Denitrification of groundwater using a biodegradable polymer as a carbon source: long-term performance and microbial diversity

Nitrate pollution in groundwater is a worldwide problem. This paper reports on the denitrifying performance of using the biodegradable polymer polybutylene succinate (PBS) as a biofilmmedium and carbon source to remove nitrate from groundwater via a packed bed bioreactor whichwas operated continuously for nearly 2 years. Results showed that the effluent nitrate concentration reached 3.3–8.8mg L 1 and 88–97% of nitrate removal was achieved. The denitrification rate range was 0.25–0.35 g N per L per d at 20–29 C and decreased to 0.12 g N per L per d at 10–18 C. According to microelectrode analysis, the nitrate consumption rate (1069 103 mmol cm 1 h ) was much higher than the ammonium production rate (74 7 mmol cm 1 h ), which proved that denitrification plays the major role in the system. A low level of DOC (1.7 0.6 mg L ) and ammonium (0.5 0.3 mg L ) was observed in the effluent, which was beneficial for practical application. The consumption rate of PBS was 2.75 0.72 g PBS/g NO3–Nremoved. In the attached biofilm, Proteobacteria, Betaproteobacteria, Burkholderiales and Comamonadaceae were the major phyla (75.6%), classes (59.8%), orders (42.3%) and families (42.2%) in each level. In the top 20 genera accounting for 25% of total sequences, 9 genera including Simplicispira, Comamonadaceae, Hydrogenophaga and Rhodocyclaceae were affiliated with denitrifying groups with an abundance of 16%, whereas the bacteria belonging to the other 11 genera including Veillonellaceae, Propionivibrio and Bdellovibrio were reported to have the function of degradation and acidification of organic substance and might serve for degrading PBS in the system. The PBS solid-phase denitrification is promising for removing nitrate from groundwater.


Introduction
Nitrate is naturally present in the environment as part of the nitrogen cycle.2][3] Drinking nitrate-contaminated groundwater has the risks of methaemoglobinaemia for infants and stomach cancer. 4The maximum permissible level of nitrate in drinking water was set as 10 mg N per L by the U.S. Environmental Protection Agency 5 and in the standard for drinking water quality of China (GB 5749-2006) to reduce the risks to human health.
Compared to the methods commonly used for nitrate removal such as ion-exchange, distillation, reverse osmosis and electrodialysis, heterotrophic biological denitrication is a cost-effective and practical on large-scale technology. 6The denitrifying microbes utilize organic substrates such as methanol, glucose and acetic acid that serve as electron donor and energy source, and convert nitrate into nitrogen gas under anoxic conditions. 7,8n recent years, solid-phase denitrication, which uses water insoluble solid materials as carbon substrates for denitrication and biolm media, has been developed to eliminate nitrate from groundwater in situ or ex situ, [9][10][11] aquaculture effluent, 12,13 and biologically treated effluent. 14There exist two kinds of solid carbon source: the natural plant materials such as woodchips, straws and reeds 15,16 and the synthetic biodegradable polymers such as polycaprolactone (PCL), polybutylene succinate (PBS), polylactic acid and polyhydroxyalkanoate. [17][18][19][20][21][22] Healy et al. 11 investigated nitrate removal from groundwater by solid-phase denitrication using woodchips, pine needles, barley straw and cardboard, respectively as carbon source and media.The nitrate removal efficiency reached 67-89% at steadystate operation and the bioreactor lled with woodchips demonstrated less adverse effects such as dissolved organic carbon (DOC) release.Gibert et al. 23 built a permeable reactive barrier lled with sowood for in situ groundwater remediation.More than 98% of nitrate removal was obtained with the a Collaborative Innovation Center for Advanced Nuclear Energy Technology, INET, Tsinghua University, Energy Science Building, Beijing 100084, P. R. China.E-mail: wangjl@tsinghua.edu.cn;Fax: +86 10 62771150; Tel: +86 10 62784843 denitrication rate of 0.067 mg N per L per d per g substrate .Wu et al. 24 reported that the nitrate removal reached 95% in a solidphase denitrication system using PBS as carbon source to treat drinking water at 25 C. Boley et al. 25 studied the treatment of wastewater from a recirculating aquaculture system with eels by a uidized bed reactor lled with PCL for denitrication carbon source.The effluent nitrate concentration remained at low levels of 3-10 mg N per L, whereas a nitrate accumulation of 200 mg N per L was observed in the control system without denitrication treatment.
Solid-phase denitrication using biopolymers as carbon source is especially attractive in groundwater treatment due to the good and stable denitrifying performance and effluent quality such as low levels of DOC and color release. 26The high production cost of the biopolymers is the major factor to limit its application.Among the biopolymers available, PBS denitri-cation is drawing increasingly attention owing to the good biodegradability and economics. 24,27In addition, most studies related to biopolymer solid-phase denitrication are operated in batch mode or continuously for a short time (3-8 months).For commercial application, the denitrifying performance and degradation properties of biopolymers during a long-term run should be studied.
In the present study, a PBS-packed bioreactor was established to eliminate nitrate in groundwater.The long-term performance of the bioreactor for nearly 2 years was evaluated in terms of nitrate removal, denitrication rate, and the quality of the treated groundwater.The characteristics of PBS degradation and the biolm attached as well as the bacterial community were analyzed.The results of this study could enrich our understanding on solid-phase denitrication and provide a reference for its practical application in the management of groundwater nitrate pollution.

PBS media
The granules of PBS used are cylindrical with an average diameter of 3.0 mm.The density and specic surface are 1.24 kg L À1 and 0.826 m 2 g À1 , respectively.The chemical structure of PBS is as follows:

Experimental set-up and procedures
A packed bed reactor consisting of a cylindrical Plexiglas vessel (working volume 3.0 L) was established (Fig. 1).The packing ratio of PBS granules was 56.3% (v/v).A sieve was inserted into the reactor to prevent washout of PBS granules.Inuent was continuously pumped into the bottom of reactor and effluent was discharged from overow.The denitrication reactor was seeded with anoxic sludge collected from a municipal wastewater treatment plant with the concentration of 1500 mg total suspended solids (TSS) per L. The initial hydraulic retention time (HRT) was 41 h and temperature was 20 C.During the long-term operation of nearly 2 years, the temperature uctuated in the range of 29-10 C with the seasons (Fig. 2).The HRT was decreased gradually to 4.5 h and then varied in 5.6-13 h, depending on the performance of the reactor at different temperatures.Samples were periodically taken throughout the experiment to evaluate NO 3 -N, NO 2 -N and NH 4 -N and DOC content in the effluent.Biolm samples were taken at about 500 days to do DNA extraction, PCR amplication and pyrosequencing.

Inuent characteristics
The groundwater was exploited from Changping district, Beijing.The content of NO 3 -N, NO 2 -N and NH 4 -N and DOC were 10-13 mg L À1 , less than 0.09 mg L À1 , 0.17 mg L À1 and 0.3 mg L À1 , respectively.The following metal ions (mg L À1 ) were detected: Ca 2+ , 69; Mg 2+ , 41; Na + , 13; K + , 2.1; Mn 2+ , 0.2.The groundwater was fed into the PBS bioreactor by spiking with NaNO 3 and kH 2 PO 4 to reach the inuent NO 3 -N concentration  of 68 AE 2 mg L À1 and TP levels of 1.1 AE 0.2 mg L À1 , respectively.The pH value of the inuent was 7.8 AE 0.1.

Batch assays
Aer a certain period (1-2 months), batch tests were conducted to determine the specic denitrication rate (SDNR) of the biolm attached to PBS media as described in previous studies. 28The temperature was kept at 28.5 C. The SDNR was determined using linear regressions tted to plots of decreasing nitrate concentration vs. time divided by the initial volatile suspended solid (VSS) content (mg N/g VSS per d).The changes in the weight per piece of PBS granule and the biolm concentration (g VSS per L) were also evaluated.

Analytic methods
NH 4 -N, NO 2 -N and NO 3 -N were measured according to Chinese SEPA Standard Methods. 29DOC was determined by a multi N/C 2100 model TOC/TN analyzer (Analytic Jena, Germany).Three-dimensional excitation-emission matrix (3D-EEM) uorescence spectroscopy was applied to analyze the characterization of dissolved organic matter in the effluent using a spectrometry (F-7000 uorescence spectrophotometer, Hitachi Japan).
The molecular weight (MW) distribution of organic matters in the effluent was evaluated by a HPLC system (LC-20A, Shimadzu, Japan) equipped with a UV detector and an SEC column (TSK-GEL G3000PWXL).Sodium polystyrene sulphonate (3-30 kDa) and acetone (58 Da) were used as the standard solutions for M W calibration curve construction.
The functional groups of PBS granules were characterized using one NTS FT-IR spectrometer (PerkinElmer Spectrum).The structure of the PBS granules and biolm attached was observed by a JSM-6700F scanning electron microscopy (SEM, JEOL, Japan).
Biolm adhered to the PBS granules was collected by ultrasonic (40 kHz, 5 min).The content of TSS and VSS of the biolm samples were evaluated using gravimetric method. 29The extracellular polymer substances (EPS) were analyzed by the formaldehyde-NaOH extraction method. 30The content of carbon, oxygen, nitrogen and hydrogen element was quantied by an Vario EL element analyzer (Elementar, Germany).
For analysis of bacterial communities in the biolm samples, aer DNA extraction and purication, PCR amplication, amplicon quantitation and pooling, the pyrosequencing was conducted using a 454 Life Sciences Genome Sequencer FLX instrument (Roche, NJ, USA) by Majorbio Bio-pharm Biotechnology Company (Shanghai, China).Analysis of sequencing data were performed using Mothur soware.
The liquid-membrane microelectrodes sensitive to nitrate anions and ammonium ions were prepared, respectively to detect the proles of nitrate and ammonium within biolm.Briey, the nitrate and ammonium microelectrodes with a tip diameter of 5 mm and 15 mm, respectively were moved from the surface to the inner part of biolm by a motor-driven micromanipulator.The output signals were transformed and recorded by computer using SensorTtrace Pro 2.0 soware (Unisense, Denmark).The nitrate consumption rate and ammonium production rate were calculated using Fick's second law of diffusion. 31The applied molecular diffusion coefficients of nitrate and ammonium were 1.1 Â 10 À5 and 1.38 Â 10 À5 cm 2 s À1 , respectively. 32

Results and discussion
Performance of PBS-packed bed bioreactor Fig. 2 shows the variation of operational parameters and the overall performance of the PBS-packed bed reactor with respect to the concentrations of effluent NO 3 -N, NH 4 -N and NO 2 -N during the long-term operation of 620 d.The operational conditions and denitrication rate in the different phases were summarized in Table 1.
The effluent NO 3 -N concentration decreased on the third day from start-up and then HRT was reduced gradually.Aer 2 months of operation (Phase 1), the reactor was started up successfully and NO 3 -N removal reached 90%.Thereaer, the reactor was operated at temperatures of 20-28 C and HRT of 5-7 h (Phase 2), the effluent nitrate maintained at low levels of around 3.0 mg L À1 with NO 3 -N removal efficiency of higher than 95%.The denitrication rate reached the highest level of 0.35 g N per L per d.As the operation temperature dropped to 10-18 C (Phase 3), the nitrate removal efficiency decreased to 87.7% even extending HRT to 12-13 h.A nitrite accumulation was also found.The denitrication rate decreased by 65% compared to that in phase 2. When the temperature of the reactor rose to more than 20 C (Phase 4), the nitrate removal efficiency and denitrication rate recovered to more than 95% and 0.25 g N per L per d, respectively.Aer 1.5 years of experiment (Phase 5), the concentration of effluent NO 3 -N increased remarkably, which might be attributed to the great degradation of PBS carbon source and the increased biolm density which limited the mass transfer of both nitrate and substrate.Therefore, 200 g of fresh PBS granules, which equals to 15% of the initial amount, was replenished to the reactor (Phase 6).The effluent nitrate decreased rapidly and 94% of nitrate removal was obtained aer PBS addition.It is obvious that renewal of the fresh carbon source aer some period of time is necessary for the continuous operation.
Only a small increase in effluent DOC of 1.7 AE 0.6 mg L À1 was observed during the long-term operation, which is much lower than that using PCL of 1.7-5.2mg L À1 (ref.9) and poly-3hydroxybutyrate-co-hyroxyvelate (PHBV) of 14.9 AE 5.5 mg L À1 (ref.22) as carbon source for denitrication in treating the similar groundwater.This is promising for practical applications because the burden of post treatment to polish the effluent is reduced.The MW distributions of the DOC in the effluent revealed four fractions, 3.31, 2.32, 1.27 and dominant 0.83 kDa.The calculated M w and M n were 1.55 and 1.14 kDa, respectively.The effluent mainly contained soluble microbial products-like substances from 3D-EEM spectrum (spectrum not shown).The pH values and TP concentration in the effluent were 8.0 AE 0.1 and 0.5 AE 0.2 mg L À1 , respectively.
It is reported that temperature is one of the important factors controlling solid-phase denitrication, in addition to HRT, inuent nitrate concentration and pH, dissolved oxygen (DO), etc. 26,33 The DO concentration detected in the inuent was around 1.5 mg L À1 , which was consumed quickly though PBSpacked bioreactor, leading to low DO levels in the effluent (less than 0.3 mg L À1 ) that was appropriate for denitrication.The denitrication rate in the PBS-packed reactor was lower by 52-66% at temperatures of 10-18 C than that at 20-29 C. The solid carbon source could not be utilized directly by denitrifying bacteria but needs to be hydrolyzed into soluble and micromolecular substrates.Both the hydrolysis efficiency of PBS and the activities of the denitrifying microbes declined with decreasing temperature, 34 leading to a reduction in denitrication rate.Our previous studies showed that the denitrication rate decreased by around 50% with a 5 C reduction in PCLpacked denitrication reactor. 9Cameron and Schipper 35 documented that the NO 3 -N removal rate was raised by 1.7 times when temperature was increased by 10 C using the sowoodspacked denitrication bed.
Nitrite is an intermediate of denitrication process.As shown in Fig. 2 and Table 1, NO 2 -N accumulation was observed in the effluent during the initial stage (Phase 1, around 1.3 mg L À1 ) and the operation at the lower temperatures (Phase 3, around 1.8 mg L À1 ) owing to the incomplete denitrication.In the most period, the NO 2 -N concentration in the effluent were less than 0.9 mg L À1 .The concentration of leached ammonium in the effluent was at low levels (0.5 AE 0.3 mg L À1 ) throughout the total operation.This indicates that heterotrophic denitrication of reducing nitrate to nitrogen gas was predominant in the system using PBS as carbon source.It is known that there is another pathway, dissimilatory nitrate reduction to ammonium (DNRA), which competes with denitrication for nitrate. 36Microelectrode data indicated that ammonium production was detected at the biolm-media interface and NH 4 -N concentration decreased toward the bulk surface, while NO 3 -N concentration decreased gradually from the biolm-bulk interface toward the biolm bottom (Fig. 3).The calculated nitrate consumption rate (1069 AE 103 mmol cm À1 h À1 ) was much higher than the ammonium production rate (74 AE 7 mmol cm À1 h À1 ), which proved that denitrication plays the major role in the system.

Characterization of PBS degradation and biolm attachment
From batch test, the weight of each piece of PBS granule declined almost linearly over the course of time (Fig. 4).The degradation rate of PBS was around 0.04 mg d À1 .If the useful life of the solid carbon source is 50% of its initial weight, the PBS granules (with the initial weight of around 0.0289 g per piece) would last around 1 year, aer that period refreshing some carbon source seems to be needed.
From SEM observation, the fresh PBS granule has relatively smooth surface with some protuberances (Fig. 5a), while the used PBS has concave cavities induced by microbial corrosion (Fig. 5b).According to FT-IR spectra (Fig. 6), PBS exhibits the characteristic adsorption at 2961 cm À1 , 2944 cm À1 and 2850 cm À1 due to CH 2 stretching vibration, 1713 cm À1 assigned to carbonyl group, 1174 cm À1 and 955 cm À1 due to C-O stretching vibration and 1044 cm À1 assigned to O[CH 2 ] 4 O group.Aer PBS was used as carbon source, a broad peak in 3000-3750 cm À1 region assigned to hydroxyl group has become obviously, indicating that there existed small fragments from PBS hydrolysis.The shape and position of the characteristic adsorption of the used PBS were similar to that of the fresh PBS, whereas the peak intensities declined greatly due to biodegradation.
When acting as carriers, PBS favors the attachment of bio-lm which was composed of cocci and rod shape bacteria with some lamentous bacteria (Fig. 5c).The amount of biolm  attached to PBS media increased gradually with the prolonged operating time and reached around 16 g VSS per L aer 1 year of operation.With the increase in the biolm density the mass transfer of both nitrate and substrate might be limited.Consequently, the SDNR of biolm attached initially decreased remarkably and then stabilized at around 0.03 mg N/g VSS per d (Fig. 7).It is noted that the VSS/TSS ratio of biolm reached more than 90%, indicating the high activity of biolm attached to the biodegradable media.Similar results were found in a denitrication reactor using PCL as carbon source. 9In the EPS composition, the content of protein (129 AE 12 mg per g VSS) was much higher than that of polysaccharides (41 AE 5 mg per g VSS), which might be owing to the excretion of cytosolic proteins or cell lysis. 37hen PBS was utilized as carbon source for denitrication, the reaction of PBS monomer with nitrate leads to the production of carbon dioxide, nitrogen gas and biomass.The elemental composition of biomass was C 3.87 H 6.86 N 0.67 O 2.14 from the elemental analysis of biolm.Assuming a yield 0.45 g biomass/g PBS, 38 the equation of denitrication reaction can be given as: The theoretical amount of PBS consumed in removing a gram of NO 3 -N was 2.68 g PBS/g NO 3 -N.The practical PBS consumption rate (2.75 AE 0.72 g PBS/g NO 3 -N) was slightly higher than the theoretical value.Similar results were also found by other researchers who utilized PCL and poly-3hydroxybutyric acid (PHB) as denitrifying carbon source. 13,25his difference might be due to the residual oxygen consumption on carbon source.In the presence of dissolved oxygen, a portion of carbon source was consumed by aerobic respiration instead of denitrication. 25he unit price of the domestic PBS products in China ranges 18-25 CNY/kg.The calculated denitrication cost using PBS as carbon source was 49-68 CNY/kg NO 3 -N, which are higher than using methanol. 22If the economic PBS polymers were available with the development of bioplastic industry, PBS solid-phase denitrication might be competitive in practical application.

Microbial community analysis
The numbers of effective sequence tags obtained from pyrosequencing were 13 950.Fig. 8 shows the bacterial community and abundance identied at the phylum, class, order and family levels in biolm attached.At the phylum level, Proteobacteria was predominant with relative abundance of 75.6%, followed by  Bacteroidetes (5.62%), Firmicutes (4.32%), Chlorobi (4.14%) and Spirochaetes (0.8%).Betaproteobacteria, Burkholderiales and Comamonadaceae were the major classes (59.8%), orders (42.3%) and families (42.2%) in each level, respectively.Hiraishi and Khan 38 have documented that most of the PHA-degrading denitrifying bacteria were affiliated to Betaproteobacteria and family Comamonadaceae.Khan et al. 39 have isolated 23 strains of PHBV-degrading denitrifying bacteria from different activated sludge.16S ribosomal DNA sequence analysis demonstrated that 20 of the strains belonged to the family Comamonadaceae.
At the genus level, the identied bacteria of the major OTUs, the relative abundance and function reported were shown in Table 2.It is worthwhile to note that there were 71% of sequences unclassied in the biolm samples.In the top 20 OTUs accounting for 25% of the total sequence, 9 OTUs involving Simplicispira, Comamonadaceae, Hydrogenophaga and Rhodocyclaceae belonged to denitrifying groups with an abundance of 16%.All the genera of identied denitriers affiliated with phylum Proteobacteria.The abundance of genus Simplicispira was the highest.It was reported bacteria belonging to Simplicispira are able to reduce nitrate to nitrite and have been isolated from activated sludge in treating wastewater for inorganic nutrient removal. 40Ruan et al. 41 also reported that the denitriers Azoarcus and Simplicispira were dominant in a recirculating aquaculture system lled with PBS as the biolm carrier and carbon source for simultaneous removal of ammonium and nitrate.In addition, genus Desulfovibrio was found in the pyrosequencing analysis (0.6%), which was reported to be able to reduce nitrate to ammonium. 42This is the reason why ammonium was detected in the effluent.
The denitriers using the solid carbon source were found to be different from that using the soluble carbon substrate.The denitrifying bacteria in the genera Diaphorobacter, Simplicispira and Comamonas which were found in the present study and Acidovorax belonging to family Comamonadaceae were commonly dominant in the solid-phase denitrication system. 12,43,44][47] As shown in Table 2, the bacteria including Veillonellaceae, Propionivibrio and Bdellovibrio, belonging to other 11 genera with an abundance of 9% were reported to have the function of degradation and acidication of organics.In the present study, they might serve for degrading PBS.The denitrifying microbes are not capable of utilizing the macromolecular PBS directly.In the solid-phase denitrication system there are microbes which secrete some hydrolases such as protease and lipases to decompose PBS to small soluble monomers. 17,26The degraded products were then utilized by denitrifying bacteria to achieve biological denitrication.It should be noted that some species of genus Diaphorobacter which was detected in the PBS solid-phase denitrication system, were reported to be characterized with both degrading PHBV and denitrication. 48In addition, no methanogen microbes were observed in the current work.It is reported that in the solid-phase denitrication process, the organic solid substances might be degraded by anaerobic digestion, leading to the production of CO 2 and CH 4 . 26In such cases, the methanogens might develop in the inner part of the carriers, which compete with denitries for carbon source.

Conclusions
The long-term performance of a PBS-packed bed bioreactor for nearly 2 years demonstrated that the system is promising to remove nitrate from groundwater.The effluent nitrate concentration reached 3.3-8.8mg L À1 and 88-97% of nitrate removal was achieved.The denitrication rate at temperatures of 10-18 C decreased by 52-66% compared to that at 20-29 C.Only a small increase of DOC (1.7 AE 0.6 mg L À1 ) and ammonium (0.5 AE 0.3 mg L À1 ) was observed in effluent which reduce the burden of post treatment.The practical PBS consumption rate (2.75 AE 0.72 g PBS/g NO 3 -N) was slightly higher than the theoretical value.Renewal of the fresh carbon source aer certain period was necessary for the continuous operation.Bacteria belonging to genera Simplicispira, Veillonellaceae uncultured, Comamonadaceae uncultured and Hydrogenophaga were highly enriched in PBS biolm.In the top 20 OTUs accounting for 25% of the total sequence, 9 OTUs belonged to denitrifying groups with an abundance of 16%.Some members belonging to other 11 genera were reported to be capable of degradation and acidication of the organic substances.

Fig. 1
Fig. 1 The schematic diagram of the experimental set-up.

Fig. 2
Fig. 2 Variation of temperature, HRT and effluent nitrogen concentrations with time.

Fig. 4
Fig. 4 Changes in weight per piece of PBS with operation time.

Fig. 6
Fig.6FT-IR spectrum of the fresh and used PBS.

Fig. 7 Fig. 8
Fig. 7 Changes in SDNR and the amount of attached biomass with time.

Table 2
Bacterial identification of the major OTUs at genus level, the relative abundance, the phylogenetic affiliation and function a + Proteobacteria/Thiotrichaceae Reduce nitrate to nitrite and/or further to N 2 O 63 a The bacteria with + represent denitriers.