Bioresource Technology 175 (2015) 239–244

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Enhanced removal of nitrate using starch/PCL blends as solid carbon source in a constructed wetland Zhiqiang Shen a,b, Yuexi Zhou a,b,⇑, Jia Liu a,b,c, Yu Xiao a,b,d, Rong Cao c, Fuping Wu d a

State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China Research Center of Water Pollution Control Technology, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China c School of Urban Construction, Hebei University of Engineering, Handan 056038, PR China d School of Environmental and Municipal Engineering, LanZhou JiaoTong University, Lanzhou 730070, PR China b

h i g h l i g h t s  Enhanced removal of nitrate in constructed wetland was investigated using cornstarch/PCL blends as external carbon sources.  The major component of DOM was polysaccharides which mainly consisted of reducing sugar.  Denitrifying bacteria Bacillus (24.25%) and Thauera (9.36%) were the most abundant genera.

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Article history: Received 12 August 2014 Received in revised form 29 September 2014 Accepted 1 October 2014 Available online 24 October 2014 Keywords: Denitrification Solid carbon source Starch Constructed wetland Microbial community

a b s t r a c t Cornstarch/polycaprolactone (SPCL) blends were prepared and used as external carbon source for biological denitrification in a constructed wetland. The denitrification performances, components of dissolved organic matter (DOM) and microbial diversity were investigated. The results showed that nitrate was removed mainly in the layer filled with SPCL, and the average denitrification rate was 0.069 kg/m3 d (nitrate removal efficiency was 98.23%). The major component of DOM was polysaccharides which mainly consisted of reducing sugar. Besides, the concentrations of polysaccharides and reducing sugar decreased along the height of the constructed wetland. Therefore, the dissolved organic carbon (DOC) of effluent decreased to 6.54 mg/L. Denitrifying bacteria Bacillus (24.25%) and Thauera (9.36%) were the most abundant genera in the biofilm attached on the surface of SPCL. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Constructed wetlands are typical natural and environmental friendly systems with the unique advantages of higher effluent quality and low operation costs (Saeed and Sun, 2012). Nitrogen, phosphorus and organics can be removed in constructed wetlands by interactions of plants, media and microbes. Among various nitrogen removal routes in constructed wetlands, microbial nitrification and subsequent denitrification is the main route (Stottmeister et al., 2003). It is difficult to achieve efficient denitrification in constructed wetlands when the influent has low C/N ratio or the major labile

⇑ Corresponding author at: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China. Research Center of Water Pollution Control Technology, Chinese Research Academy of Environment Sciences, Beijing 100012, PR China. Tel./fax: +86 10 84915311. E-mail address: [email protected] (Y. Zhou). http://dx.doi.org/10.1016/j.biortech.2014.10.006 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

organic matters are eliminated via microbial oxidation (Wen et al., 2010). Thus, methanol (Huett et al., 2005), fructose (Lin et al., 2002) and glucose (Caselles-Osorio and García, 2006) were used as external carbon sources to enhance the denitrification efficiencies in low C/N ratio constructed wetlands. However, there is a risk of overdosing with liquid carbon sources as well as a sophisticated and costly control system (Boley et al., 2000). Besides, most liquid carbon sources which are readily biodegraded would be lost via aerobic decomposition under field conditions. Therefore, insoluble biodegradable polymers were chosen as the alternative external carbon sources for denitrification (Boley et al., 2000). The insoluble biodegradable polymers are accessible by extracellular enzyme in this type of heterotrophic biological denitrification process (‘‘solid-phase denitrification’’). Two kinds of solid carbon sources have been investigated for ‘‘solid-phase denitrification’’, synthetic polymers (such as polycaprolactone and polyhydroxyalkanoates) and natural materials (such as wheat straw, cotton, pine bark and crab-shell chitin), especially the cellulose-rich materials (Shen et al., 2013a). In

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organic carbon-limited constructed wetlands, cattail litter (Wen et al., 2010), wheat straw (Camerona and Schipper, 2012), eucalypt wood mulch (Saeed and Sun, 2011), rice husk (Tee et al., 2012) and maize cobs (Camerona and Schipper, 2012) were used as external solid carbon sources to enhance the denitrification efficiencies. Cellulose-rich materials are cheap, renewable and widely available, however, which may bring the increase of DOC and color problem in effluent. Synthetic polymers (such as PCL and PHAs) are expensive with high denitrification efficiency (Shen et al., 2013b). Therefore, the key issue of ‘‘solid-phase denitrification’’ is to develop new solid substrates with low denitrification costs and without deterioration of effluent quality (Shen et al., 2013a). Effluents from biological processes contain a variety of complex organic compounds, including residual influent substrate, substrate intermediates and end products, and complex organic compounds were formed through condensation reactions (Barker and Stuckey, 1999). It is important to clearly identify the primary organic components of effluents in order to understand the fundamental mechanisms of biological activity that create these compounds, and how to reduce these compounds in the effluent (Kunacheva and Stuckey, 2014). However, the organic components of effluents are still not completely understood in solid carbon sources supporting denitrification system. Therefore, the main objectives of this study were: (1) to investigate the feasibility and efficiency of using a novel kind of insoluble starch/polycaprolactone (SPCL) blends as external solid carbon sources for denitrification in a constructed wetland; (2) to identify the primary organic components along the height of constructed wetland; (3) to analyze the microbial community structure of biofilm attached on the SPCL.

NaNO3 and KH2PO4 into the tap water to provide nitrate-N (NO3-N) and P concentrations of about 50 mg/L and 0.5 mg/L, respectively. The DO and pH of the synthetic wastewater were not controlled during the tests. The inoculated activated sludge was collected from a municipal wastewater treatment plant. 2.2. Experimental apparatus and procedure Continuous experiments were carried out in a laboratory scale simulated vertical constructed wetland. This reactor is a cylindrical Plexiglas with 80 mm inner diameter and 500 mm height (Fig. 1). A Plexiglas mesh disc (75 mm diameter, 3 mm pore size) was placed at the low end of the column as the support for packing material. The filter layer of gravel (5–8 mm) was laid at the disc at the depth of 200 mm. The enhanced denitrification layer was composed by gravel (5–8 mm) and SPCL (3–5 mm), 119 g SPCL mixed with 304.5 g gravel (v/v = 1:1) and laid at the top of filter layer at the depth of 100 mm. Addition, 50 mm height gravel (5–8 mm) was filled at the top layer. Three side sampling points were placed at different heights (from the disc 100, 200 and 300 mm, respectively, Fig. 1) to determine the nitrate concentration profile along the height of the reactor. To focus on the effect of SPCL on enhanced removal of nitrate, the reactor was unplanted. The synthetic nitrate-contaminated wastewater seeded with activated sludge (with the final concentration of 800 mg/L MLSS) was fed to the top of the reactor at a flow rate of 1.4 mL/min (Empty Bed Residence Time (EBRT) = 0.82 d). The liquid level was controlled to just exceed the top layer of gravel (Fig. 1), and the reactor was operated under environmental temperature. After being seeded for 3 d, the synthetic nitrate-contaminated wastewater was fed to the reactor without adding activated sludge.

2. Methods 2.3. Analytical methods 2.1. Materials Cornstarch/PCL (SPCL) blends were prepared by twin-screw extruder, which contains 55.44% starch, 30.00% PCL and 14.56% additives (plasticizer and coupling agent) (Shen et al., 2013a). Synthetic nitrate-contaminated wastewater was prepared by adding

Water samples were taken from the reactor, and filtered through 0.45 lm cellulose acetate membrane before analysis. NO3-N, nitrite-N (NO2-N) and ammonia-N (NH4-N) were assayed according to Chinese SEPA Standard Methods (SEPA, 2002). Dissolved organic carbon (DOC) was measured using a TOC analyzer

Fig. 1. Schematic diagram of the simulated vertical constructed wetland.

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6 4 2

40

60 Time (d)

80

ð2Þ

where NO3-Nin is the influent concentration of NO3-N; NO3-Nef, NO2-Nef and NH3-Nef are the effluent concentration of NO3-N, NO2-N and NH3-N, respectively (negligible accumulation of gaseous-N by-products and organic-N). 3. Results and discussion 3.1. Denitrification performances in constructed wetland The denitrification performances of SPCL supporting simulated vertical constructed wetland are shown in Fig. 2. In the period of days 0–28, though the environmental temperature was ranged from 18 to 26 °C, nitrate removal efficiency kept stable and the average nitrate removal efficiency reached 98.23%, the average NO3-N concentration of effluent was only 1.015 mg/L. In this period, the average denitrification rate was 0.069 kg/m3d under the influent NO3-N loading rate of 0.070 kg/m3 d. The denitrification rate was 0.64 kg/m3 d in the SPCL supporting packed-bed system which was filled with SPCL (Shen et al., 2013a). The difference of denitrification rate between this study and the packed-bed system which both supported by SPCL can probably be explained by the difference of filled SPCL quantity. There was very little NO2-N accumulation (0.008 mg/L). However, about 1.873 mg/L NH4-N was accumulated in the effluent, which probably resulted from the dissimilatory nitrate reduction to ammonia (DNRA) reaction. Vymazal and Kröpfelová (2011) also reported that NH4-N increased across the effluent in horizontal flow constructed wetlands. In other solid carbon sources supporting denitrification systems, the formation of ammonium were also observed (Wu et al., 2012; Shen et al., 2013a). When the NO3-N loading rate increased to 0.127 kg/m3 d (on the 30th d) by elevating the NO3-N concentration to about 100 mg/L, the effluent NO3-N and NO2-N concentrations increased significantly. Though, the average denitrification rate increased from 0.069 kg/m3 d to 0.092 kg/m3 d, the average NO3-N removal rate decreased from 98.23% to 72.24%, which indicated that the NO3-N loading rate of 0.127 kg/m3d exceeded the denitrification capability of the reactor. In unpretreated cattail litter and alkalipretreated cattail litter supporting constructed wetlands, the denitrification rates were 0.014 and 0.029 kg/m3 d in the initial stage,

100

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60 Influent NO3-N Effluent NO3-N Effluent DOC

80 60

50 40 30

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0

0 20

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60 Time (d)

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DOC (mg/L)

20

ð1Þ

where Cin is the influent NO3-N concentration (mg/L) and Cef is the effluent NO3-N concentration. EBRT is the empty bed residence time (d). Nitrate removal efficiency (Nre) is defined by the Eq. (2):

Nre ¼ 100  ðNO3 -Nin -NO3 -Nef -NO2 -Nef -NH3 -Nef Þ=NO3 -Nin

Effluent NO2-N Effluent NH4-N

8

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Nitrate (mg/L)

Rvd ¼ 0:001  ðC in  C ef Þ=EBRT

10 Nitrogen concentration (mg/L)

(Shimadzu, TOC-VCPH/CPN), and the samples were acidified and sparged before analysis. The pH value was determined with pH meter (METTLER TOLEDO). The polysaccharides were analyzed by phenol–sulfuric acid method. The proteins were determined according to a modified Lowry method. Reducing sugar was analyzed by 3,5-dinitrosalicylic acid method. After operation, biofilm was sampled from the reactor, and the microbial community was analyzed by pyrosequencing method (Shen et al., 2013a). Three-dimensional fluorescence measurements were conducted using a fluorescence spectrophotometer (F-7000, HITACHI, Japan). The excitation and emission slits width were set into 5 nm. To obtain fluorescence spectra, excitation wavelength was incrementally increased from 200 to 500 nm, and emission wavelength was set from 200 to 550 nm, which applied a bandwidth of 10 nm. The volumetric denitrification rate Rvd in kg/(m3 d) of the reactor is given by the Eq. (1):

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Fig. 2. Denitrification performances of the reactor.

and it decreased to 0.002 and 0.003 kg/m3 d after being operated 75 d, respectively (Wen et al., 2010). Average TN removal rates ranged 0.009–0.020 kg/m3 d in eucalypt wood mulch supporting vertical flow wetland (Saeed and Sun, 2011). In solid carbon sources supporting systems, denitrification rate is strongly dependent on the biodegradability of solid carbon sources. A higher denitrification rate will be received when the solid carbon sources have better biodegradability. Camerona and Schipper (2012) reported that nitrate removal rates were significantly higher for the labile carbon media (maize cobs, wheat straw and green waste) than for both soft-wood and hard-wood media. Compared with lignocellulosic materials, SPCL containing starch should be degraded easily by bacteria. Thus, the denitrification rate in this study was higher than that of cattail litter and eucalypt wood mulch supporting systems. As can be seen from Fig. 2, the effluent DOC increased at first accompanied with the formation of biofilm, and then it decreased quickly. In solid carbon sources supporting denitrification systems, DOC was eventually released from the solid carbon sources surface by enzymic hydrolysis, and the amount of released DOC mainly depends on the biodegradability of solid carbon sources and the amount of biomass. Starch is more biosusceptible than PCL in starch/PCL blends. Therefore, starch on the surface of the SPCL will be biodegraded more quickly than PCL. Meanwhile, the amount of biomass increased with the formation of biofilm. Hence, a high release of DOC will occur during the startup period. DOC will accumulate in the effluent when the amount of released DOC exceed the need of microbes for both growth and denitrification (Shen et al., 2013a). During the first 30 d, the amount of DOC for denitrification should be stable since a pseudo-stable nitrate removal was

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received, and the amount of DOC for microbes growth should be elevated since the increase of biomass was received. In this study, the increase of released DOC should exceed the increase of utilized DOC for microbes growth. As a result, the increase of accumulated DOC was observed during the startup period. As the biodegradation of starch on the surface of the SPCL progressed, the biodegraded rate of the SPCL should be decreased, resulting in a decrease of released DOC. In addition, the amount of DOC for microbes both growth and denitrification was stable after the mature of biofilm. Thus, the amount of DOC accumulated in the effluent decreased rapidly (Fig. 2). The feature of DOC accumulation during the start-up period was also observed in cross-linked starch/PCL blends (Shen and Wang, 2011), starch/PCL blends (Shen et al., 2013a) and starch/PLA blends (Shen et al., 2013b). With the NO3-N loading rate increasing from 0.070 kg/m3 d to 0.127 kg/m3 d, the denitrification rate elevate. Meanwhile, the amount of DOC consumed for denitrification should be increased, and the amount of released DOC should be stable under the pseudo-stable operation. As a result, the average effluent DOC decreased from 38.95 mg/L to 7.29 mg/L. 3.2. Nitrate removal profile along the height of the reactor Under the pseudo-stable operation, the nitrate removal profile along the height of the reactor was studied at the NO3-N loading rate of 0.127 kg/m3 d, and the result is shown in Table 1. Nitrate was removed mainly from the height of 20–30 cm, which was filled with SPCL. Though only gravel was filled in the height of 10–20 cm, there was also 14.22% nitrate removal rate, which resulted from the denitrification and assimilation of nitrate into biomass. In this layer, denitrifiers can only utilize DOM to reduce nitrate, so the DOC decreased from 15.70 mg/L to 9.83 mg/L. In the height of 10 cm, the DOC was low, so the removal of nitrate was not obvious in the height of 0–10 cm. Saeed and Sun (2011) also reported that COD in the effluent of vertical flow wetlands (with eucalypt wood-mulch media) increased and then reduced in the following horizontal flow wetlands (with gravel media). Since the NO3-N loading rate of 0.127 kg/m3 d exceeded the denitrification capability, the nitrite concentration increased along the height of the reactor. From the top to the low end of the reactor, the pH decreased at first and then increased (Table 1). In solid carbon sources supporting denitrification system, some organic acids and CO2 were produced by biodegradation of solid carbon sources (Wu et al., 2012; Shen et al., 2013a), and pH of effluent should be a net result of the acidic products and alkalinity derived from denitrification. The pH decreased slightly from the height of 30 cm to 20 cm, indicating that the produced acidity (by biodegradation of SPCL) was greater than the alkalinity (by denitrification) in this layer. The pH increased slightly in the low layer of the reactor (0–20 cm), in which only gravel was filled, which indicated alkalinity were produced by denitrification. Schiener et al. (1998) reported that the major component of the soluble organic matter in effluents from biological treatment processes is actually soluble microbial products (SMP). SMP are believed to contain polysaccharides, proteins, humic substances, nucleic acid substances, and other carbohydrates and small molecules (Kunacheva and Stuckey, 2014; Barker and Stuckey, 1999). To

2500 Fluorescence intensity (a.u.)

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Height of the reactor Fig. 3. Fluorescence intensity at the kEx/Em of 275/330 nm along the height of the reactor.

release the components of dissolved organic matter (DOM), polysaccharides, proteins and reducing sugar were detected along the height of the reactor, and the results also are shown in Table 1. As can be seen from Table 1, the concentrations of polysaccharides and reducing sugar decreased from the top to the low end of the constructed wetland. Actually, in the reactor the major component of DOM was polysaccharides. From the height of 20 cm to 0 cm, the ratios of polysaccharides contained carbon (1 g polysaccharides contains 0.4 g carbon) to DOC were 42.73%, 55.63% and 70.46%, respectively. Reducing sugar was the major component of polysaccharides, and the ratios of reducing sugar to polysaccharides were 59.69%, 46.45% and 33.94% from the height of 20 cm to 0 cm, respectively. Compared with proteins and other polysaccharides, reducing sugar may be easily utilized by denitrifiers. So, not only the concentrations of reducing sugar, but also the ratios of reducing sugar to polysaccharides decreased from the top to the low end of the constructed wetland. The change of reducing sugar confirmed the further reduction of nitrate from the height of 20 cm to 0 cm (Table 1). Three-dimensional fluorescence spectroscopy is useful technology to indentify the fluorescence properties of DOM (Chen et al., 2003; Wu et al., 2003). Usually, peaks at intermediate excitation wavelengths (250–280 nm) and shorter emission wavelength (

PCL blends as solid carbon source in a constructed wetland.

Cornstarch/polycaprolactone (SPCL) blends were prepared and used as external carbon source for biological denitrification in a constructed wetland. Th...
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