d. During the respirometry, a small amount of CO2 is produced by heterotrophic oxidation of the supplied acetate. However, in L2, which was most affected by varying IC levels, the maximum possible production of IC was calculated to be 0.72 mgTIC l−1. According to the kinetic data of Guisasola et al. (2007), this would increase nitrifier activity by 4.6% at most when the highest possible gradient is considered at IC concentration levels of 1.2 mmol l−1. This case, however, never occurred in this study.

nitrifiers possess the ability to adapt to different forms of IC. Mellbye et al. (2016) stated that Nitrosomonas europaea can increase its capacity to utilize atmospheric carbon. While Jiang et al. (2015) reported that the supply of gaseous IC negatively impacted reactor performance, Mellbye et al. (2016) were able to keep steady state conditions with the same sole carbon source. In combination with the present study, these findings support a potential use for CO2 enriched exhaust gas streams as sole carbon source for nitrifiers. The conversion into biomass will decrease the amount of emitted atmospheric CO2 and no additional dosing of chemicals is needed to maintain a high performing nitrification. Since limiting IC conditions also promotes the emission of NO and N2O (Jiang et al., 2015; Khunjar et al., 2011; Peng et al., 2015), controlling IC levels in the bulk phase by aeration with external CO2 could also be a valid strategy to reduce greenhouse gas emissions. 3.4. IC-limitation of nitrifiers

3.5. Active nitrifier ratio and abundance of nitrifiers

The effect of supply and deprivation of CO2 as sole carbon source on a biomass dominated by nitrifiers is illustrated in Fig. 3. A strong correlation between sOUR and TIC with r = 0.7191 and a p-value of 0.0037 was observed in L2. An increase in TIC concentration before and after the C-starvation period yielded an elevated nitrifier activity. Vadivelu et al. (2006) attributed up to 65% of oxygen uptake by nitrifiers to their maintenance processes even in the absence of inorganic carbon. However, their activity measurements were conducted immediately after CO2 was stripped from the batch reactor and only lasted up to 100 min. Chain et al. (2003) suspect Nitrosomonas europaea to have transporter enzymes for inorganic carbon and have identified genes encoding carbonic anhydrases. In the present study, the biocenoses were introduced to a carbon deprivation period of 34 days, forcing nitrifiers to utilize possibly accumulated carbon dioxide in cells. As a result, ammonia concentration in L2 increased to the overall highest value of 640 mgN L−1 and the respective nitrifying activity decreased to 25.85% of the maximum value measured during excess CO2 supply. Denecke and Liebig (2003) found that an autotrophically dominated sludge deprived of CO2 for six days yielded growth rates of only 24–30% compared to non-carbon-limited conditions. Guisasola et al. (2007) discussed the presence of residual activity by nitrifying bacteria in activated sludge at TIC concentrations below 1.2 mg L−1, which was also estimated to be around a quarter of the non-limited activity. They propose three hypotheses, namely the possible utilization of organic substrates by nitrifiers, oxygen uptake due to maintenance purposes and an internally increased TIC concentration obtained by heterotrophic metabolism of biomass decay products. However, results of Guisasola et al. (2007) as well as this study reflect the strong dependency of nitrifier OUR on the available anabolism substrate in both batch and continuous activated sludge cultures. In this study, the inoculation with activated sludge introduced heterotrophs to the system and although no readily available organic substrate was added, soluble microbial products (SMP) of thriving autotrophs possibly posed as a substrate for heterotrophs (Kindaichi et al., 2004). Reliable ammonia degradation over 93% was only observed at TIC concentrations above

A comparison of the OURn/OURt values for L1 and L2 (Fig. 4a) revealed the effect of carbon sources supplied to a mixed culture on its active nitrifier fraction. As the most significant changes were observed during phases I and III, AOB and NOB abundance was quantified in the respective samples (Fig. 4b). NOB abundance was constantly below detection limit in L1 for every sample point. In the absence of organic substrate in L2, GU-concentrations of targeted NOB proofed to be quantifiable and are presented in the Supplementary materials (A.7). Before bicarbonate was supplied to L1, the oxygen uptake ratio contributed by nitrogen oxidation was generally lower compared to L2 in both carbon-supply and starvation periods. Also, L2 primarily showed higher AOB abundance and OURn/OURt values than L1. At day 35, OURn/OURt dropped below 0.1 and AOB abundance below detection limit in L1 (Fig. 4) which coincided with an increased ammonia effluent concentration (Supplementary materials A.5). Ammonia was not oxidized sufficiently at sludge loading rates of 35 ± 5 mgNH4N (gMLSS d)−1. This condition persisted until the end of Phase I which shows that growth disadvantages of nitrifiers due to excess OC supply impairs nitrification performance. Sheng et al. (2018) reported similar dynamics between autotrophs and heterotrophs in a simultaneous anammox and denitrification (SAD) process, where an increased influent C:N ratio resulted in a severe drop of autotrophic bacteria abundance. Using glucose and acetate as COD source in synthetic wastewater, Lin et al. (2016) demonstrated that AOB and NOB activity was inversely related to the COD influent concentration with dropping OURn/OURt ratios from 0.87 to 0.37 when the COD:N was increased from 3 to 10. In this study, only after the transition to an inorganic carbon source and without any added COD, OURn/OURt increased to 0.86 and AOB abundance constantly increased from 5.5 × 107 to 1.06 × 108 GU μL−1 in L1. With increasing levels of AOB abundance and activity in L1, OURn/OURt values reached their maximum of 0.89 (Fig. 4) and complete ammonia oxidation was recovered even at a higher average sludge loading rate of 66 ± 13 mgNH4N (gMLSS d)−1. This increase in sludge loading is a result of a decrease in biomass concentration by 40% observed in L1. Complete ammonia oxidation in L2 was achieved at even higher sludge loading rates of 185 ± 42 mgNH4N (gMLSS d)−1 before and 337 ± 77 mgNH4N (gMLSS d)−1 after C-starvation with a respective OURn/OURt ratio of 0.93 and 0.87. The overall higher sludge loading in L2 is a result of the suppressed growth of heterotrophs from the beginning of the experiment, resulting in biomass concentrations of only one third compared to L1. Despite the lower suspended solids content, L2 consistently showed significantly higher AOB abundance (1.04 × 106 to 2.65 × 108) and OURn/OURt values (0.56–0.93) than L1 during the first trial phase. After day 28, abundance and OURn/OURt of L2 follow a similar trend but because no abundance measurements were conducted during Phase II, a statistical analysis regarding correlation between the two values was not considered. While CO2 was not supplied during Phase II, OURn/OURt values decreased in L2, possibly due

Fig. 3. Specific oxygen uptake rate by nitrifiers and corresponding TIC bulk concentrations of L2.

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Fig. 4. OURn/OURt (a) and abundance of AOB (b) during Phase I and III.

energy generated from NH4+ oxidation might be spent to convert HCO3− into CO2 via carbonic anhydrase activity. This would also explain why L2 shows elevated activity and AOB abundance despite higher IC levels in L1 during Phase III. Due to the varying biomass concentrations and different regimes of carbon supply, the presented values for sludge loading can only serve as an indication that sludges with an elevated ratio of active nitrifiers withstand higher ammonia sludge loadings. To validate these findings, a comprehensive study of maximum ammonia sludge loadings in relation to the sludge's OURn/ OURt ratio is necessary. However, although the same mass flow of ammonia was supplied to both experimental lines, changes in the active nitrifier fraction and abundance determined the performance of ammonia degradation. Through qPCR analysis and respirometry it was possible to distinguish between two systems with differing growth advantages for autotrophic nitrifiers. Both approaches clearly identified the nitrification step with CO2 as sole carbon source as the system with a higher nitrification performance potential. The course of AOB abundance, nitrifier-activity and ammonia effluent concentration of both experimental lines clearly reflects the prominent growth advantages for nitrifiers when no COD is added to the system. A comparison of those values between L1 and L2 during Phase III suggests that gaseous CO2 is the preferred inorganic carbon source over bicarbonate. This finding differs from the results of Jiang et al. (2015). Although the applied ICsources are the same, our study compares AOB abundance and activity of a CO2-adapted culture (L2) to a culture with a recently changed ICsource (L1) which possibly has an impact on nitrifiers.

to the severe reduction of nitrifying activity while other oxygen consuming processes e.g. endogenous respiration and heterotrophic metabolism remained at mostly unvaried levels. As mentioned before, an accumulation of ammonium in the reactor occurred during that time as well. This shows that the controlled deprivation of CO2 has a direct effect on the active nitrifier fraction of the sludge which leads to a reduced reactor performance. When CO2 was resupplied, yielding the highest IC concentrations during the experiment nitrifier abundance peaked. An up to 100-fold increase in GU-concentration was observed compared to Phase I with a maximum GU-level of 2.65 × 108 GU μL−1. This coincided with the highest nitrifier oxygen uptake ratio of 0.89 and ammonia degradation rates over 95%. Although both experimental lines were supplied with a purely inorganic carbon source during Phase III and OURn/OURt values were very similar, the abundance of AOB in L2 was almost double compared to L1. The sufficient ammonium degradation suggests that the required nitrifying performance was met in both lines. However, the higher abundance and sOUR of L2 suggests that the culture supplied with CO2 has the potential of degrading ammonia at even higher levels. Jiang et al. (2015) investigated the relation between sOUR and enzymes involved in autotrophic carbon fixation, namely RubisCO and carbonic anhydrase (CA). In their study, an increasing oxygen consumption was accompanied by an increased RubisCO activity while CA activity was not influenced. In this study, the AOB culture in L2, which was directly supplied with CO2, possibly has a higher cell yield because the anabolism substrate is more readily available. In the reactor supplied with bicarbonate, a portion of the 138

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This study proves the feasibility of continuous nitrification without any added COD and the potential of gaseous CO2 as the sole carbon source. To estimate changes in the biomass composition, the influent carbon to nitrogen ratio should be revised as OC:IC:N instead of C:N. Excess OC causes a shift towards heterotrophically dominated biomass which disfavors nitrification. In the absence of OC, the supply of IC leads to the selection of nitrifiers, resulting in nitrogen-oxidation rates 8-times higher compared to carbon oxidation. These findings might benefit applications treating wastewater with low OC:N ratios e.g. leachate treatment, deammonification or nitrification of industrial effluents. Acknowledgements This work has been supported by the Förderverein der Siedlungswasser-, Wasser-und Abfallwirtschaft (SiWaWi e.V.), Essen, Germany and Currenta GmbH & Co. OHG, Dormagen, Germany. The authors are grateful for the advisory aid of Ludwika Nieradzik and Stephanie Steuernagel. Natascha Rossi and Tobias Hesse are thanked for their contribution in the lab. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.ibiod.2018.02.001. References Bitton, G., 2005. In: Wiley-Liss (Ed.), Wastewater Microbiology, third ed. John Wiley & Sons, Hoboken, N.J. Bothe, H., Jost, G., Schloter, M., Ward, B.B., Witzel, K., 2000. Molecular analysis of ammonia oxidation and denitrification in natural environments. FEMS Microbiol. Rev. 24, 673–690. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622. https://doi.org/10.1373/clinchem.2008. 112797. Chain, P., Lamerdin, J., Larimer, F., Regala, W., Lao, V., Land, M., Hauser, L., Hooper, A., Klotz, M., Norton, J., Sayavedra-Soto, L., Arciero, D., Hommes, N., Whittaker, M., Arp, D., 2003. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 185, 2759–2773. https://doi.org/10.1128/JB.185.9.2759-2773.2003. Contreras, E.M., Ruiz, F., Bertola, N.C., 2008. Kinetic modeling of inhibition of ammonia oxidation by nitrite under low dissolved oxygen conditions. J. Environ. Eng. 134, 184–190. Daims, H., Lücker, S., Wagner, M., 2006. daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8, 200–213. https://doi. org/10.1111/j.1462-2920.2005.00880.x. Daims, H., Nielsen, J.L., Nielsen, P.H., Schleifer, K.-H., Wagner, M., 2001. In Situ characterization of nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67, 5273–5284. https://doi.org/10.1128/ AEM.67.11.5273-5284.2001. Denecke, M., Liebig, T., 2003. Effect of carbon dioxide on nitrification rates. Bioproc. Biosyst. Eng. 25, 249–253. https://doi.org/10.1007/s00449-002-0303-z. DIN, 1987. Bestimmung des Gesamttrocken-rückstandes, des Filtrattrockenrückstandes und des Glührückstandes (H 1), DIN 38409–1. Dunkel, T., de León Gallegos, E.L., Schönsee, C.D., Hesse, T., Jochmann, M., Wingender, J., Denecke, M., 2016. Evaluating the influence of wastewater composition on the growth of Microthrix parvicella by GCxGC/qMS and real-time PCR. Water Res. 88, 510–523. https://doi.org/10.1016/j.watres.2015.10.027. Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H., Stackebrandt, E. (Eds.), 2006. The Prokaryotes. Springer New York, New York, NY. https://doi.org/10.1007/0387-30742-7. Forster, C.F., 2003. Wastewater Treatment and Technology. Thomas Telford. Gerardi, M.H., 2002. Nitrification and Denitrification in the Activated Sludge Process, Wastewater Microbiology Series. Wiley-Interscience, New York. Guisasola, A., Petzet, S., Baeza, J.A., Carrera, J., Lafuente, J., 2007. Inorganic carbon limitations on nitrification: experimental assessment and modelling. Water Res. 41, 277–286. https://doi.org/10.1016/j.watres.2006.10.030. Hommes, N.G., Sayavedra-Soto, L.A., Arp, D.J., 2003. Chemolithoorganotrophic growth of Nitrosomonas europaea on fructose. J. Bacteriol. 185, 6809–6814. https://doi.org/ 10.1128/JB.185.23.6809-6814.2003. Jahnke, L.S., Lyman, C., Hooper, A.B., 1984. Carbonic anhydrase, carbon dioxide levels

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