In-situ fixed bed denitrification in sequential biofiltration: laboratory testing of solid substrates

High nitrate concentrations in wastewater treatment plant effluents and aquifers can challenge sequential biofiltration systems in preventing nitrite and gas formation in the sand bed, as well as to achieve the regulated limit value for nitrate in potable water reuse applications. This study investigates the introduction of electron donors in the form of organic fixed bed materials as an in-situ anoxic zone into sequential biofiltration systems. Laboratory batch and column tests with straw, soft wood, peat, polylactic acid (PLA), and polycaprolacton (PCL) revealed incomplete denitrification with a hydraulic retention time below 10 h, high organic carbon leaching, especially during the first three months, and gas accumulation within the filter bed. Therefore, ex-situ denitrification prior to oxic biofilters or in a defined side-stream treatment is recommended. No enhanced transformation of trace organic chemicals was observed under nitrate reducing conditions. Peat revealed a sorption potential for 5-methyl-benzotriazole, carbamazepine, benzotriazole, and metoprolol. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wrd.2020.005 om http://iwaponline.com/jwrd/article-pdf/10/4/394/831447/jwrd0100394.pdf er 2021 Josefine Filter (corresponding author) Christin Bosinsky Sefine Oksal Kilinc Aki Sebastian Ruhl Martin Jekel Chair of Water Quality Control, Technische Universität Berlin, KF4, Straße des 17. Juni 135, 10623 Berlin, Germany E-mail: josefine.filter@tu-berlin.de Aki Sebastian Ruhl German Environment Agency, Section II 3.1, Schichauweg 58, 12307 Berlin, Germany


INTRODUCTION
Surface water used for potable water reclamation can be influenced by upstream discharges of waste water treatment plant (WWTP) effluents (Karakurt et al. ).Besides this de facto potable reuse, (semi-)closed water cycles can lead to the accumulation of poorly degradable or even persistent trace organic chemicals (TOrCs) in the aquatic environment.Managed aquifer recharge (MAR) systems allow a brine-free removal of pathogens and dissolved organic compounds like various TOrCs with low operational costs.
Sequential biofiltration (SBF) systems or sequential managed aquifer recharge technologies (SMART) represent an advancement of conventional biofilters and MAR systems.
They are characterized by two infiltration steps with an intermediate aeration or introduction of oxygen.While in the first filtration step, easily biodegradable DOC is removed associated with the consumption of oxygen, beneficial carbon limiting and oxic conditions are established in the second filtration step after aeration for enhanced TOrC removal.Those SBF systems can be realized in different forms.A pilot scale vertical column set up was tested and described by Müller et al. ().A further technical pilot application consists of a horizontal plug-flow reactor filled with technical sand and an in-situ introduction of oxygen (Karakurt-Fischer et al. ).Since these systems are promoting oxic conditions, contaminants such as nitrate, carbamazepine, or iodinated x-ray contrast agents which are predominantly removed or dehalogenated under anoxic and anaerobic conditions (König et al. ; Redeker et al. ), might not be removed.This can be critical in potable reuse with a lack of dilution by natural waters.
The EU water framework directive seeks a good status of all waters by 2027 (The Council of the European Union ).Therefore, the limit of discharged total nitrogen into waterbodies sensitive to eutrophication is set below 15 mg/L for WWTPs exceeding 10,000 population equivalents (European Council ).

Materials
Straw, pine woodchips (soft wood), peat, PCL, and PLA were applied as fixed bed materials.Sand from a MAR site located at Lake Tegel in Berlin (Germany) was used as a reference filling material.Detailed specifications of the substrates are given in Table S1 in the supporting information.

Data processing
The mean nitrate removal rate R N (mg N/(L•d)) was calculated according to Equation ( 1) and the theoretical gas formation rate R N2 (mL/d) was calculated according to Equation (2) assuming a complete reduction of nitrate to molecular nitrogen:

Nitrate removal
The batch tests showed a nitrate removal within 30 days for PCL, straw, soft wood, peat, and H 2 .The nitrate removal rate constant k correlates with the DOC released from the materials (Figure S2, supporting information).
In the column experiment, a mean nitrogen removal R N of 4-21 mg N/(L•d) was calculated according to Equation (1) for the tested substrates, except for the reference sand and PLA columns, which showed no nitrate removal (Figure S3).As presented in Figure 2, the highest removals were achieved with rye straw (30 g filter material) and PCL (153 g), which was already indicated by the batch tests.
The relative effluent concentrations of nitrate and nitrite This decline was also observed with straw in this study.
During the first month nitrate removal rates reached 29 mg N/(L•d) and steadily decreased to 14 mg N/(L•d).
Reasons might be the decreasing pore volume and HRT due to gas entrapment as well as the washout of readily degradable BDOC during the first month.
The observed average nitrate removal for soft wood of A direct comparison of the observed nitrate removal rates with literature data is not possible and always needs to be seen within the context of operational conditions, since HRTs are usually longer, temperatures differ, or the content of BDOC in the influent water is much higher.

Gas formation
The gas formation in the column experiments was quantified by measuring the weight loss of the replaced water in column and gas capture bottles.To transfer mass of replaced water, into a nitrogen equivalent volume, the mass was divided by 0.998 g/mL reflecting the density of water.The obtained gas formation rates correlate with the theoretical gas formation rates R N2 calculated from nitrogen removal, indicating that molecular nitrogen was the main gas formed during the experiment (Figure 3).Only a minor gas formation was observed for sand and PLA.In contrast, the gas retention capacity of the porous media was exceeded  for straw, PCL, wood, and peat, as more than 70% of the formed gas was released from those columns into the gas capture bottles (Figure S4).
In a biofiltration system with horizontal or downstream flow conditions the gas retention capacity can be higher, since gas entrapments would not be 'flushed' out with the water stream as in the up-stream operated column tests.
This might lead to a further decrease of the hydraulic con- Released DOC from peat was found to be mainly hardly biodegradable humic substances.

Removal of TOrCs
Data reported in the literature often show different observations for the removal of TOrCs under nitrate reducing conditions.Due to potential micro-milieus and insufficient resolution, it can be hard to distinguish between processes under micro-aerophilic and nitrate reducing conditions in field as well as in column studies; especially, since denitrifying bacteria are commonly facultative aerobes which can switch between aerobic and anoxic respiration (Madigan & Brock ).
Therefore, removal data from the batch experiments were evaluated to verify biotransformation under nitrate reducing conditions.To eliminate oxic transformation with residual oxygen, data from the first five days was excluded, as well as data when sulfate was reduced.The mean relative removal under nitrate reducing conditions, which can be  Denitrification with H 2 as electron donor did not show an enhanced removal of the investigated TOrCs in batch experiments (Figure S9).amounts that ranged between 1.6 and 3.7 g PCL/g NO 3 --N for their experiments.

CONCLUSIONS
An effective nitrate removal was achieved with PCL and straw at HRTs below 10 hours.Whereas the nitrogen removal with straw decreased over time, a complete removal was achieved with PCL after an adaptation time of 125 days.
With a better adjustment of HRT, influent oxygen concentrations or even temperature, this time could be decreased and PCL might be a possible substrate for long-term fixed bed denitrification.Only a minor removal of TOrCs was observed under denitrifying conditions, except for peat, which partly removed benzotriazole, 5-methy-benzotriazole, metoprolol, carbamazepine, and acesulfame.
Especially during the initial phase, high DOC leaching from the materials and nitrite formation can occur, which would cause additional oxygen consumption in a subsequent oxic zone in an SBF system.In-situ fixed bed denitrification seems to be difficult to control, especially in a technical system with limited HRT or space.A better option might be the combination of denitrification with total suspended solids removal in the rapid sand filter prior to the technical SBF system (Figure S1).For this purpose, the rapid sand filter needs to include additional biofilm carriers for the denitrifiers and an optional dosage of easily degradable organic carbon.Treating just a part of the influent as a recirculated side-stream in a separated and controlled denitrification unit could be another possibility to achieve the required threshold values.This option allows for better control of the system conditions and the application of fixed bed materials as carbon and electron source can be reconsidered.By optimizing the HRT, the leaching of DOC and nitrite formation could be minimized.
Figure 1 | Scheme of the column setup.
2) with: ΔN ¼ c NO3-N;out À c NO3-N;out À c NO2-N;out Q volume flow (L/d) BV bed volume of the column (L) c NO 3 -N;in nitrate concentration in the column influent (mg/L) c NO 3 -N;out nitrate concentration in the column effluent (mg/L) c NO 2 -N;out nitrite nitrogen calculated as nitrate (mg/L) δ N2 density of molecular nitrogen at 20 C (1.16 mg/mL).The Student's t-test was carried out with unpaired samples with differing variance.The probability for a similar behavior of two samples is expressed by the p-value.A p-value smaller than 0.05 is considered as significant difference between the samples.To examine if the observed removal in batch tests is significant, the dataset of relative removals was tested against 0 (¼ no removal).
reveal an incomplete denitrification to nitrite with PCL during the first four months of operation.The residual oxygen in the influent water as well as the short hydraulic retention time of only 3 h might have provoked incomplete denitrification and nitrite formation.Furthermore, biofilm seems to develop, since the total nitrogen removal increases with time.After day 125, an almost complete nitrogen removal is achieved resulting in a removal rate of 23 ± 5 mg N/(L•d) (n ¼ 9).In contrast, Chu & Wang () achieved a high removal rate of 182 mg N/(L•d) in a PCL reactor at 20 C and 6-9 h HRT with spiked groundwater after 300 days of operation.They recommend high temperature and HRT during the initial phase.However, this could be difficult to implement in an in-situ application.Furthermore, they conducted microbial analyses of the biofilm and found denitrifying processes in deeper layers of the biofilm, requiring a certain biofilm thickness.In the column experiments, straw achieved the highest average nitrate removal rate of 21 mg N/(L•d) during the first four months of operation.The rates were calculated only from measurements up to day 128, since air was accidentally pumped into the column due to a broken pumping hose.However, Cameron & Schipper () reported comparable values for wheat straw during the first ten months after start up but a significantly lower average nitrate removal of 7.8 mg N/(L•d) between 10 and 23 months, indicating a decline in nitrate removal over time.

5
mg N/(L•d) during six months of operation corresponds to the reported value of 4.9 g N/m 3 d by Cameron & Schipper ().The high removal rates reported by Ghane et al. () can be explained by the carbon turn-over of wastewater DOC.Therefore, soft wood seems to be a suitable fixed bed material for treating carbon-rich influents even under short HRT (Christianson et al. ).The results of Halaburka et al. () suggest that denitrification in an aged woodchip bioreactor at constant temperature can effectively be modeled using even zero-order kinetics when nitrate concentrations are exceeding 2 mg N/L.

Figure 3 |
Figure 3 | Theoretical gas formation calculated from nitrogen removal compared to the gas formation quantified as replaced water in column and gas capture bottles for PCL and straw during the column experiment.

Figure 2 |
Figure 2 | Stacked effluent concentration of NO 3 --N and NO 2 --N relative to the NO 3 --N influent concentration.The mean volumetric nitrogen removal rate R N with standard deviation was calculated between days 14 and 188 (only until day 128 for straw due to technical problems).
ductivity, as reported by Cameron & Schipper (), especially for coarser grain size media.DOC release All materials that induced denitrification in the experiments also released DOC.Batch tests were able to indicate the amount of released DOC from the different substrates, especially for straw and peat (Figure 4(a)).A higher DOC release was observed for PCL and soft wood in the column tests compared to batch experiments.In the column tests, especially PCL and straw showed an excessive release of DOC.While DOC release from straw mainly occurred within the first 10 days, DOC washout from PCL was observed for several weeks, as can be seen in Figure 4(b).LC-OCD analyses revealed a release of low molecular weight compounds from PCL (Figure S5) which were found to be biodegradable in batch tests.In contrast, Boley et al. () reported only a slow DOC increase in the effluent of a PCL reactor.A high DOC release from wheat straw was also observed by Aslan & Türkman ().Although the DOC release from the PCL column decreased with time, denitrifying activity increased.This might be another indication for an incomplete biofilm development in the PCL column.In contrast to PCL, straw revealed a full nitrate removal during the first weeks, associated with the washout of DOC.Possibly, the DOC was more diverse and more easily accessible for microorganisms than the PCL polymers.Furthermore, straw as a natural material already might have contained microorganisms, which initiated denitrification after a short runtime.

Figure 4
Figure 4 | (a) Comparison of specific DOC release from PCL, straw, soft wood, and peat in batch and column experiments; (b) DOC released during column experiments.

Table 1 |
Operational details of the packed columns