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Denitrification in biofilm configured horizontal flow woodchip bioreactor: effect of hydraulic retention time and biomass growth
Ecohydrology & Hydrobiology 15 (2015) 39–48
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Original Research Article
Denitrification in biofilm configured horizontal flow woodchip bioreactor: effect of hydraulic retention time and biomass growth Sivaramakrishna Damaraju a,*, Umesh Kumar Singh a,b, Desi Sreekanth c, Alok Bhandari a a b c
Department of Civil Engineering, Kansas State University, Manhattan, KS 66503, USA ISERC, Visva-Bharati University, Santhinikethan 731235, India Department of Botany, Osmania University, Hyderabad 500007, Telangana, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 February 2014 Accepted 19 November 2014 Available online 5 December 2014
Removal of nitrates and nitrogen (NO3-N) from surface and groundwater was a matter of special importance, and there was a need for sustaining the public health problems and aquatic life related to the presence of NO3-N in drinking water supplies. The influence of hydraulic retention time (HRT), volumetric loading rates (VLRs) and temperature on denitrification were investigated in a biofilm configured woodchip bioreactor (WBR) from synthetic tile water. Three HRTs were examined, ranging from 4 to 12 h. The denitrification removal efficiency (RE) >99% was obtained in WBR operated at 8-h HRT. The WBR was operated with VLR ranging from 49 to 198 g NO3-N/L/day for denitrification study. The maximum load reduction of the WBR showed 99% in second level of reactor operation with biomass concentration of 12.3 g/L. The pH (6.5–7.2) and temperature (17–20 8C) under the unique study profoundly impact on denitrification. The coefficient factors of 0.8 and 1.05 have been reported in studies with a temperature change from 4 to 20 8C. Scanning electron microscope (SEM) analysis documented formation of biofilm on woodchips in each level of reactor operation study. Therefore we recommend from this study, that the horizontal flow based WBR to enhance denitrification while minimizing higher operating conditions in denitrification beds. ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Denitrification Biomass Nitrate HRT Temperature Scanning electron microscope
1. Introduction Nitrate-nitrogen (NO3-N) concentration in aquatic environments has been increasing in recent years as a result of the intensive application of nitrogen fertilizers
* Corresponding author at: Department of Civil Engineering, 2118 Fiedler Hall, Kansas State University, Manhattan, KS 66506, USA. Tel.: +1 785 340 5969. E-mail address: [email protected] (S. Damaraju).
and animal manure to agricultural land, septic tanks, and land disposal of wastes (Gee-Bong and Jai-koo Park, 2012). Nationwide studies have detected nitrate levels in water below agricultural fields to be in the range of 5–100 mg/L, with regular sampling between 20 and 40 mg/L (Maria Osiadacz et al., 2010). Accumulation of various forms of nitrogen compounds in ground and surface water bodies, could lead to adverse effects including eutrophication in receiving waters, ammonia toxicity to aquatic life, and public health problems related to the presence of NO3-N in drinking water supplies (Elefsiniotis and Li, 2006).
http://dx.doi.org/10.1016/j.ecohyd.2014.11.001 1642-3593/ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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Therefore, the removal of nitrogen compounds from wastewater would be of increasing importance. Treatment of waters contaminated by nitrate or nitrite could be achieved through physicochemical or biological processes. Physicochemical methods such as ion exchange and reverse osmosis, however, were not selective and resulted in secondary wastes with high levels of nitrate or nitrite. Conventional biological processes for the removal of nitrate and nitrite were referred to as denitrification (Yimin and Mehdi, 2012). Under anoxic microenvironment conditions, heterotrophic denitrifying bacteria Bacillus (Flores et al., 2007) and Corynebacterium (Ines et al., 2011) convert nitrate (NO3) or nitrite (NO2) into gaseous forms, either dinitrogen (N2) or nitrous oxide (N2O). This microbialmediated process plays a particularly important role in nitrogen-enriched coastal systems, since it diminishes the amount of fixed nitrogen transported to the ocean, and consequently reduces the nitrogen available for primary production (Beman et al., 2005; Catarina et al., 2010). As a heterotrophic process, denitrification tends to be less sensitive to environmental parameters than, for example, nitrification; however, it is still affected by variations in carbon source, dissolved oxygen (DO), pH, temperature, presence or accumulation of NO2 and NH4+ during the process (Xu et al., 2009; Cameron and Schipper, 2010). The availability of organic carbon source was important in heterotrophic denitrification. The characteristics of the added carbon sources have been found to have major effects on important parameters of the denitrification process such as the denitrification rate, COD demand, biomass yield and biomass composition (Schipper et al., 2010). For these reasons, denitrification woodchip-based bioreactors were of low cost with high C/N ratio and thus come under a relatively new technology for edge of field removal of nitrates (Gibert et al., 2008). These engineered denitrification systems provide an environment that supports the growth of denitrifying bacteria, and compensate for the reduced interaction of tile drainage with natural biological soil processes. In addition, they do not require replenishment as C is not rapidly depleted from them, although the duration of their effectiveness will be affected by the longevity of the C supply to the denitrifying microorganisms (Moorman et al., 2010). In wastewater treatment applications include using wood based bioreactors in subsurface barriers to treat storm water runoff, acid mine drainage and groundwater contamination. This water treatment was achieved through the well studied ability of woody organics to sorb dissolved petroleum hydrocarbons as well as metals (Robert and Othman, 2009) and the degradation of chlorinated and aromatic hydrocarbons. In a bioreactor, the synthesis of microbial mass (biomass) by the mineralization of biodegradable pollutants leads to biomass growth and accumulation over time, its effect on the denitrification removal rate (Mohseni et al., 1998). Biomass accumulation, which was greater at the inlet sections of bioreactor lead to a change in bed characteristics, such as the reduction of the inter particle void space and the compaction of natural packing materials, which caused channeling and increased pressure drops. This translates into higher operating and
maintenance costs that become significant for long-term bioreactor operation (Swanson and Loehr, 1997). This study aimed to develop optimal operation strategies for an efficient and stable performance of NO3-N removal in the four step level biofilm configured wood chip bioreactor (WBR). The information obtained from this study was expected to provide basic knowledge for the bioreactor design of practical bioprocesses capable of long-term NO3N removal from wastewaters. The effect of HRT on the performance of WBR treating synthetic tile water containing high concentration of NO3-N (15–50 mg/L) was also investigated. Furthermore, the attached biomass developed in the reactors was characterized using SEM analysis. 2. Methods and materials 2.1. Bioreactor Bench scale biofilm configuration anoxic woodchip bioreactor (WBR) was fabricated in the laboratory using acrylic material with a working volume of 7 L, liquid volume of 4 L and diameter of 7 in. (17.8 cm), and a height of 20 in. (50.8 cm). In the bioreactor system, perforated acrylic plates were fit at each end of the reactors to diffuse the flow of fluid into the columns. Four inlet and outlet ports were evenly fixed over the end of the reactor. The WBR was operated in continuous flow mode. The influent pH was kept in the range of 6.5–7.5 by addition of 0.1 N NaOH or HCl. The volumetric flow rate of the feed in reactor was adjusted using three Masterflex (C/L 77122-22) variable speed peristaltic pumps. The reactors were operated with a hydraulic retention time (HRT) of 4– 12 h, based on working volumes of the four levels. The temperature was maintained at ambient condition. Schematic details of the horizontal flow WBR used in this study along with photograph of the experimental setup were depicted in Fig. 1. 2.2. Support matrices and carbon source The reactor was packed with Standard hardwood landscape woodchips (Golden Valley Hardscapes, located in Story City, Iowa) which were used to support the cell growth. The bed zone of each reactor was packed with 192 g woodchips/L, which resulted in bed porosities of 48%. Wood chips cut into small cylindrical shapes with average diameters of 11.2 mm and heights of approximately 24 mm were used. 2.3. Synthetic tile water The concentrations of the NO3-N in synthetic tile water shown in Table 1 were very similar to the characteristics of agricultural runoff. The synthetic tile water was pumped directly from the influent container to each column. Electrical conductivity of the prepared synthetic tile drainage was monitored and maintained within a range consistent with values observed under average field conditions. Potassium chloride (KCl) was added to the influent solution to maintain an electrical conductivity within a range observed in tile drainage (600–800 mS). The
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Fig. 1. (a) Photograph of a wood packed denitrification bioreactor. (b) Schematic of bioreactor end-plate. (c) Schematic of diffuser column bioreactor. The schematic images are modified from work done by L. Christianson.
influent nitrate nitrogen concentration varies from 15 to 50 mg/L. 2.4. Wastewater-Manhattan The wastewater collected from the local wastewater treatment plant in Manhattan was additionally seeded into the reactor. Characteristically, the wastewater was highly biodegradable (BOD/COD / 0.6). The wastewater was pumped into reactors and operated for 25 days to ensure adequate contact time between the wastewater and the woodchips for attachment of the biomass enhancing the growth of denitrifying microflora on media. The wastewater characteristics were given in Table 2.
filtrate was analyzed by a Cd-reduction method for NO3N + NO2-N (Lachaat Quick – Chem 20 Genesis spectrophotometer). Volatile suspended solids (VSS), COD, pH, ORP, DO and conductivity were measured according to Standard Methods (APHA, 1998). Oxidation–reduction potential (ORP) and pH values were determined by a pH meter (Corning 450, USA). Dissolved oxygen (DO) was measured by a DO meter (YSI 550A, USA). Electrical conductivity was measured using a Fisher Scientific accumet1 AR 20 conductivity meter. The biofilm formed on the woodchips and dominant colonies were subjected to scanning electron microscopy (SEM) and light microscope. Each experimental condition was carried out in triplicate.
2.5. Analytical methods
3. Results and discussions
The samples collected at periodic time intervals were filtered through a 0.45 ml Millex filter, and nitrate in the
3.1. Start-up performance of woodchip bioreactor
Table 1 Constituents of designed synthetic wastewater used in this study. Constituents
Concentration (mg/L)
CaCl22H2O CoCl26H2O MgSO47H2O MnCl24H2O CuSO45H2O FeCl36H2O KH2PO4 ZnCl2 (NH4)6Mo7O244H2O NaCl NH4+-N NaNO3
1.2 0.1 4.3 0.04 0.03 1.21 8.2 0.03 0.04 13.5 0.2–1.4 15–50
In bioreactor accelerated startup process, the reactor was initially operated with wastewater as supply of additional heterotrophic bacteria along with woodchips at strength of COD 2.1–2.4 g/L and the concentrations of Table 2 Characteristics of wastewater used as feed for startup study. Parameters pH COD (mg/L) BOD5 (mg/L) Chlorides (mg/L) NH4+-N Nitrate-nitrogen (mg/L) Microbial composition (CFU/ml)
7.2 2182 1316 48 0.2–1.2 13 1.2 102
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influent 13 mg/L NO3-N with an HRT of 24 h during the steady period of 26 days. Table 3 shows the daily variations in pH, ORP, COD removal and NO3-N removal efficiency during the course of acclimatization period. Before reaching the stable reactor performance, the pH, COD removal and NO3-N removal efficiency fluctuated greatly in the first 8 days (Table 3). NO3-N and COD removal efficiency reached up to 48% and 58% respectively on the 8th day, but gradually decreased to 23% and 36% on the 13th day with effluent concentrations of 10.3 mg/L NO3-N and 0.88 g COD/L. After 14 days, NO3-N and COD removal efficiency increased reaching 98% and 84% on the 20th day. Based on the removal efficiency of NO3-N and COD in WBR, there were three stages for acclimatization; with 1–13 days being lag stage, 14–20 days of acclimatization and 21–26 days as stabilized stage. The NO3-N removal efficiency in reactor kept increasing at the lag phase (1–13 days); Table 3 indicates that the denitrifying bacteria started adapting to the woodchip reactor system. Both NO3-N and COD removal efficiency percentage started to increase significantly from the 15th day and stabilized on the 20th day. During the stabilized stage (21–26 days), NO3-N and COD removal efficiency was 98% and 87% in the reactor. This high NO3-N removal efficiency was possible in start-up period due to the large column length (L/D > 4) which provides long flowing pathway during NO3-N from inlet to outlet. The VSS concentration declined during the start-up period and became the lowest on the 5th day with 2.4 g VSS/L. Afterwards, it gradually increased to 5.8 g VSS/L on the 14th day in the WBR and stabilized in the later period. This horizontal flow WBR retained a high level of VSS which was far higher than the average VSS concentrations in other bioreactor studies (Guilherme et al., 2011, Ling et al., 2012). The pH values of the effluents were maintained between 6.8 and 7.2 indicating that pH was maintained stably throughout the experiment. This was the optimum pH range for denitrifying bacteria in WBR systems. The ORPs of the effluents were between 28 and 71 mV (Table 3) and DO values were below 2 mg/L. Based on the measurement of NO3-N removal efficiency, pH and ORP values (Table 3), the WBR system reached a stabilized stage on the 20th day. However, biomass concentration (VSS) showed a different trend. These experiments revealed that wastewater addition in start-up process could influence anoxic WBR which possessed a quite stable and good NO3-N removal efficiency ability, with a good acclimatization.
3.2. Effect of HRT on denitrification It was apparent from Fig. 2(a)–(d) that for NO3-N percent removal, various HRTs in continuous WBR operation mode were obtained under steady-state conditions for the four levels of reactor study. The NO3-N concentrations were increased to 14.8 1.28, 31 0.56 and 52 1.2 mg/L under various HRTs increased from 4 h to 8 h to 12 h respectively. The same trend of NO3-N concentration profiles were followed in each level of reactor study. As illustrated in Fig. 2(a), in first level of reactor study, influent and effluent NO3-N concentrations were 50 mg/L and 20.3 mg/L with 59.3% NO3-N removal at an HRT of 4h, respectively. The NO3-N percent removal was gradually increased with increasing HRT at 8-h, which was operated at the same influent concentration at 50 mg/L resulted in complete (>99%) removal of NO3-N in effluent profiles respectively. Further increasing the HRT from 8 to 12 h with influent concentration of 50 mg/L, resulted in (>99%) removal of NO3-N similar to that at HRT 8 h. Our results proved that NO3-N removal efficiency was positively related with HRT. This was possible considering that more substrate was solubilized and consumed under the longer HRTs (Ping et al., 2011). According to Yusoff et al. (2010) who reported that, at lower HRTs, the biomass was washed-out and therefore the denitrifying bacteria had a short period of contact time with surfaces of woodchips (Natasha et al., 2012). The reduced physical contact of the solution may have limited the percent of nitrate removal possible at lower HRTs. From this view point of denitrification, at 8 h HRT was found to be more suitable operation condition than HRT 4 h for the woodchip reactor. As shown in Fig. 2(b), the system commenced at HRT 4 h and was then adjusted to 8 h until steady state condition was reached in second level of reactor operation. In the beginning HRT 4 h, influent concentration NO3-N with 50 mg/L resulted in effluent NO3-N concentration of 28 mg/L, demonstrating a 56% mean removal in effluent profiles respectively. Further increasing HRT from 4 to 8 h, the influent NO3-N percent removal at this HRT was complete (>99%) in effluent NO3-N concentrations. So Fig. 2(a) and (b), illustrates that second level experiments showed more stable and fast removal of NO3-N and cumulative HRT days were shorter (20 days) in comparison with first level (26 days) of reactor experiments. This may be due to the denitrifying microflora density increasing significantly as a result of the formation of sufficient amount of biomass in the second level reactor study.
Table 3 Operating conditions during the accelerated start-up of WBR treating wastewater. Days
COD (g/L/day)a
NO3-N (mg/L/day)
COD reduction (%)
NO3-N removal (%)
VSS (g/L)b
pH
ORP (mV)c
1–8 8–13 13–20 20–26
2.1 2.0 2.1 2. 0
13 13 13 13
16–58 28–36 58–84 84–87
21–48 12–23 92–98 94–98
2.4 5.8 5.8 5.8
6.4–6.6 6.8–7.2 6.9–7.0 6.8–6.9
28 52 71 69
a b c
Chemical oxygen demand. Volatile suspended solids. Oxidation–reduction potential.
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Fig. 2. The effect of HRT on NO3-N percent removal in four levels of reactor operation.
From Fig. 2(c), the NO3-N removal efficiency in third level at HRT 4 h, with influent concentrations at 15, 30 and 50 mg/L resulted in effluent concentrations of 11.3 mg/L, 24.3 mg/L and 44 2 mg/L, which showed 24 2%, 17 1% and 11 2% mean removal efficiencies. Then the HRT were set at 8 h and 12 h with the same influent concentration, so that the effluent NO3-N removal efficiencies increased to 15 mg/L (>98%), 30 mg/ L (52%) and 50 mg/L (21%) respectively. Fig. 2(d) depicts the influent concentration of NO3-N (15, 30 and 50 mg/L) and percent removal (17%, 11% and 8%) at an HRT of 4, 8 and 12h, which indicated low NO3-N removal efficiency at fourth level reactor operation study. This was due to the excessive formation of biomass growth in the third and fourth of the reactor operation, resulting in the uneven flow of influent NO3-N concentrations between woodchips in the reactor and also higher flow rate and loading rate at third and fourth levels causing the bacterial community being washed out. Media (woodchips) in the reactor may have swollen due to absorption of water, decreasing the inter particle porosity. Ovez et al. (2006) reported that, for higher NO3-N removal efficiencies, experiments may need to be operated under high HRTs which would end up with the utilization of larger reactors, since the
available organic compounds in the substrates were decreasing with time. 3.3. Effect of volumetric loading rate (VLR) on denitrification and biomass growth The combined effects of both NO3-N concentration and HRT on denitrification could be represented by volumetric loading rate (VLR). Variations of VLR with mass load reductions were shown in Fig. 3(a)–(d). Referring to the results, during first and second level reactor operation, highest total percent load reductions at greater than 59 3% and 99 1% at optimal HRT 12 h with bioreactor influent VLR ranged from 49 to 94.9 g NO3-N/L/ day. From the third and fourth level reactor studies, the average load reductions dropped to 24 2% and 13 2% at VLR which ranged from 138 to 173 g NO3-N/L/day with effluent loads of 52 4 and 150 2 g NO3-N/L/day. These results made clear that when influent loads exceeded approximately 140 g NO3-N/L/day in reactor, the load reductions performance in this study began to decline greatly. Using higher VLR could have led to a higher load reduction rate, but much more VLR would result in inhibition of load reduction activity.
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Fig. 3. The effect of volumetric loading rate on NO3-N load reductions in four levels of reactor operation.
The above correlation between load reductions could be explained by the reason that, the growth of biomass (VSS) was closely associated with VLR, and was also influenced greatly by packed bed volume occupied in each level of reactor. As illustrated in Fig. 4, in first and second level reactor bed volume, the biomass concentration in the reactor was maintained between 7.2 and 15 g VSS/L during the first 75 days, while in third and fourth studies the concentration of biomass increased from 5.8 g VSS/L at start-up to approximately 24.3 g VSS/L after 115 days of reactor operation and remained constant thereafter with an average value of 21.3 g VSS/L. Comparison of four levels of reactor volume operation, which worked at different VLRs (see Fig. 3) showed that the system with the higher VLR (third and fourth) levels could sustain more biomass; this was due to high VLRs and nutrient availability in excess of what was needed for denitrification. The inverse relationship between the biomass and load reductions emphatically reported that the higher biomass yield was attributed to different bacteria other than denitrifying bacteria. These results were in agreement with other studies (Fernando et al., 2001). Auria et al. (1993) found that pressure losses in a solid state fermenter packed with wheat bran and cane bagasse were also controlled by mold growth, although these supports underwent changes due to microbial growth. Higher decay rates were usually
reported for cells in biofilm systems than for cells in suspension, as stated by Okkerse et al. (1999); therefore, lower net yield coefficients were expected in biofilm systems. This showed that the performance of the WBR was effectively enhanced by applying favorable HRT, biomass growth and NO3-N concentration.
Fig. 4. Variations of biomass with time in the denitrification systems.
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3.4. Bioprocess monitoring Along with NO3-N removal efficiency, parameters such as pH, ORP and temperature were also monitored during the bioreactor operation to evaluate the bioprocess mechanism during denitrification. In this study from Fig. 5(a)–(d), the pH increased slightly from inlet to outlet in all four levels of reactor operation. In overall study, the optimal pH was maintained from 6.5 to 7.5. The above results depicted that, denitrifying microorganisms function effectively within a pH range from 6.0 to 8.0, with the microbial activity being at an optimum around neutral reported in other studies (Mathava Kumar and Jih-Gaw, 2010). Consequently, in order to improve the potential of the denitrification process, the initial pH in each level was adjusted to an approximate value of 6.5–6.8 for the WBR study. During denitrification process, ORP values and DO concentration reflect the oxidizing or reducing conditions in a reactor and were used to evaluate the direction of biological and chemical process in reactors. The average values of ORP were 35 8.2 (mV) to 70 18.4 (mV) which confirmed the anoxic condition in WBR. The dissolved oxygen (DO) results of WBR effluent also varied between 1.2 and 2 mg/L (data not shown) and
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consistently maintained the same concentration throughout the study period, which was lower than that of influent (6.2 mg/L) concentration. However, some reports showed that concentrations of up to 4.2 mg/L could facilitate denitrification (Mark et al., 2012). 3.5. Effect of temperature Operation temperature effect on the NO3-N removal percentage was investigated in a temperature range of 4– 20 8C, representing the actual average temperature change throughout a year, in order to identify the best temperature for NO3-N percent removal. Influent NO3-N concentrations from 28.3 to 31.8 mg/L, with a mean value of 30.42 0.8 mg/L were achieved at 4 8C and 20 8C with 8 h HRT operated at second level of reactor study. Fig. 6 shows that NO3-N removal at the controlled temperature of 4 8C was 15% with mean effluent concentration of 24.82 mg/L, respectively, after approximately 22 days of continuous operation. At 20 8C, NO3-N removal was relatively complete (effluent <1 mg L1) with optimum HRT of 8 h. Conclusively, increasing temperature gave rise to an increase in average cellular denitrification activity. This may be due to the fact that reaction at kinetically-favorable high temperatures
Fig. 5. Daily variations in pH and oxidation–reduction potential (ORP) in four levels of reactor operation.
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Fig. 6. Variations of NO3-N percent removal with temperature.
Table 4 Temperature coefficients for different study reactors. Temperature range (8C)
Reactor type
Initial NO3-N concentration (mg/L)
Temperature coefficient
Source
20–30 15–25 16–25 5–15 4–20
Erlenmeyer flasks SBRa SBR Shake flasks WBR
100 600 100 50 30
1.08 1.12 1.01 1.14 1.05
Elefsiniotis and Li (2006) Christensson et al. (1994) Rostron et al. (2001) Vackova´ et al. (2011) This study
a
Sequencing batch reactor.
Fig. 7. SEM images of (a) biofilm (50), (b) molds growth, (c) crystals formation and (d) isolated strain.
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induces high conversion efficiency of substrate (wood) by the denitrifying bacteria in the reactor operation. The nitrate removal efficiencies showed an exponential trend, consistent with trends observed in a sawdust filled column study for nitrate removal in septic systems (Robertson, 2010). The effect of temperature on the rate of nitrate removal could be expressed by means of temperature coefficient (Ø). The temperature dependence of rate of nitrate removal could be described by using Arrhenius-type Eq. (2) (Sperling, 2007). Results obtained at this study were variable coefficient factors with comparing the rate of removal at 4 and 20 8C. The coefficient factors of 0.8 and 1.05 have been reported in studies with a temperature change from 4 and 20 8C. Other studies also refer to Table 4, at temperatures range between 15 and 20 8C, however the coefficient values showed to be near to 1.14, regardless of the type of reactor used. 3.6. Microscopic studies At the end of the 90 days operation the woodchip samples were collected from the bottom of the reactor for the measurement of its characteristics. Biofilm formation and molds growth were observed under scanning electron microscope (SEM, 50,000). The SEM examination of wood chips showed a white color mate formation with large cavities which were present on the surface (Fig. 7(a) and (b)) of woodchips. Fig. 7(c) shows the presence of some inorganic crystals (phosphorus, calcium and iron) which played an important role in formation of biofilm on support media. Inorganic crystals were stimulated by the alkaline pH due to the degradation of acidic substances in the wastewater (Sreekanth et al., 2009). Fig. 7(d) also revealed that morphological examination (under light microscope) of the culture acquired from WBR were visualized as slightly bent, scattered, long chain rod shaped (predominant; streptobacillus; 10–20 mm in length) bacteria present in the reactor. These bacteria images showed comparatively similar morphology of denitrifying group bacteria proliferated in the WBR for removal of nitrates. The most important denitrifying bacteria are Micrococcus denitrificans, Thiobacillus denitrificans, and species of Pseudomonas, Bacillus, Paracoccus denitrificans (Robertson et al., 1995) etc. This species reduces NO3 even in the presence of a saturating concentration of O2. More recent studies of aerobic denitrifiers have revealed some novel species, such as Thaurea mechernichensis and Microvirgula aerodenitrificans; the former organism denitrifies as efficiently as P. denitrificans under aerobic conditions (Naoki et al., 2003). 4. Conclusion The conclusions drawn by the study of this four level biofilm configured WBR system for removal of NO3-N using synthetic tile water. The NO3-N removal efficiency was found to be dependent on the applied volumetric loading rate (VLR) and biomass concentration in the reactor. Increase in VLR and biomass concentration showed a marked drop in NO3-N removal efficiency. The pH variation during NO3-N removal efficiency was in
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the range of 6.5–7.5, which was favorable for effective function of denitrifying bacteria. The adopted WBR configuration, accelerated start-up study and operating conditions along with VLR have influenced on the overall NO3-N removal efficiency. This study could provide some valuable information in order to design a more effective NO3-N removal efficiency system from agricultural drainage. Conflict of interest None declared. Financial disclosure None declared. Acknowledgements The authors acknowledge the financial support from the Department of Civil Engineering, Kansas State University, USA for carrying out this research work in the form of project and also to the Department of Science and Technology (DST), Govt. of India, to provide BOYSCAST Fellowship to Dr. Umesh Kumar Singh. References APHA, 1998. Standard Methods for Examination of Water and Wastewater, 19th ed. American Public Health Association, Washington. Auria, R., Morales, M., Villegas, E., Revah, S., 1993. Influence of mold growth on the pressure drop in aerated solid state fermenters. Biotechnol. Bioeng. 41, 1007–1013. Beman, J.M., Arrigo, K.R., Matson, P.A., 2005. Agricultural runoff fuels large phytoplankton blooms in vulnerable areas of the ocean. Nature 434, 211–214. Cameron, S.C., Schipper, L.A., 2010. Nitrate removal and hydraulic performance of carbon substrates for potential use in denitrification beds. Ecol. Eng. 36, 1588–1595. Catarina, T., Catarina, M., Rui, A.R.B., Adriano, A.B., 2010. Potential rates and environmental controls of denitrification and nitrous oxide production in a temperate urbanized estuary. Mar. Environ. Res. 70, 336– 342. Christensson, M., Lie, E., Welander, T., 1994. A comparison between ethanol and methanol as carbon sources for denitrification. Water Sci. Technol. 30, 83–90. Elefsiniotis, P., Li, D., 2006. The effect of temperature and carbon source on denitrification using volatile fatty acids. Biochem. Eng. J. 28, 148–155. Fernando, M.S., Brent, E.S., Grant, A., 2001. Effects of biomass growth on gas pressure drop in biofilters. J. Environ. Eng. 127, 1–9. Flores, L., Winans, S.C., Holguin, G., 2007. Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots. Appl. Environ. Microbiol. 73, 7308–7321. Gee-Bong, H., Jai-koo Park, M., 2012. NO3-N removal with sulfur-lime porous ceramic carrier (SLPC) in the packed-bed bioreactors by autosulfurotrophic denitrification. J. Taiwan Inst. Chem. Eng. 43, 591–596. Gibert, O., Pomierny, S., Rowe, I., Kalin, R.M., 2008. Selection of organic substrates as potential reactive materials for use in a denitrification permeable reactive barrier (PRB). Bioresour. Technol. 99, 7587–7596. Guilherme, P., Nora, K.S., Varesche, B.A.M., Marcelo, Z., 2011. Hydrogen production from soft-drink wastewater in an up flow anaerobic packed-bed reactor. Int. J. Hydrogen Energy 36, 8953–8966. Ines, V., Nico, B., Paul De, V., Kim, H., 2011. Denitrification is a common feature among members of the genus Bacillus. Syst. Appl. Microbiol. 34, 385–391. Ling, X., Li, J., Qing, Y., Shuying, W., Bin, M., Yongzhen, P., 2012. Biomass characteristics and simultaneous nitrification–denitrification under long sludge retention time in an integrated reactor treating rural domestic sewage. Bioresour. Technol. 119, 277–284.
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Maria Osiadacz, et al., 2010. Successful treatment systems for removing nitrate concentrations from agricultural runoff. The Watershed Institute, Division of Science and Environmental Policy, California State University Monterey Bay. Publication No. WI2010-07. Mark, G.H., Tristan, G., Ibrahim, G.J., Lanigan, J.S., Owen, F., 2012. Nitrate removal rate, efficiency and pollution swapping potential of different organic carbon media in laboratory denitrification bioreactors. Ecol. Eng. 40, 198–209. Mathava Kumar, Jih-Gaw, L., 2010. Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal – strategies and issues. J. Hazard. Mater. 178, 1–9. Mohseni, M., Allen, D.G., Nichols, K.M., 1998. Biofiltration of a-pinene and its application to the treatment of pulp and paper air emissions. TAPPI J. 81, 205–211. Moorman, T.B., Parkin, T., Kaspar, T.C., Jaynes, D.B., 2010. Denitrification activity, wood loss, and N2O emissions over 9 years from a wood chip bioreactor. Ecol. Eng. 36, 1567–1574. Naoki, T., Maria, A.B., Catalan, S., Yasushi, S., Isao, K., Zhemin, Z., Hirofumi, S., 2003. Aerobic denitrifying bacteria that produce low levels of nitrous oxide. Appl. Environ. Microbiol. 69, 3152–3157. Natasha, L.H., Alok, B., Michelle, L., Soupir, Matthew, H., Tom, M., 2012. Denitrification woodchip bioreactor two-phase column study: evaluation of nitrate removal at various hydraulic retention times and effect of temperature on denitrification rates (Unpublished results). Okkerse, W., Ottengraf, S.P., Osinga-Kuipers, B., Okkerse, M., 1999. Biomass accumulation and clogging in biotrickling filters for waste gas treatment evaluation of a dynamic model using dichloromethane as a model pollutant. Biotechnol. Bioeng. 63, 418–430. Ovez, B., Ozgen, S., Yuksel, M., 2006. Biological denitrification in drinking water using Glycyrrhiza glabra and Arunda donax as the carbon source. Process Biochem. 41, 1539–1544. Ping, J.L., Jo, S.C., Lee, H.Y., Chiu, Y., Shu, Y.W., Kuo, S.L., 2011. Enhancing the performance of pilot-scale fermentative hydrogen production by proper combinations of HRT and substrate concentration. Int. J. Hydrogen Energy 36, 14289–14294.
Robert, G.M., Othman, A., 2009. Simple models for the release kinetics of dissolved organic carbon from woody filtration media. Bioresour. Technol. 100, 2588–2593. Robertson, W.D., 2010. Rates of nitrate removal in woodchip media of varying age. Ecol. Eng. 36, 1581–1587. Robertson, L.A., Dalsgaar, T., Revsbech, N.P., Kuenen, J.G., 1995. Confirmation of aerobic denitrification in batch cultures, using gas chromatography and 15N mass spectrometry. FEMS Microbiol. Ecol. 18, 113–120. Rostron, W.M., Stuckey, D.C., Young, A.A., 2001. Nitrification of high strength ammonia wastewaters: comparative study of immobilisation media. Water Res. 35, 1169–1178. Schipper, L.A., Robertson, W.D., Gold, A.J., Jaynes, D.B., Cameron, S.C., 2010. Denitrifying bioreactors – an approach for reducing nitrate loads to receiving waters. Ecol. Eng. 36, 1532–1543. Sperling, M.V., 2007. Activated Sludge and Aerobic Biofilm Reactors. IWA Publishing, London. Sreekanth, D., Sivaramakrishna, D., Himabindu, V., Anjaneyulu, Y., 2009. Thermophilic degradation of phenolic compounds in lab scale hybrid up flow anaerobic sludge blanket reactors. J. Hazard. Mater. 164, 1532–1539. Swanson, W.G., Loehr, R.C., 1997. Biofiltration: fundamentals, design and operations principles, and applications of biological APC technology. J. Environ. Eng. 123, 538–546. Vackova´, L., Martin, Srb., Radek, S., Jiri, W., 2011. Comparison of denitrification at low temperature using encapsulated Paracoccus denitrificans, Pseudomonas fluorescens and mixed culture. Bioresour. Technol. 102, 4661–4666. Xu, Z., Shao, L., Yin, H., Chu, H., Yao, Y., 2009. Biological denitrification using corncobs as a carbon source and biofilm carrier. Water Environ. Res. 81, 242–247. Yimin, S., Mehdi, N., 2012. Evaluation of sulfur-based autotrophic denitrification and denitritation for biological removal of nitrate and nitrite from contaminated waters. Bioresour. Technol. 114, 207–216. Yusoff, Z.M., Rahman, N.A., Hassan, M.A., Shirai, Y., 2010. The effect of hydraulic retention time and volatile fatty acids on biohydrogen production from palm oil mill effluent under non-sterile condition. Aust. J. Basic Appl. Sci. 4, 577–587.
Contents lists available at ScienceDirect
Ecohydrology & Hydrobiology journal homepage: www.elsevier.com/locate/ecohyd
Original Research Article
Denitrification in biofilm configured horizontal flow woodchip bioreactor: effect of hydraulic retention time and biomass growth Sivaramakrishna Damaraju a,*, Umesh Kumar Singh a,b, Desi Sreekanth c, Alok Bhandari a a b c
Department of Civil Engineering, Kansas State University, Manhattan, KS 66503, USA ISERC, Visva-Bharati University, Santhinikethan 731235, India Department of Botany, Osmania University, Hyderabad 500007, Telangana, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 February 2014 Accepted 19 November 2014 Available online 5 December 2014
Removal of nitrates and nitrogen (NO3-N) from surface and groundwater was a matter of special importance, and there was a need for sustaining the public health problems and aquatic life related to the presence of NO3-N in drinking water supplies. The influence of hydraulic retention time (HRT), volumetric loading rates (VLRs) and temperature on denitrification were investigated in a biofilm configured woodchip bioreactor (WBR) from synthetic tile water. Three HRTs were examined, ranging from 4 to 12 h. The denitrification removal efficiency (RE) >99% was obtained in WBR operated at 8-h HRT. The WBR was operated with VLR ranging from 49 to 198 g NO3-N/L/day for denitrification study. The maximum load reduction of the WBR showed 99% in second level of reactor operation with biomass concentration of 12.3 g/L. The pH (6.5–7.2) and temperature (17–20 8C) under the unique study profoundly impact on denitrification. The coefficient factors of 0.8 and 1.05 have been reported in studies with a temperature change from 4 to 20 8C. Scanning electron microscope (SEM) analysis documented formation of biofilm on woodchips in each level of reactor operation study. Therefore we recommend from this study, that the horizontal flow based WBR to enhance denitrification while minimizing higher operating conditions in denitrification beds. ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Denitrification Biomass Nitrate HRT Temperature Scanning electron microscope
1. Introduction Nitrate-nitrogen (NO3-N) concentration in aquatic environments has been increasing in recent years as a result of the intensive application of nitrogen fertilizers
* Corresponding author at: Department of Civil Engineering, 2118 Fiedler Hall, Kansas State University, Manhattan, KS 66506, USA. Tel.: +1 785 340 5969. E-mail address: [email protected] (S. Damaraju).
and animal manure to agricultural land, septic tanks, and land disposal of wastes (Gee-Bong and Jai-koo Park, 2012). Nationwide studies have detected nitrate levels in water below agricultural fields to be in the range of 5–100 mg/L, with regular sampling between 20 and 40 mg/L (Maria Osiadacz et al., 2010). Accumulation of various forms of nitrogen compounds in ground and surface water bodies, could lead to adverse effects including eutrophication in receiving waters, ammonia toxicity to aquatic life, and public health problems related to the presence of NO3-N in drinking water supplies (Elefsiniotis and Li, 2006).
http://dx.doi.org/10.1016/j.ecohyd.2014.11.001 1642-3593/ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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Therefore, the removal of nitrogen compounds from wastewater would be of increasing importance. Treatment of waters contaminated by nitrate or nitrite could be achieved through physicochemical or biological processes. Physicochemical methods such as ion exchange and reverse osmosis, however, were not selective and resulted in secondary wastes with high levels of nitrate or nitrite. Conventional biological processes for the removal of nitrate and nitrite were referred to as denitrification (Yimin and Mehdi, 2012). Under anoxic microenvironment conditions, heterotrophic denitrifying bacteria Bacillus (Flores et al., 2007) and Corynebacterium (Ines et al., 2011) convert nitrate (NO3) or nitrite (NO2) into gaseous forms, either dinitrogen (N2) or nitrous oxide (N2O). This microbialmediated process plays a particularly important role in nitrogen-enriched coastal systems, since it diminishes the amount of fixed nitrogen transported to the ocean, and consequently reduces the nitrogen available for primary production (Beman et al., 2005; Catarina et al., 2010). As a heterotrophic process, denitrification tends to be less sensitive to environmental parameters than, for example, nitrification; however, it is still affected by variations in carbon source, dissolved oxygen (DO), pH, temperature, presence or accumulation of NO2 and NH4+ during the process (Xu et al., 2009; Cameron and Schipper, 2010). The availability of organic carbon source was important in heterotrophic denitrification. The characteristics of the added carbon sources have been found to have major effects on important parameters of the denitrification process such as the denitrification rate, COD demand, biomass yield and biomass composition (Schipper et al., 2010). For these reasons, denitrification woodchip-based bioreactors were of low cost with high C/N ratio and thus come under a relatively new technology for edge of field removal of nitrates (Gibert et al., 2008). These engineered denitrification systems provide an environment that supports the growth of denitrifying bacteria, and compensate for the reduced interaction of tile drainage with natural biological soil processes. In addition, they do not require replenishment as C is not rapidly depleted from them, although the duration of their effectiveness will be affected by the longevity of the C supply to the denitrifying microorganisms (Moorman et al., 2010). In wastewater treatment applications include using wood based bioreactors in subsurface barriers to treat storm water runoff, acid mine drainage and groundwater contamination. This water treatment was achieved through the well studied ability of woody organics to sorb dissolved petroleum hydrocarbons as well as metals (Robert and Othman, 2009) and the degradation of chlorinated and aromatic hydrocarbons. In a bioreactor, the synthesis of microbial mass (biomass) by the mineralization of biodegradable pollutants leads to biomass growth and accumulation over time, its effect on the denitrification removal rate (Mohseni et al., 1998). Biomass accumulation, which was greater at the inlet sections of bioreactor lead to a change in bed characteristics, such as the reduction of the inter particle void space and the compaction of natural packing materials, which caused channeling and increased pressure drops. This translates into higher operating and
maintenance costs that become significant for long-term bioreactor operation (Swanson and Loehr, 1997). This study aimed to develop optimal operation strategies for an efficient and stable performance of NO3-N removal in the four step level biofilm configured wood chip bioreactor (WBR). The information obtained from this study was expected to provide basic knowledge for the bioreactor design of practical bioprocesses capable of long-term NO3N removal from wastewaters. The effect of HRT on the performance of WBR treating synthetic tile water containing high concentration of NO3-N (15–50 mg/L) was also investigated. Furthermore, the attached biomass developed in the reactors was characterized using SEM analysis. 2. Methods and materials 2.1. Bioreactor Bench scale biofilm configuration anoxic woodchip bioreactor (WBR) was fabricated in the laboratory using acrylic material with a working volume of 7 L, liquid volume of 4 L and diameter of 7 in. (17.8 cm), and a height of 20 in. (50.8 cm). In the bioreactor system, perforated acrylic plates were fit at each end of the reactors to diffuse the flow of fluid into the columns. Four inlet and outlet ports were evenly fixed over the end of the reactor. The WBR was operated in continuous flow mode. The influent pH was kept in the range of 6.5–7.5 by addition of 0.1 N NaOH or HCl. The volumetric flow rate of the feed in reactor was adjusted using three Masterflex (C/L 77122-22) variable speed peristaltic pumps. The reactors were operated with a hydraulic retention time (HRT) of 4– 12 h, based on working volumes of the four levels. The temperature was maintained at ambient condition. Schematic details of the horizontal flow WBR used in this study along with photograph of the experimental setup were depicted in Fig. 1. 2.2. Support matrices and carbon source The reactor was packed with Standard hardwood landscape woodchips (Golden Valley Hardscapes, located in Story City, Iowa) which were used to support the cell growth. The bed zone of each reactor was packed with 192 g woodchips/L, which resulted in bed porosities of 48%. Wood chips cut into small cylindrical shapes with average diameters of 11.2 mm and heights of approximately 24 mm were used. 2.3. Synthetic tile water The concentrations of the NO3-N in synthetic tile water shown in Table 1 were very similar to the characteristics of agricultural runoff. The synthetic tile water was pumped directly from the influent container to each column. Electrical conductivity of the prepared synthetic tile drainage was monitored and maintained within a range consistent with values observed under average field conditions. Potassium chloride (KCl) was added to the influent solution to maintain an electrical conductivity within a range observed in tile drainage (600–800 mS). The
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Fig. 1. (a) Photograph of a wood packed denitrification bioreactor. (b) Schematic of bioreactor end-plate. (c) Schematic of diffuser column bioreactor. The schematic images are modified from work done by L. Christianson.
influent nitrate nitrogen concentration varies from 15 to 50 mg/L. 2.4. Wastewater-Manhattan The wastewater collected from the local wastewater treatment plant in Manhattan was additionally seeded into the reactor. Characteristically, the wastewater was highly biodegradable (BOD/COD / 0.6). The wastewater was pumped into reactors and operated for 25 days to ensure adequate contact time between the wastewater and the woodchips for attachment of the biomass enhancing the growth of denitrifying microflora on media. The wastewater characteristics were given in Table 2.
filtrate was analyzed by a Cd-reduction method for NO3N + NO2-N (Lachaat Quick – Chem 20 Genesis spectrophotometer). Volatile suspended solids (VSS), COD, pH, ORP, DO and conductivity were measured according to Standard Methods (APHA, 1998). Oxidation–reduction potential (ORP) and pH values were determined by a pH meter (Corning 450, USA). Dissolved oxygen (DO) was measured by a DO meter (YSI 550A, USA). Electrical conductivity was measured using a Fisher Scientific accumet1 AR 20 conductivity meter. The biofilm formed on the woodchips and dominant colonies were subjected to scanning electron microscopy (SEM) and light microscope. Each experimental condition was carried out in triplicate.
2.5. Analytical methods
3. Results and discussions
The samples collected at periodic time intervals were filtered through a 0.45 ml Millex filter, and nitrate in the
3.1. Start-up performance of woodchip bioreactor
Table 1 Constituents of designed synthetic wastewater used in this study. Constituents
Concentration (mg/L)
CaCl22H2O CoCl26H2O MgSO47H2O MnCl24H2O CuSO45H2O FeCl36H2O KH2PO4 ZnCl2 (NH4)6Mo7O244H2O NaCl NH4+-N NaNO3
1.2 0.1 4.3 0.04 0.03 1.21 8.2 0.03 0.04 13.5 0.2–1.4 15–50
In bioreactor accelerated startup process, the reactor was initially operated with wastewater as supply of additional heterotrophic bacteria along with woodchips at strength of COD 2.1–2.4 g/L and the concentrations of Table 2 Characteristics of wastewater used as feed for startup study. Parameters pH COD (mg/L) BOD5 (mg/L) Chlorides (mg/L) NH4+-N Nitrate-nitrogen (mg/L) Microbial composition (CFU/ml)
7.2 2182 1316 48 0.2–1.2 13 1.2 102
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influent 13 mg/L NO3-N with an HRT of 24 h during the steady period of 26 days. Table 3 shows the daily variations in pH, ORP, COD removal and NO3-N removal efficiency during the course of acclimatization period. Before reaching the stable reactor performance, the pH, COD removal and NO3-N removal efficiency fluctuated greatly in the first 8 days (Table 3). NO3-N and COD removal efficiency reached up to 48% and 58% respectively on the 8th day, but gradually decreased to 23% and 36% on the 13th day with effluent concentrations of 10.3 mg/L NO3-N and 0.88 g COD/L. After 14 days, NO3-N and COD removal efficiency increased reaching 98% and 84% on the 20th day. Based on the removal efficiency of NO3-N and COD in WBR, there were three stages for acclimatization; with 1–13 days being lag stage, 14–20 days of acclimatization and 21–26 days as stabilized stage. The NO3-N removal efficiency in reactor kept increasing at the lag phase (1–13 days); Table 3 indicates that the denitrifying bacteria started adapting to the woodchip reactor system. Both NO3-N and COD removal efficiency percentage started to increase significantly from the 15th day and stabilized on the 20th day. During the stabilized stage (21–26 days), NO3-N and COD removal efficiency was 98% and 87% in the reactor. This high NO3-N removal efficiency was possible in start-up period due to the large column length (L/D > 4) which provides long flowing pathway during NO3-N from inlet to outlet. The VSS concentration declined during the start-up period and became the lowest on the 5th day with 2.4 g VSS/L. Afterwards, it gradually increased to 5.8 g VSS/L on the 14th day in the WBR and stabilized in the later period. This horizontal flow WBR retained a high level of VSS which was far higher than the average VSS concentrations in other bioreactor studies (Guilherme et al., 2011, Ling et al., 2012). The pH values of the effluents were maintained between 6.8 and 7.2 indicating that pH was maintained stably throughout the experiment. This was the optimum pH range for denitrifying bacteria in WBR systems. The ORPs of the effluents were between 28 and 71 mV (Table 3) and DO values were below 2 mg/L. Based on the measurement of NO3-N removal efficiency, pH and ORP values (Table 3), the WBR system reached a stabilized stage on the 20th day. However, biomass concentration (VSS) showed a different trend. These experiments revealed that wastewater addition in start-up process could influence anoxic WBR which possessed a quite stable and good NO3-N removal efficiency ability, with a good acclimatization.
3.2. Effect of HRT on denitrification It was apparent from Fig. 2(a)–(d) that for NO3-N percent removal, various HRTs in continuous WBR operation mode were obtained under steady-state conditions for the four levels of reactor study. The NO3-N concentrations were increased to 14.8 1.28, 31 0.56 and 52 1.2 mg/L under various HRTs increased from 4 h to 8 h to 12 h respectively. The same trend of NO3-N concentration profiles were followed in each level of reactor study. As illustrated in Fig. 2(a), in first level of reactor study, influent and effluent NO3-N concentrations were 50 mg/L and 20.3 mg/L with 59.3% NO3-N removal at an HRT of 4h, respectively. The NO3-N percent removal was gradually increased with increasing HRT at 8-h, which was operated at the same influent concentration at 50 mg/L resulted in complete (>99%) removal of NO3-N in effluent profiles respectively. Further increasing the HRT from 8 to 12 h with influent concentration of 50 mg/L, resulted in (>99%) removal of NO3-N similar to that at HRT 8 h. Our results proved that NO3-N removal efficiency was positively related with HRT. This was possible considering that more substrate was solubilized and consumed under the longer HRTs (Ping et al., 2011). According to Yusoff et al. (2010) who reported that, at lower HRTs, the biomass was washed-out and therefore the denitrifying bacteria had a short period of contact time with surfaces of woodchips (Natasha et al., 2012). The reduced physical contact of the solution may have limited the percent of nitrate removal possible at lower HRTs. From this view point of denitrification, at 8 h HRT was found to be more suitable operation condition than HRT 4 h for the woodchip reactor. As shown in Fig. 2(b), the system commenced at HRT 4 h and was then adjusted to 8 h until steady state condition was reached in second level of reactor operation. In the beginning HRT 4 h, influent concentration NO3-N with 50 mg/L resulted in effluent NO3-N concentration of 28 mg/L, demonstrating a 56% mean removal in effluent profiles respectively. Further increasing HRT from 4 to 8 h, the influent NO3-N percent removal at this HRT was complete (>99%) in effluent NO3-N concentrations. So Fig. 2(a) and (b), illustrates that second level experiments showed more stable and fast removal of NO3-N and cumulative HRT days were shorter (20 days) in comparison with first level (26 days) of reactor experiments. This may be due to the denitrifying microflora density increasing significantly as a result of the formation of sufficient amount of biomass in the second level reactor study.
Table 3 Operating conditions during the accelerated start-up of WBR treating wastewater. Days
COD (g/L/day)a
NO3-N (mg/L/day)
COD reduction (%)
NO3-N removal (%)
VSS (g/L)b
pH
ORP (mV)c
1–8 8–13 13–20 20–26
2.1 2.0 2.1 2. 0
13 13 13 13
16–58 28–36 58–84 84–87
21–48 12–23 92–98 94–98
2.4 5.8 5.8 5.8
6.4–6.6 6.8–7.2 6.9–7.0 6.8–6.9
28 52 71 69
a b c
Chemical oxygen demand. Volatile suspended solids. Oxidation–reduction potential.
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Fig. 2. The effect of HRT on NO3-N percent removal in four levels of reactor operation.
From Fig. 2(c), the NO3-N removal efficiency in third level at HRT 4 h, with influent concentrations at 15, 30 and 50 mg/L resulted in effluent concentrations of 11.3 mg/L, 24.3 mg/L and 44 2 mg/L, which showed 24 2%, 17 1% and 11 2% mean removal efficiencies. Then the HRT were set at 8 h and 12 h with the same influent concentration, so that the effluent NO3-N removal efficiencies increased to 15 mg/L (>98%), 30 mg/ L (52%) and 50 mg/L (21%) respectively. Fig. 2(d) depicts the influent concentration of NO3-N (15, 30 and 50 mg/L) and percent removal (17%, 11% and 8%) at an HRT of 4, 8 and 12h, which indicated low NO3-N removal efficiency at fourth level reactor operation study. This was due to the excessive formation of biomass growth in the third and fourth of the reactor operation, resulting in the uneven flow of influent NO3-N concentrations between woodchips in the reactor and also higher flow rate and loading rate at third and fourth levels causing the bacterial community being washed out. Media (woodchips) in the reactor may have swollen due to absorption of water, decreasing the inter particle porosity. Ovez et al. (2006) reported that, for higher NO3-N removal efficiencies, experiments may need to be operated under high HRTs which would end up with the utilization of larger reactors, since the
available organic compounds in the substrates were decreasing with time. 3.3. Effect of volumetric loading rate (VLR) on denitrification and biomass growth The combined effects of both NO3-N concentration and HRT on denitrification could be represented by volumetric loading rate (VLR). Variations of VLR with mass load reductions were shown in Fig. 3(a)–(d). Referring to the results, during first and second level reactor operation, highest total percent load reductions at greater than 59 3% and 99 1% at optimal HRT 12 h with bioreactor influent VLR ranged from 49 to 94.9 g NO3-N/L/ day. From the third and fourth level reactor studies, the average load reductions dropped to 24 2% and 13 2% at VLR which ranged from 138 to 173 g NO3-N/L/day with effluent loads of 52 4 and 150 2 g NO3-N/L/day. These results made clear that when influent loads exceeded approximately 140 g NO3-N/L/day in reactor, the load reductions performance in this study began to decline greatly. Using higher VLR could have led to a higher load reduction rate, but much more VLR would result in inhibition of load reduction activity.
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Fig. 3. The effect of volumetric loading rate on NO3-N load reductions in four levels of reactor operation.
The above correlation between load reductions could be explained by the reason that, the growth of biomass (VSS) was closely associated with VLR, and was also influenced greatly by packed bed volume occupied in each level of reactor. As illustrated in Fig. 4, in first and second level reactor bed volume, the biomass concentration in the reactor was maintained between 7.2 and 15 g VSS/L during the first 75 days, while in third and fourth studies the concentration of biomass increased from 5.8 g VSS/L at start-up to approximately 24.3 g VSS/L after 115 days of reactor operation and remained constant thereafter with an average value of 21.3 g VSS/L. Comparison of four levels of reactor volume operation, which worked at different VLRs (see Fig. 3) showed that the system with the higher VLR (third and fourth) levels could sustain more biomass; this was due to high VLRs and nutrient availability in excess of what was needed for denitrification. The inverse relationship between the biomass and load reductions emphatically reported that the higher biomass yield was attributed to different bacteria other than denitrifying bacteria. These results were in agreement with other studies (Fernando et al., 2001). Auria et al. (1993) found that pressure losses in a solid state fermenter packed with wheat bran and cane bagasse were also controlled by mold growth, although these supports underwent changes due to microbial growth. Higher decay rates were usually
reported for cells in biofilm systems than for cells in suspension, as stated by Okkerse et al. (1999); therefore, lower net yield coefficients were expected in biofilm systems. This showed that the performance of the WBR was effectively enhanced by applying favorable HRT, biomass growth and NO3-N concentration.
Fig. 4. Variations of biomass with time in the denitrification systems.
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3.4. Bioprocess monitoring Along with NO3-N removal efficiency, parameters such as pH, ORP and temperature were also monitored during the bioreactor operation to evaluate the bioprocess mechanism during denitrification. In this study from Fig. 5(a)–(d), the pH increased slightly from inlet to outlet in all four levels of reactor operation. In overall study, the optimal pH was maintained from 6.5 to 7.5. The above results depicted that, denitrifying microorganisms function effectively within a pH range from 6.0 to 8.0, with the microbial activity being at an optimum around neutral reported in other studies (Mathava Kumar and Jih-Gaw, 2010). Consequently, in order to improve the potential of the denitrification process, the initial pH in each level was adjusted to an approximate value of 6.5–6.8 for the WBR study. During denitrification process, ORP values and DO concentration reflect the oxidizing or reducing conditions in a reactor and were used to evaluate the direction of biological and chemical process in reactors. The average values of ORP were 35 8.2 (mV) to 70 18.4 (mV) which confirmed the anoxic condition in WBR. The dissolved oxygen (DO) results of WBR effluent also varied between 1.2 and 2 mg/L (data not shown) and
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consistently maintained the same concentration throughout the study period, which was lower than that of influent (6.2 mg/L) concentration. However, some reports showed that concentrations of up to 4.2 mg/L could facilitate denitrification (Mark et al., 2012). 3.5. Effect of temperature Operation temperature effect on the NO3-N removal percentage was investigated in a temperature range of 4– 20 8C, representing the actual average temperature change throughout a year, in order to identify the best temperature for NO3-N percent removal. Influent NO3-N concentrations from 28.3 to 31.8 mg/L, with a mean value of 30.42 0.8 mg/L were achieved at 4 8C and 20 8C with 8 h HRT operated at second level of reactor study. Fig. 6 shows that NO3-N removal at the controlled temperature of 4 8C was 15% with mean effluent concentration of 24.82 mg/L, respectively, after approximately 22 days of continuous operation. At 20 8C, NO3-N removal was relatively complete (effluent <1 mg L1) with optimum HRT of 8 h. Conclusively, increasing temperature gave rise to an increase in average cellular denitrification activity. This may be due to the fact that reaction at kinetically-favorable high temperatures
Fig. 5. Daily variations in pH and oxidation–reduction potential (ORP) in four levels of reactor operation.
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Fig. 6. Variations of NO3-N percent removal with temperature.
Table 4 Temperature coefficients for different study reactors. Temperature range (8C)
Reactor type
Initial NO3-N concentration (mg/L)
Temperature coefficient
Source
20–30 15–25 16–25 5–15 4–20
Erlenmeyer flasks SBRa SBR Shake flasks WBR
100 600 100 50 30
1.08 1.12 1.01 1.14 1.05
Elefsiniotis and Li (2006) Christensson et al. (1994) Rostron et al. (2001) Vackova´ et al. (2011) This study
a
Sequencing batch reactor.
Fig. 7. SEM images of (a) biofilm (50), (b) molds growth, (c) crystals formation and (d) isolated strain.
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induces high conversion efficiency of substrate (wood) by the denitrifying bacteria in the reactor operation. The nitrate removal efficiencies showed an exponential trend, consistent with trends observed in a sawdust filled column study for nitrate removal in septic systems (Robertson, 2010). The effect of temperature on the rate of nitrate removal could be expressed by means of temperature coefficient (Ø). The temperature dependence of rate of nitrate removal could be described by using Arrhenius-type Eq. (2) (Sperling, 2007). Results obtained at this study were variable coefficient factors with comparing the rate of removal at 4 and 20 8C. The coefficient factors of 0.8 and 1.05 have been reported in studies with a temperature change from 4 and 20 8C. Other studies also refer to Table 4, at temperatures range between 15 and 20 8C, however the coefficient values showed to be near to 1.14, regardless of the type of reactor used. 3.6. Microscopic studies At the end of the 90 days operation the woodchip samples were collected from the bottom of the reactor for the measurement of its characteristics. Biofilm formation and molds growth were observed under scanning electron microscope (SEM, 50,000). The SEM examination of wood chips showed a white color mate formation with large cavities which were present on the surface (Fig. 7(a) and (b)) of woodchips. Fig. 7(c) shows the presence of some inorganic crystals (phosphorus, calcium and iron) which played an important role in formation of biofilm on support media. Inorganic crystals were stimulated by the alkaline pH due to the degradation of acidic substances in the wastewater (Sreekanth et al., 2009). Fig. 7(d) also revealed that morphological examination (under light microscope) of the culture acquired from WBR were visualized as slightly bent, scattered, long chain rod shaped (predominant; streptobacillus; 10–20 mm in length) bacteria present in the reactor. These bacteria images showed comparatively similar morphology of denitrifying group bacteria proliferated in the WBR for removal of nitrates. The most important denitrifying bacteria are Micrococcus denitrificans, Thiobacillus denitrificans, and species of Pseudomonas, Bacillus, Paracoccus denitrificans (Robertson et al., 1995) etc. This species reduces NO3 even in the presence of a saturating concentration of O2. More recent studies of aerobic denitrifiers have revealed some novel species, such as Thaurea mechernichensis and Microvirgula aerodenitrificans; the former organism denitrifies as efficiently as P. denitrificans under aerobic conditions (Naoki et al., 2003). 4. Conclusion The conclusions drawn by the study of this four level biofilm configured WBR system for removal of NO3-N using synthetic tile water. The NO3-N removal efficiency was found to be dependent on the applied volumetric loading rate (VLR) and biomass concentration in the reactor. Increase in VLR and biomass concentration showed a marked drop in NO3-N removal efficiency. The pH variation during NO3-N removal efficiency was in
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the range of 6.5–7.5, which was favorable for effective function of denitrifying bacteria. The adopted WBR configuration, accelerated start-up study and operating conditions along with VLR have influenced on the overall NO3-N removal efficiency. This study could provide some valuable information in order to design a more effective NO3-N removal efficiency system from agricultural drainage. Conflict of interest None declared. Financial disclosure None declared. Acknowledgements The authors acknowledge the financial support from the Department of Civil Engineering, Kansas State University, USA for carrying out this research work in the form of project and also to the Department of Science and Technology (DST), Govt. of India, to provide BOYSCAST Fellowship to Dr. Umesh Kumar Singh. References APHA, 1998. Standard Methods for Examination of Water and Wastewater, 19th ed. American Public Health Association, Washington. Auria, R., Morales, M., Villegas, E., Revah, S., 1993. Influence of mold growth on the pressure drop in aerated solid state fermenters. Biotechnol. Bioeng. 41, 1007–1013. Beman, J.M., Arrigo, K.R., Matson, P.A., 2005. Agricultural runoff fuels large phytoplankton blooms in vulnerable areas of the ocean. Nature 434, 211–214. Cameron, S.C., Schipper, L.A., 2010. Nitrate removal and hydraulic performance of carbon substrates for potential use in denitrification beds. Ecol. Eng. 36, 1588–1595. Catarina, T., Catarina, M., Rui, A.R.B., Adriano, A.B., 2010. Potential rates and environmental controls of denitrification and nitrous oxide production in a temperate urbanized estuary. Mar. Environ. Res. 70, 336– 342. Christensson, M., Lie, E., Welander, T., 1994. A comparison between ethanol and methanol as carbon sources for denitrification. Water Sci. Technol. 30, 83–90. Elefsiniotis, P., Li, D., 2006. The effect of temperature and carbon source on denitrification using volatile fatty acids. Biochem. Eng. J. 28, 148–155. Fernando, M.S., Brent, E.S., Grant, A., 2001. Effects of biomass growth on gas pressure drop in biofilters. J. Environ. Eng. 127, 1–9. Flores, L., Winans, S.C., Holguin, G., 2007. Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots. Appl. Environ. Microbiol. 73, 7308–7321. Gee-Bong, H., Jai-koo Park, M., 2012. NO3-N removal with sulfur-lime porous ceramic carrier (SLPC) in the packed-bed bioreactors by autosulfurotrophic denitrification. J. Taiwan Inst. Chem. Eng. 43, 591–596. Gibert, O., Pomierny, S., Rowe, I., Kalin, R.M., 2008. Selection of organic substrates as potential reactive materials for use in a denitrification permeable reactive barrier (PRB). Bioresour. Technol. 99, 7587–7596. Guilherme, P., Nora, K.S., Varesche, B.A.M., Marcelo, Z., 2011. Hydrogen production from soft-drink wastewater in an up flow anaerobic packed-bed reactor. Int. J. Hydrogen Energy 36, 8953–8966. Ines, V., Nico, B., Paul De, V., Kim, H., 2011. Denitrification is a common feature among members of the genus Bacillus. Syst. Appl. Microbiol. 34, 385–391. Ling, X., Li, J., Qing, Y., Shuying, W., Bin, M., Yongzhen, P., 2012. Biomass characteristics and simultaneous nitrification–denitrification under long sludge retention time in an integrated reactor treating rural domestic sewage. Bioresour. Technol. 119, 277–284.
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S. Damaraju et al. / Ecohydrology & Hydrobiology 15 (2015) 39–48
Maria Osiadacz, et al., 2010. Successful treatment systems for removing nitrate concentrations from agricultural runoff. The Watershed Institute, Division of Science and Environmental Policy, California State University Monterey Bay. Publication No. WI2010-07. Mark, G.H., Tristan, G., Ibrahim, G.J., Lanigan, J.S., Owen, F., 2012. Nitrate removal rate, efficiency and pollution swapping potential of different organic carbon media in laboratory denitrification bioreactors. Ecol. Eng. 40, 198–209. Mathava Kumar, Jih-Gaw, L., 2010. Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal – strategies and issues. J. Hazard. Mater. 178, 1–9. Mohseni, M., Allen, D.G., Nichols, K.M., 1998. Biofiltration of a-pinene and its application to the treatment of pulp and paper air emissions. TAPPI J. 81, 205–211. Moorman, T.B., Parkin, T., Kaspar, T.C., Jaynes, D.B., 2010. Denitrification activity, wood loss, and N2O emissions over 9 years from a wood chip bioreactor. Ecol. Eng. 36, 1567–1574. Naoki, T., Maria, A.B., Catalan, S., Yasushi, S., Isao, K., Zhemin, Z., Hirofumi, S., 2003. Aerobic denitrifying bacteria that produce low levels of nitrous oxide. Appl. Environ. Microbiol. 69, 3152–3157. Natasha, L.H., Alok, B., Michelle, L., Soupir, Matthew, H., Tom, M., 2012. Denitrification woodchip bioreactor two-phase column study: evaluation of nitrate removal at various hydraulic retention times and effect of temperature on denitrification rates (Unpublished results). Okkerse, W., Ottengraf, S.P., Osinga-Kuipers, B., Okkerse, M., 1999. Biomass accumulation and clogging in biotrickling filters for waste gas treatment evaluation of a dynamic model using dichloromethane as a model pollutant. Biotechnol. Bioeng. 63, 418–430. Ovez, B., Ozgen, S., Yuksel, M., 2006. Biological denitrification in drinking water using Glycyrrhiza glabra and Arunda donax as the carbon source. Process Biochem. 41, 1539–1544. Ping, J.L., Jo, S.C., Lee, H.Y., Chiu, Y., Shu, Y.W., Kuo, S.L., 2011. Enhancing the performance of pilot-scale fermentative hydrogen production by proper combinations of HRT and substrate concentration. Int. J. Hydrogen Energy 36, 14289–14294.
Robert, G.M., Othman, A., 2009. Simple models for the release kinetics of dissolved organic carbon from woody filtration media. Bioresour. Technol. 100, 2588–2593. Robertson, W.D., 2010. Rates of nitrate removal in woodchip media of varying age. Ecol. Eng. 36, 1581–1587. Robertson, L.A., Dalsgaar, T., Revsbech, N.P., Kuenen, J.G., 1995. Confirmation of aerobic denitrification in batch cultures, using gas chromatography and 15N mass spectrometry. FEMS Microbiol. Ecol. 18, 113–120. Rostron, W.M., Stuckey, D.C., Young, A.A., 2001. Nitrification of high strength ammonia wastewaters: comparative study of immobilisation media. Water Res. 35, 1169–1178. Schipper, L.A., Robertson, W.D., Gold, A.J., Jaynes, D.B., Cameron, S.C., 2010. Denitrifying bioreactors – an approach for reducing nitrate loads to receiving waters. Ecol. Eng. 36, 1532–1543. Sperling, M.V., 2007. Activated Sludge and Aerobic Biofilm Reactors. IWA Publishing, London. Sreekanth, D., Sivaramakrishna, D., Himabindu, V., Anjaneyulu, Y., 2009. Thermophilic degradation of phenolic compounds in lab scale hybrid up flow anaerobic sludge blanket reactors. J. Hazard. Mater. 164, 1532–1539. Swanson, W.G., Loehr, R.C., 1997. Biofiltration: fundamentals, design and operations principles, and applications of biological APC technology. J. Environ. Eng. 123, 538–546. Vackova´, L., Martin, Srb., Radek, S., Jiri, W., 2011. Comparison of denitrification at low temperature using encapsulated Paracoccus denitrificans, Pseudomonas fluorescens and mixed culture. Bioresour. Technol. 102, 4661–4666. Xu, Z., Shao, L., Yin, H., Chu, H., Yao, Y., 2009. Biological denitrification using corncobs as a carbon source and biofilm carrier. Water Environ. Res. 81, 242–247. Yimin, S., Mehdi, N., 2012. Evaluation of sulfur-based autotrophic denitrification and denitritation for biological removal of nitrate and nitrite from contaminated waters. Bioresour. Technol. 114, 207–216. Yusoff, Z.M., Rahman, N.A., Hassan, M.A., Shirai, Y., 2010. The effect of hydraulic retention time and volatile fatty acids on biohydrogen production from palm oil mill effluent under non-sterile condition. Aust. J. Basic Appl. Sci. 4, 577–587.