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Effect of potassium on nitrate removal from groundwater in agricultural waste-based heterotrophic denitrification system
Science of the Total Environment 703 (2020) 134830
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Effect of potassium on nitrate removal from groundwater in agricultural waste-based heterotrophic denitrification system Haishuang Wang, Chuanping Feng ⇑, Yang Deng School of Water Resources and Environment, MOE Key Laboratory of Groundwater Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing 100083, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
+
The threshold of K concentration for
nitrate reduction was 229.78 ± 25.80 mg-K/L. 1.15–1.88 fold denitrification rate was improved under different K+ concentration. The evolution pathway and utilization sequence were revealed. Pseudomonas and Thiobacillus are the unique species in 229.78 ± 25.80 mgK/L.
a r t i c l e
i n f o
Article history: Received 23 July 2019 Received in revised form 11 September 2019 Accepted 3 October 2019 Available online 3 November 2019 Keywords: Potassium Denitrification Hydrolysis Nitrate Banana peel
a b s t r a c t Heterotrophic denitrification based on solid carbon sources has been widely investigated for nitrogen removal in recent years. In this study, the response of the heterotrophic denitrification process under different K+ concentrations was clarified. Additionally, the denitrification enhancement mechanism was revealed and resource utilization of agricultural waste was achieved. A series of batch tests were conducted to study the effect of different K+ concentrations on the denitrification performance, dissolved organic matter (DOM) dissolution and microbial community structure. Results demonstrate that the threshold of K+ concentration for the NO 3 -N and NO2 -N reduction rates were 229.78 ± 25.80 and 159.10 ± 24.60 mg-K/ L, respectively. Excitation-emission matrix (EEM) analysis identified the main DOM components associated with tyrosine-like, tryptophan-like and humic-like substances, as well as illustrated the evolutionary behavior and utilization of DOM. High throughput 16S rRNA gene sequencing indicates that a K+ concentration of 229.78 ± 25.80 mg-K/L exhibited the highest diversity of functional species associated with fermentation and denitrification. The genera Pseudomonas and Thiobacillus were the unique denitrifiers at this K+ concentration. The correlation of K+ concentration, DOM dissolution of different regions and microorganism structure were analyzed using correlation matrix and PCA, and the appropriate K+ concentration of different functional microorganisms survival was optimized by this analysis method. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction
⇑ Corresponding author. E-mail address: [email protected] (C. Feng). https://doi.org/10.1016/j.scitotenv.2019.134830 0048-9697/Ó 2019 Elsevier B.V. All rights reserved.
Nitrate pollution has become a serious issue in groundwater worldwide due to the increasing use of nitrogenous fertilizers; the discharge of domestic and industrial wastewater; and the leak-
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age of septic tanks (Jafari et al., 2015). Nitrate can induce a series of human diseases, including methemoglobinemia in infants and cancer in adults, and can endanger the ecological environment. Heterotrophic denitrification, a cost-effective method to remove nitrate from groundwater, has been extensively studied in recent years, while the characteristics of eutrophication are detrimental to denitrification in groundwater. Selection of suitable carbon sources and additional metal elements of suitable concentrations are important means to enhance the heterotrophic denitrification process. Current research on denitrification enhancement generally includes pretreatment of solid carbon sources (Feng et al., 2017) and the effects of trace elements (e.g. Mg, Cu, Fe, Na, Ca) (Wang et al., 2018). K+ has become a common metal detected in groundwater due to the large amount of K+-containing fertilizers used in agriculture (Melo et al., 2012) that seeps into the groundwater through the soil. Heterotrophic denitrification is a biological process, and K+ is an important parameter affecting the growth and removal of microorganisms (Cyplik et al., 2007). The accumulation of K+ within cells reportedly counteracts the high level of brine osmotic potential in which halophilic microorganisms live (Cyplik et al., 2007). In addition, K+ is a major essential macronutrient for biological growth and development (Sarikhani et al., 2016), playing an important role in plant cellular homeostasis by contributing to charge balance, osmotic adjustment and enzyme catalysis (Zhou et al., 2018). K+ can also enhance microbial activity by affecting potassium channel proteins and altering the intracellular and extraosseous osmotic pressure (Sarikhani et al., 2016). In recent decades, several studies have identified at least four different uses for K+: as a promoter for the anaerobic digestion process (Li et al., 2018); as an adsorbent for NH+4-N removal; as an anode material for K+ batteries (Liu et al., 2018); and as a necessary element for the growth of plants and animals (Zhou et al., 2018). Although, elemental potassium is a necessary macroelement for microorganisms, there is a lack of studies regarding the influence of K+ on biological processes, especially heterotrophic denitrification. Furthermore, the enhancement or inhibition of K+ on heterotrophic denitrification and its mechanisms are still unclear. K+ can help the denitrifier Haloferax survive unfavorable environmental conditions characterized by a high osmotic potential (Cyplik et al., 2007). In order to understand the mechanism of K+ on denitrification, it is necessary to study the functional effects of K+ during the denitrification process; explore the optimum K+ concentration for different functional denitrifying and fermenting bacteria; and reveal the relationship among different K+ concentrations, denitrification properties, DOM dissolution and functional species. Changes in DOM during composting can reflect the conversion process of organic matter. The reaction mechanism of the organic carbon source during the fermentation phase of the heterotrophic denitrification process is similar to that during the composting process (Chen et al., 2003; He et al., 2011). Therefore, in this study, the changes in DOM were used to characterize the change in organic matter during the experiment. Compared to the relatively wellcharacterized toxic effects of high concentrations of organic acids on the activity of microorganisms (Cheung et al., 2010), there is a lack of studies on the effects of K+ during DOM dissolution. The activity and abundance of functional species also play a crucial role in the rate and efficiency of denitrification. Furthermore, the optimal K+ concentration for functional species, including denitrifiers and fermentative bacteria, remains unknown. In this study, the mechanism of nitrate removal enhancement was studied. Banana peel, a typical agricultural waste product, was selected to evaluate the performance of denitrification because it has almost all the characteristics of agricultural waste (further details are provided in the supplementary materials). Therefore, the overarching goal of this study was to examine the
role of K+ on banana peel-driven heterotrophic denitrification and the possible underlying mechanism within a wide range of dissolved K+ concentrations. A series of well-designed batch tests were conducted to: (1) determine the threshold and optimal K+ concentration for denitrification; (2) identify the relationship between K+ concentration and DOM dissolution; (3) evaluate the abundance and structure of microorganisms and dominant functional species under different K+ concentrations; (4) explore the possible mechanism of dissolved K+ during the denitrification process; and (5) perform multivariate statistical analysis and correlation analysis between the denitrification performance, DOM dissolution and functional species relative abundance.
2. Materials and methods 2.1. Microorganism cultivation and bioreactor setup Sludge for the acclimation of denitrification was obtained from the pre-anoxic zone of the Qinghe Wastewater Treatment Plant in Beijing, China. It was then cultured in a 2-L breaker containing culture medium composed of the following mineral substances: 2.7 g/ L CH3COONa, 0.3 g/L NH4Cl, 0.85 g/L NaNO3, 0.15 g/L KH2PO4, 0.1 g/ L MgSO47H2O and 0.005 g/L FeSO47H2O (Yao et al., 2013). The sludge was incubated for one month and the medium was refreshed every 4 days. The initial soluble organics in the inoculum were depleted after cultivation. Five 1-L conical flasks with rubber plugs were employed as the batch reactors. Two holes existed in the rubber plugs for N2 stripping and sampling. Each flask was purged with N2 and sealed with rubber plugs to maintain anaerobic condition. Each bioreactor, with different K+ concentrations, was filled with 1 L of synthetic nitrate wastewater, 10 mL of anaerobic sludge and 1 g of dried banana peel as the fixed carbon source. Nitrate-N was supplied in the form of NaNO3 at a concentration of 50 mg/L and soluble K+ was supplied in the form of KCl. The reactor without additional KCl was designated as control group B-0, while the bioreactors containing different concentrations of additional K+, namely 50, 100, 150 and 200 mg/L, were designed as B-5, B-10, B-15 and B-20, respectively. All bioreactors were provided with 1 g of fixed banana peel (size of 4.75 mm, dried at 105 °C for 24 h in an oven) as the carbon source. In addition, phosphorus used for bacterial growth originated from the dissolution of banana peel during these experiments. The dissolved COD of the banana peel was about 240.83 ± 7.50 mg/g, which meets the C/N ratio required for heterotrophic denitrification for three cycles (the initial nitrate concentration was 50 ± 5 mg-N/L at each cycle). All reactors were operated at 30 ± 2 °C with shaking at 150 rpm. Treatments were performed in duplicate and the mean values of the experimental data are reported. Finally, NO 3 -N reduction with banana peel as the carbon source under different K+ concentrations were individually assessed over three consecutive operating cycles. The end of an operating cycle was defined by the complete reduction of NO 2 -N and only NO3 -N was added, without carbon source, at the beginning of the next cycle. The suitable concentration range of soluble K+ during the denitrification process was also evaluated. Synthetic groundwater was prepared by amending deionized water with NaNO3, maintaining the nitrate concentration at 50 mg-N/L (Zhang et al., 2012). The phosphorus requirement for denitrification was approximately 0.02–0.03 g PO3 4 -P/g NO3 -N. The banana peel provided enough phosphorus to support the requirement of the microorganisms throughout the experiment. Thus, there was no need to add external phosphorus to the synthetic groundwater. In addition, the nitrogen content of the banana
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peel was 0.36 ± 0.02% and no accumulation of NO 3 -N occurred in the dissolution test. 2.2. Chemical and biological analyses Water samples taken from the reactors were immediately filtered through 0.22 lm membranes prior to analysis. The pH was immediately determined with a pH meter (Seven Multi S40, Met tler Toledo, Switzerland). NO 3 -N and NO2 -N were measured using a spectrophotometer (DR6000, HACH, USA) according to the Chinese NEPA standard methods. NH+4-N, PO3 4 -P and CODCr were monitored using phenol-sodium hypochlorite, the water and wastewater monitoring analysis method and, respectively (SEPA, 2002). K+ in the aqueous solution was analyzed by ICP-AES (ICAP 6000 SERIES, Thermo Fisher, Germany). The denitrification performance of the bioreactor was evaluated based on the NO 3 -N removal efficiency (NRE) and NO 3 -N removal rate (NRR), which are defined by Eqs. (1) and (2) (Zhao et al., 2018):
NREð%Þ ¼ ½ðC 0 C t Þ 100 =C 0
ð1Þ
NRRðmg N=ðL hÞÞ ¼ ðC 0 C t Þ =t
ð2Þ
where C0 is the initial NO 3 -N concentration (mg/L); Ct is the NO3 -N concentration at time t (mg/L); and t is the reaction time (h). DOM was characterized by three-dimensional excitationemission matrix (EEM) fluorescence spectroscopy using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) (All samples were diluted 5 times.). The slit width for both the excitation and emission was 10 nm and the scanning speed was set at 40 nm/s. Excitation and emission were simultaneously scanned at wavelengths ranging from 200 to 450 nm. After regulating scattering using interpolation in areas affected by Rayleigh and Raman scattering (He et al., 2011), the fluorescence regional integration (FRI) technique was used for analysis (Wei et al., 2014). Microbial samples from each bioreactor were collected and pretreated ultrasonically. The total genomic DNA was extracted using the FastDNAÒ SPIN Kit (MP Biomedicals, USA) following the manufacturer’s instructions. The extracted DNA was amplified with PCR primers 338F (50-ACTCCTACGGGAGGCAGCAG- 30) and 806R (50GGACTACHVGGGTWTC TAAT-30). A mixture of amplified samples was used for high throughput 16S rRNA gene analysis using the MiSeq platform (Illumina, USA), conducted by Shanghai Majorbio Technology (Shanghai, China). The sequences are available on the NCBI Sequence Read Archive under accession number PRJNA491377.
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2.3. Statistical analysis + The variation in NO 3 -N, NO2 -N, NH4-N and COD, as well as the area of different DOM, were analyzed using Origin 9.1. Principal component analysis (PCA) was used for data reduction via SPSS version 20.0. In addition, the relationship between bacterial species, DOM dissolution, denitrification performance and K+ concentrations were analyzed.
3. Results and discussion 3.1. Denitrification performance Nitrate-N was gradually removed during the three consecutive operating cycles and microbial NO 3 -N reduction was clearly affected in the presence of different K+ concentrations (Fig. 1). Due to the promotion of microbial activity, NO 3 -N reduction was positively correlated with the K+ concentration up to 229.78 ± 25.80 mg-K/L (Table 1). The observed boundary condition for the K+ concentration was lower than the 1000 mg/L K+ obtained in a previous report (Cyplik et al., 2007). The reasons for this phenomenon are attributed to the different phase of carbon sources and bacterial species because the H. denitrificans used in the previous study is a halotolerant microorganism (Cyplik et al., 2007). The effect of the K+ concentration on banana peel-based heterotrophic denitrification transitioned from stimulation (<229.78 ± 25.80 mg-K/L) to inhibition (>229.78 ± 25.80 mg-K/L). When the K+ concentration exceeded the boundary condition, the B-20 bioreactor only reduced 85.73 ± 0.54 mg NO 3 -N during the three consecutive operating cycles, exhibiting the lowest NO 3 -N reduction rate (1.10 mg/(Lh)) in the first cycle (Table 1). This result clearly confirms that excessive K+ suppresses NO 3 -N reduction. Compared with the NO 3 -N removal efficiencies of B-0 (67.3% ± 0.6%), B-5 (80.7% ± 0.9%) and B-10 (93.9% ± 0.5%), B-15 exhibited the highest NO 3 -N removal efficiency (98.1% ± 0.2%) at 502 h during the three consecutive cycles. This indicates that the K+ concentration of 229.78 ± 25.80 mg-K/L can enhance the continuous denitrification ability with the same amount of solid phase carbon source. The obtained denitrification rates of the banana peel-based system during the first cycle was higher than the denitrification rates obtained in a methanol system (0.98 mg-N/(Lh)) (He et al., 2018) and a rice bran system (0.73–1.05 mg-N/(Lh)) (Hou, 2018). The denitrification rate during the second cycle was higher than the rates obtained in a fish waste system (0.19 mg-N/(Lh))
+ Fig. 1. Variations of NO 3 -N concentration in banana peel-based heterotrophic denitrification system with different K concentrations of three consecutive cycles.
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Table 1 Zero-order kinetic parameters of NO 3 -N removal in bioreactors over typical operating cycles. Reaction system
K1 (mg N/Lh) R2
B-0 B-5 B-10 B-15 B-20
1.9007 1.8931 1.8714 1.8581 1.1044
K2 (mg N/Lh) R2 0.9779 0.9745 0.9720 0.9709 0.9197
0.3268 0.3558 0.4521 0.4515 –
K3 (mg N/Lh) R2 0.9794 0.9866 0.9864 0.9562 –
0.0825 0.1205 0.1290 0.1456 –
0.9447 0.9829 0.9327 0.9011 –
* K1: reduction rate of nitrate in the first cycle; K2: reduction rate of nitrate in the second cycle; K3: Reduction rate of nitrate in the third cycle.
Table 2 Zero-order kinetic equation and parameters of the production and reduction of NO 2 -N in bioreactors over the first typical operating cycle, C0 (mg/L) and Ct (mg/L) are the concentrations of NO 2 -N at initial condition and time t (h), respectively. NO 2 -N production
Reaction system
B-0 B-5 B-10 B-15 B-20
NO 2 -N reduction 2
K0 (mg N/Lh)
R
K (mg N/Lh)
R2
1.1683 1.1797 1.1349 1.0992 0.8584
0.9587 0.9800 0.9550 0.9526 0.9186
0.6676 0.6871 1.0518 0.8604 0.5613
0.9865 0.9801 0.9926 0.9810 0.9371
* K0: the production of NO2-N in bioreactors over the first typical operating cycle; K: the reduction of NO2-N in bioreactors over the first typical operating cycle.
(He et al., 2018) and a eucalyptus mulch system (0.11 mg-N/(Lh)) (He et al., 2018). Due to inhibition by excessive K+, the NO 3 -N reduction capacity of the banana peel in B-20 was significantly lower than the amount that could be reduced in the banana peel-based denitrification system (164.42 ± 1.15 mg-N/g-BP). Excessive K+ may have caused the cell salinity to be so high that it inhibited ion exchange, thereby permeating between intra- and extra-cellular spaces, and the microorganism in our system was not halotolerant. During the first typical operating cycle, 50 ± 2 mg-N/L was completely bioreduced within 24 h in B-0, B-5, B-10 and B-15, which all showed similar NO 3 -N reduction rates (Table 1), while a removal efficiency of 63.4% ± 0.0% was obtained in B-20 (p < 0.05). This reveals that the threshold K+ concentration for NO 3 -N reduction in the banana peel-based denitrification system was around 229.78 ± 25.80 mg-K/L and that the denitrification process would be restrained if this value exceeded the boundary condition. However, in the following operating cycle, both B-10 and B-15 exhibited the same and highest NO 3 -N reduction rate (0.45 mg/(Lh)), followed by B-5 (0.36 mg/(Lh)) and B-0 (0.33 mg/(Lh)), while B-20 could not continue to degrade NO 3 -N after 166 h. During the third operating cycle, B-15 showed the highest NO 3 -N removal rate (0.15 ± 0.03 mg/(Lh)), followed by B-10 (0.13 ± 0.03 mg/(Lh)), B-5 (0.12 ± 0.07 mg/(Lh)) and B-0 (0.08 ± 0.02 mg/(Lh)) (p < 0.05) (Table 1). This further indicates that the K+ concentration of 229.78 ± 25.80 mg-K/L had a lasting promotion effect on denitrification. During the denitrification process, some intermediate reactions may occur synchronously, as follows (Eqs. (3)–(7)) (Li et al., 2016):
3.2. The evolution of dissolved organic matter (DOM)
2NO3 + 4Hþ + 4e !2NO2 + 2H2 O
ð3Þ
2NO2 + 4Hþ + 4e !2NO + 2H2 O
ð4Þ
2NO + 2Hþ + 2e !N2 O + H2 O
ð5Þ
N2 O + 2Hþ + 2e !N2 + H2 O
ð6Þ
2NO3 + C6 H12 O6 + 6Hþ !3NH4 þ + 6CO2 + 3H2 O NO 2 -N
NH+4-N
2002), while little accumulation of NO 2 -N (below the detection limit) and NH+4-N (2.61–3.09 mg-N/L) appeared at the end of the reaction. In addition, B-10 showed the highest NO 2 -N reduction rate (1.05 mg/(Lh)) during the NO 2 -N reduction process, followed by B-15 (0.86 ± 0.03 mg/(Lh)), B-5 (0.69 ± 0.05 mg/(Lh)) and B-0 (0.67 ± 0.04 mg/(Lh)), while B-20 exhibited a rate of 0.56 ± 0.05 mg/(Lh) (p < 0.05). B-10 showed the highest NO 2 -N reduction rate among all the reactors, as excessively high or low K+ concentrations decreased NO 2 -N reduction (Fig. S3). Results demonstrate that the K+ boundary conditions for NO 3 -N and NO 2 -N reduction were 229.78 ± 25.80 and 159.10 ± 24.60 mg-K/L, respectively, in the banana peel-based denitrification system. Furthermore, the banana peel could achieve efficient nitrate removal during long-term operation (398 h; Fig. S6). Variation of pH in five bio-reactors (Fig. S7) showed the same sharply decreasing trend from 0 to 28 h, but then gradually increased and finally stabilized at 7.0–7.5. The optimum pH for nitrate and nitrite reduction ranges from 7 to 8, which is a range that is most suitable for the survival and function of denitrifying microorganisms. During the reaction process, the carbon source was first fermented to produce acid under the action of fermentative microorganisms. The denitrifying bacteria could then use the small molecular acid produced by carbon source fermentation to achieve nitrate reduction. Denitrification is a biological process of alkali production. The OH generated during the denitrification process can neutralize the acid produced by fermentation, thereby increasing the pH during the later stage of the reaction.
ð7Þ
The small amount of and implies the presence of multiple metabolic pathways, including denitrification and dissimilatory nitrate reduction to ammonium (Fig. S2) (Patterson et al.,
The main banana peel components included 12.50% cellulose, 19.90% hemicellulose and 14.10% lignin. To study the changes of these components during the reaction, the DOM was analyzed by EEM. DOM, a heterogeneous mixture of complex organic compounds, includes organic acids, amino acids, humic substances and enzymes (He et al., 2011). It can work as an electron shuttle and mediate electron transfer between reducing agents and electron acceptors (Dawson and Hilton, 2011). These actions could accelerate the composting process by accelerating the degradation of persistent organic matter, such as chloro-organics and nitrophenols (Zhu et al., 2013). Nutrients such as cellulose, hemicellulose and lignin in banana peel can be degraded to glucose, hexose and
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pentose under the action of hydrolases (Jönsson et al., 2013). In addition, these nutrients can dissolve a large amount of DOM; however, the evolutionary pathway of specific DOM components remains to be further clarified. Therefore, the DOM in this study was analyzed by EEM to illustrate the evolution of carbon compounds. Regions I, II and IV were associated with protein-like substances (tyrosine-like, tryptophan-like and soluble microbial byproduct-like substances), whereas regions III and V represented fulvic acid-like and humic acid-like substances, respectively (Chen et al., 2003). A dissolution phenomenon was observed at the beginning of the reaction, with the main peak appearing in region V and an increased integral area (Fig. 2). As the reaction proceeded, the main peaks shifted to regions I and II, and the integral area decreased, meaning that the substances of all regions decreased and the main humic-like components were degraded into low molecular substances (confirmed in Fig. S3). The migration and transformation of the EEM fluorescence spectra reflected the order of DOM utilization (Fig. 2). Protein-like substances (tyrosine-like, tryptophan-like and soluble microbial by-product-like substances) were easily utilized by the denitrifiers due to their easily degradable structures, and the substances of region V were degraded into low molecular weight compounds after being used by the microorganisms (Fig. 2). This result agrees with a previous study, which reported that microorganisms preferentially use labile organic substances in the DOM (e.g. aliphatics, polysaccharides and proteins) during the reaction process, whereas DOM compounds with aromatic structural units were not easily degraded and generally accumulated (Said-Pullicino and Gigliotti, 2007). The change in the amount of DOM during the reaction was measured by the change in COD. The dissolution of organic matter is of positive significance in promoting the heterotrophic denitrification process. However, it is undeniable that residual organic matter could cause secondary
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COD pollution. At present, physical adsorption methods, such as activated carbon, anthracite and bamboo charcoal, can achieve efficient COD removal (Hata et al., 2016; Mueller et al., 2001). Therefore, in practical engineering applications, COD adsorption could be integrated as a back-end process in denitrification units. Alternatively, COD concentration in wastewater can be reduced by optimizing reactor design including size and packing addition. B-15 exhibited the largest degradation of humic-like substances of 10.59% at the end of the reaction, followed by B-10 (8.17%), B-5 (5.50%), B-0 (4.88%) and B-20 (2.25%) (Fig. S3). Meanwhile, the largest protein-like substance generation occurred in B-15 (7.69%), followed by B-10 (6.78%), B-5 (5.46%), B-0 (3.00%) and B-20 (2.45%) (Fig. 4). The substances of regions I and II were derived from the inherent self-dissolution of the banana peel and the degradation of region V substances. These changes indicate that the utilization degree of DOM was positively correlated with the K+ concentration up to 229.78 ± 25.80 mg/L. Thus, the higher K+ concentration, the more humic-like substances are converted to protein-like compounds within the boundary condition. In addition, correlation analysis was used to evaluate the correlation between the K+ concentration and the change in different regions (regions I + II and region V) (Table S2). There was a significant positive correlation between the change in different regions (the rate of decline in the percentage of region V and increase in regions I + II) and K+ concentration when K+ was below the boundary condition. When the concentration of K+ was above the boundary condition, the degradation of organic matter was suppressed, which had a positive correlation with the denitrification performance under different K+ concentrations, especially during cycles 2 and 3 (confirmed in Table S3). This conclusion indicates that additional K+ enhances the degree of DOM degradation and utilization in the banana peel, and simultaneously improves the denitrification rate. Banana peel fermentation is similar to the composting process,
Fig. 2. EEM spectra of each cycle of different K+ concentration in banana peel-based denitrification system (all samples were diluted 5-fold). (the EEM image represent B-0 (a), B-5 (b), B-10 (c), B-15 (d) and B-20 (e), there were only two cycles in B-20 and then keep stable nitrate concentration without any reaction).
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with both processes achieving humification of waste organic compounds. From this, we can infer that additional K+ can promote composting to some extent. The optimized K+ concentration for the degradation of organic matter was around 229.78 ± 25.80 mg/ L in the banana peel-based denitrification system, and the optimized K+ concentration of DOM degradation was consistent with + the reduction of NO 3 -N. Moreover, the K concentration experiment further confirmed that K+ addition (Fig. 1) accelerated the denitrification rate by promoting the dissolution of organic substances within the appropriate K+ concentration range (Fig. S3). This result is consistent with a previously reported study indicating that K+ can effectively enhance the degradation of organics (Li et al., 2018). Analysis from compound 1 indicates that the concentration of K+ was negatively correlated with the dissolution of organic substances from the banana peel (Fig. 3), which further indicates that addition K+ can improve the dissolution of organic matter from the banana peel. According to the calculation and PCA of different regions in banana peel dissolution, the amount of dissolution change in regions I, II and III had a negative effect with the amount in regions IV and V in compound 2 (Fig. 3). This result demonstrates that the reduction of regions IV and V transferred into regions I, II and III, which were more easily degraded in subsequent reactions. Appropriate addition of K+ could promote the dissolution of organic matter from the banana peel and the increase of organic matter dissolution will increase the amount of degradable organic matter, thereby improving the efficiency of the carbon source. 3.3. Microbial community and mechanisms A total of 46743, 59973, 56112, 69810, 55,341 and 64,675 highquality reads with an average length of 442 bp were recorded from blank sample B-0, B-5, B-10, B-15 and B-20, respectively. Chao 1 and ACE indexes (Table S1) indicate that the abundances in B-0, B-5, B-10 and B-15 were higher than those of B-20 and the blank sample. This implies that the K+ concentration (below the boundary condition) may lead to an increased abundance, while excessive K+ had an inhibitory effect on abundance. The Simpson and Shannon diversity indexes can provide insights into species richness and evenness (Liu et al., 2017). The higher Shannon index and lower Simpson index suggest a higher diversity and evenness in the five samples (B-0, B-5, B-10, B-15 and B-20) compared with blank sample, indicating that K+ can weaken the environmental selectivity of
microorganisms and improve the ability of different functional species to adapt to the same environment (Table S1). It has been previously reported that denitrification can be facilitated by appropriate addition of K+ because it plays an important role in stimulating bacterial activity (Cyplik et al., 2007). Analysis of the 16S rRNA gene sequences further proved that the K+ concentration affected the community structure and relative abundance of dominant functional species. The effect of K+ on the abundance of the microbial community members apparently transitioned from stimulation (<229.78 ± 25.80 mg-K/L) to inhibition (>229.78 ± 25.80 mg-K/L) (Table S1). B-0, B-5, B-10 and B-20 exhibited the higher diversity of microbial species for fermentation and denitrification when compared with the blank sample, (Table 3, Fig. 4(a)), which also corresponded to the denitrification and fermentation performance. These results could be attributed to the interaction between performance and different functional species. A microbial community shift was observed in the five bioreactors at the genus level (Fig. 4(a)). Diaphorobacter, which is closely related to denitrification and can synthesize nitrate and nitrite reductases (Chakravarthy et al., 2011), exhibited relative abundances of 10.05%, 20.07%, 21.84%, 18.29% and 1.85% in B-0, B-5, B-10, B-15 and B-20, respectively. This indicates that the most suitable K+ concentration range for the enrichment of Diaphorobacter is around 114.04 ± 25.38 mg-K/L to 229.78 ± 25.80 mg-K/L. Paludibacter is involved in biodegradation and denitrification processes under anoxic conditions and can utilize various sugars, including glucose, starch and sucrose, while simultaneously producing propionate and acetate (Ueki, 2006; Qiu et al., 2014). The relative abundance of Paludibacter decreased from 15.22% to 1.36% as the K+ concentration increased from 81.88 ± 11.22 mg-K/L to 292.32 ± 28.20 mg-K/L (Table 3). This indicates that the relative abundance is negatively correlated with the K+ concentration. Thauera is capable of degrading isopropanol and acetone under denitrifying conditions (Fida et al., 2017). The relative abundance of Thauera increased from 1.61% (80.88 ± 21.66 mg/L of K+) to 10.15% (229.78 ± 25.80 mg-K/ L) and then decreased to 1.3% (292.32 ± 46.12 mg-K/L). These results significantly confirm that K+ concentrations below 229.78 ± 25.80 mg-K/L are beneficial to Thauera enrichment but concentrations above 229.78 ± 25.80 mg-K/L will inhibit its growth. Furthermore, the relative abundance of Anaerolineaceae, which is associated with the degradation of refractory dissolved organic matter (rDOM) (Li et al., 2018), decreased with increasing K+ concentrations, verifying that a K+ concentration above 80.88 ± 11.22 mg-K/L
Fig. 3. Correlation matrix between K+ concentration and DOM dissolution (a) and PCA of the correlation between K+ concentration and DOM dissolution of different regions (b). * R1: the change between starting and ending in Region I (DRegion I), R2: the change between starting and ending in Region I (DRegion II), R3: the change between starting and ending in Region I (DRegion III), R4: the change between starting and ending in Region I (DRegion IV), R5: the change between starting and ending in Region I (DRegion V), and Ck: different K+ concentration.
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H. Wang et al. / Science of the Total Environment 703 (2020) 134830 Table 3 Relative abundance of functional species at genus level in different reaction systems. Function
Genus
B-0
B-5
B-10
B-15
B-20
Fermentation
Dechlorobacter Paludibacter norank_f_Anaerolineaceae norank_f_Porphyromonadaceae Diaphorobacter Simplicispira Thauera Thiobacillus Pseudomonas Closlridium_sensu_stricto Lentimicrobium norank_o_Ignavibacteriales
– 15.22 10.33 2.93 10.05 8.22 1.61 – – – 8.1 3.18
3.1 11.62 2.75 5.34 20.07 10.03 2.21 – – – 6.22 1.95
8.48 10 1.77 1.78 21.48 3.13 4.44 – – – 6.69 –
8.09 4.3 2.18 1.8 18.29 12.15 10.15 1.52 1.86 1.46 4.16 –
– 1.36 1.32 – 1.85 – 1.3 – – – 1.91 –
Denitrification
Fig. 4. Microbial community structure and multivariate statistical analysis. (a) 1Heatmap to profile the most abundant genera (top 30) of five bioreactors and blank sample (The color bar indicates the range of the percentage of a genus in a sample, based on the color key at the bottom). (b) Correlation matrix between K+ concentration and functional species. (c) PCA of the correlation between K+ concentration and functional species. * K: K+ concentration, De: Dechlorobacter, P: Paludibacter, NA: norank_f_Anaerolineaceae, NP: norank_f_Porphyromonadaceae, Di: Diaphorobacter, T: Thauera, L: Lentimicrobium, S: Simplicispira.
inhibits its activity. Dechlorobacter, which can use nitrate as electron donor in polyhydroxybutyrate valerate (PHBV)-supported systems (Xu et al., 2018), was not only present in the K+ concentration range of 114.04 ± 25.38 to 229.78 ± 25.80 mg-K/L, but also increased from 3.10% to 8.09% with increasing of K+ concentrations. This indicates
that a K+ concentration between 114.04 ± 25.38 and 229.78 ± 25.80 mg-K/L is suitable for its survival and that concentrations outside this range will suppress its enrichment. Simplicispira has the ability to reduce nitrate under anoxic conditions with PBS as carbon source and a biofilm carrier for wastewater treatment (Zhu et al.,
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Fig. 5. Proposed mechanisms of nitrate reduction and DOM degradation of banana peel in B-15.
2015). Interestingly, B-15 exhibited the highest relative abundance of Simplicispira (12.15%), followed by B-5 (10.03%), B-0 (8.22%) and B-10 (3.13%), while it was not found in B-20 (Table 3). Thus, a K+ concentration exceeding the boundary condition is not conducive to the formation of Simplicispira. Lentimicrobium, whose relative abundance decreased with the increasing K+ concentration, might participate in the fermentation of complex organics in food waste (Sun et al., 2016). These enriched denitrifiers and fermentative bacteria may be responsible for the removal of nitrate and the degradation of organic matter. Moreover, Pseudomonas, Closlridium and Thiobacillus were the unique functional species in B-15. Pseudomonas exhibits high resistance to aromatic pollutant toxicity and high degradation efficiencies for a wide variety of aromatic compounds under various environmental conditions. Thiobacillus is an autotrophic denitrifying bacteria that is also is known to simultaneously reduce sulfate and nitrate in the presence of high concentration of organics (Park et al., 2015). These bacteria are beneficial to the degradation of NO 3 -N and complex organic matter (DOM), thereby explaining why the highest denitrification rate was achieved in B-15. In addition, B-15 accounted for the largest proportion of denitrifying functional species (Table 3), which is consistent with the denitrification process shown in Fig. 1. This may be attributed to the fact that denitrifiers can stimulate biodenitrification, thereby accelerating the reaction rate. Moreover, different functional species have their own optimal K+ concentration range, which was indeed demonstrated by our experiments. In addition, the data presented in Fig. 4(b) indicates the correlation between different parameters, namely the K+ concentration, Dechlorobacter, Paludibacter, norank_f_A-naerolineaceae, norank_f_Porphyromonadaceae, Diaphorobacter, Thauera, Lentimicrobium and Simplicispira. These analyses further verify that the K+ concentration was positively cor-
related with Dechlorobacter (fermentor) and Thauera (denitrifier) (p < 0.05) within the boundary condition determined by statistical analysis. The mechanisms of denitrification and fermentation in the B-15 reactor are summarized in Fig. 5. B-15 promoted the degradation of humic-like substances by 217.0%, 192.5% and 129.6% when compared with B-0, B-5 and B-10, respectively, while the excessive K+ in B-20 (above the boundary condition of 229.78 ± 25.80 mg-K/L) suppressed the degradation of humic-like substances, further proving that the K+ boundary condition for promoting fermentation is around 229.78 ± 25.80 mg-K/L. In addition, the denitrification rates of B-15 were 136.4% and 125.0% higher than B-0 and B-5, respectively, during the second cycle, and 187.5%, 125.0% and 115.4% higher than B-0, B-5 and B-10 in the third cycle, respectively, confirming that the K+ boundary condition enhanced the denitrification process in the banana peelbased system. Moreover, the detection of Diaphorobacter, Thauera, Paludibacter, norank_f_Anaerolineaceae and Lentimicrobium verified that both DOM (dissolved from the banana peel) oxidation and nitrate removal can be achieved simultaneously in a banana peel-based system. B-15 had the most abundant microbial community of all the bioreactors, with the presence of Pseudomonas and Thiobacillus being very beneficial for promoting denitrification. Furthermore, this study found that the survival of a large proportion of denitrifiers and fermentative bacteria (e.g. Diaphorobacter and Thauera) was positively correlated with the K+ concentration (below the boundary condition of 229.78 ± 25.80 mg-K/L). 4. Conclusions This study elucidated the stimulatory and inhibitory effects of K+ on denitrification, DOM degradation and microbial community structure in a banana peel-based heterotrophic denitrification sys-
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tem under the given treatment conditions. The main results were as follows: (i) the nitrate removal performance was promoted by K+ up to 229.78 ± 25.80 mg-K/L, but nitrate reduction was significantly inhibited at higher concentrations; (ii) the DOM degradation pathway was demonstrated by showing that protein-like substances are used first, followed by the conversion of humic-like substances into low-molecular weight substances for microorganisms utilization, which leaves macromolecular humic-like substances that cannot be biodegraded; and (iii) microbial community analysis of the five bioreactors showed that the genera Pseudomonas (1.86%) and Thiobacillus (1.52%) are the unique functional species in B-15, which exhibited the best nitrate bioreduction characteristics. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (NSFC) (No. 51578519; No. 21876159) and the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07 202002). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2019.134830. References Chakravarthy, S.S., Pande, S., Kapoor, A., Nerurkar, A.S., 2011. Comparison of Denitrification Between Paracoccus sp. and Diaphorobacter sp. Appl. Biochem. Biotechnol. 165 (1), 260–269. https://doi.org/10.1007/s12010-011-9248-5. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. https://doi. org/10.1021/es034354c. Cheung, H.N.B., Huang, G.H., Yu, H., 2010. Microbial-growth inhibition during composting of food waste: Effects of organic acids. Bioresour. Technol. 101 (15), 5925–5934. https://doi.org/10.1016/j.biortech.2010.02.062. Cyplik, P., Grajek, W., Marecik, R., et al., 2007. Effect of macro/micro nutrients and carbon source over the denitrification rate of Haloferax denitrificans archaeon. Enzyme Microb. Technol. 40 (2), 212–220. Dawson, C.J., Hilton, J., 2011. Fertiliser availability in a resource-limited world: Production and recycling of nitrogen and phosphorus. Food Policy 36, S14–S22. https://doi.org/10.1016/j.foodpol.2010.11.012. Feng, L., Chen, K., Han, D., Zhao, J., Lu, Y., Yang, G., Mu, J., Zhao, X., 2017. Comparison of nitrogen removal and microbial properties in solid-phase denitrification systems for water purification with various pretreated lignocellulosic carriers. Bioresour. Technol. 224, 236–245. https://doi.org/10.1016/j.biortech.2016.11.002. Fida, T.T., Gassara, F., Voordouw, G., 2017. Biodegradation of isopropanol and acetone under denitrifying conditions by Thauera sp. TK001 for nitratemediated microbially enhanced oil recovery. J. Hazard. Mater. 334, 68–75. https://doi.org/10.1016/j.jhazmat. 2017.03.061. Hata, M., Amano, Y., Thiravetyan, P., et al., 2016. Preparation of bamboo chars and bamboo activated carbons to remove color and COD from ink wastewater. Water Environ. Res. 88 (1), 87–96. He, Q., Zhang, D., Main, K., Feng, C., Ergas, S.J., 2018. Biological denitrification in marine aquaculture systems: A multiple electron donor microcosm study. Bioresour. Technol. 263, 340–349. https://doi.org/10.1016/j.biortech.2018.05.018. He, X., Xi, B., Wei, Z., Guo, X., Li, M., An, D., Liu, H., 2011. Spectroscopic characterization of water extractable organic matter during composting of municipal solid waste. Chemosphere 82, 541–548. https://doi.org/10.1016/j. chemosphere.2010.10.057. Hou, T., n.d. Enhancement of rice bran as carbon and microbial sources on the nitrate removal from groundwater. Biochemical Engineering Journal https://doi. org/10.1016/j.bej. 2018.07.010. Jafari, S.J., Moussavi, G., Yaghmaeian, K., 2015. High-rate biological denitrification in the cyclic rotating-bed biological reactor: Effect of COD/NO3, nitrate concentration
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Contents lists available at ScienceDirect
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Effect of potassium on nitrate removal from groundwater in agricultural waste-based heterotrophic denitrification system Haishuang Wang, Chuanping Feng ⇑, Yang Deng School of Water Resources and Environment, MOE Key Laboratory of Groundwater Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing 100083, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
+
The threshold of K concentration for
nitrate reduction was 229.78 ± 25.80 mg-K/L. 1.15–1.88 fold denitrification rate was improved under different K+ concentration. The evolution pathway and utilization sequence were revealed. Pseudomonas and Thiobacillus are the unique species in 229.78 ± 25.80 mgK/L.
a r t i c l e
i n f o
Article history: Received 23 July 2019 Received in revised form 11 September 2019 Accepted 3 October 2019 Available online 3 November 2019 Keywords: Potassium Denitrification Hydrolysis Nitrate Banana peel
a b s t r a c t Heterotrophic denitrification based on solid carbon sources has been widely investigated for nitrogen removal in recent years. In this study, the response of the heterotrophic denitrification process under different K+ concentrations was clarified. Additionally, the denitrification enhancement mechanism was revealed and resource utilization of agricultural waste was achieved. A series of batch tests were conducted to study the effect of different K+ concentrations on the denitrification performance, dissolved organic matter (DOM) dissolution and microbial community structure. Results demonstrate that the threshold of K+ concentration for the NO 3 -N and NO2 -N reduction rates were 229.78 ± 25.80 and 159.10 ± 24.60 mg-K/ L, respectively. Excitation-emission matrix (EEM) analysis identified the main DOM components associated with tyrosine-like, tryptophan-like and humic-like substances, as well as illustrated the evolutionary behavior and utilization of DOM. High throughput 16S rRNA gene sequencing indicates that a K+ concentration of 229.78 ± 25.80 mg-K/L exhibited the highest diversity of functional species associated with fermentation and denitrification. The genera Pseudomonas and Thiobacillus were the unique denitrifiers at this K+ concentration. The correlation of K+ concentration, DOM dissolution of different regions and microorganism structure were analyzed using correlation matrix and PCA, and the appropriate K+ concentration of different functional microorganisms survival was optimized by this analysis method. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction
⇑ Corresponding author. E-mail address: [email protected] (C. Feng). https://doi.org/10.1016/j.scitotenv.2019.134830 0048-9697/Ó 2019 Elsevier B.V. All rights reserved.
Nitrate pollution has become a serious issue in groundwater worldwide due to the increasing use of nitrogenous fertilizers; the discharge of domestic and industrial wastewater; and the leak-
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age of septic tanks (Jafari et al., 2015). Nitrate can induce a series of human diseases, including methemoglobinemia in infants and cancer in adults, and can endanger the ecological environment. Heterotrophic denitrification, a cost-effective method to remove nitrate from groundwater, has been extensively studied in recent years, while the characteristics of eutrophication are detrimental to denitrification in groundwater. Selection of suitable carbon sources and additional metal elements of suitable concentrations are important means to enhance the heterotrophic denitrification process. Current research on denitrification enhancement generally includes pretreatment of solid carbon sources (Feng et al., 2017) and the effects of trace elements (e.g. Mg, Cu, Fe, Na, Ca) (Wang et al., 2018). K+ has become a common metal detected in groundwater due to the large amount of K+-containing fertilizers used in agriculture (Melo et al., 2012) that seeps into the groundwater through the soil. Heterotrophic denitrification is a biological process, and K+ is an important parameter affecting the growth and removal of microorganisms (Cyplik et al., 2007). The accumulation of K+ within cells reportedly counteracts the high level of brine osmotic potential in which halophilic microorganisms live (Cyplik et al., 2007). In addition, K+ is a major essential macronutrient for biological growth and development (Sarikhani et al., 2016), playing an important role in plant cellular homeostasis by contributing to charge balance, osmotic adjustment and enzyme catalysis (Zhou et al., 2018). K+ can also enhance microbial activity by affecting potassium channel proteins and altering the intracellular and extraosseous osmotic pressure (Sarikhani et al., 2016). In recent decades, several studies have identified at least four different uses for K+: as a promoter for the anaerobic digestion process (Li et al., 2018); as an adsorbent for NH+4-N removal; as an anode material for K+ batteries (Liu et al., 2018); and as a necessary element for the growth of plants and animals (Zhou et al., 2018). Although, elemental potassium is a necessary macroelement for microorganisms, there is a lack of studies regarding the influence of K+ on biological processes, especially heterotrophic denitrification. Furthermore, the enhancement or inhibition of K+ on heterotrophic denitrification and its mechanisms are still unclear. K+ can help the denitrifier Haloferax survive unfavorable environmental conditions characterized by a high osmotic potential (Cyplik et al., 2007). In order to understand the mechanism of K+ on denitrification, it is necessary to study the functional effects of K+ during the denitrification process; explore the optimum K+ concentration for different functional denitrifying and fermenting bacteria; and reveal the relationship among different K+ concentrations, denitrification properties, DOM dissolution and functional species. Changes in DOM during composting can reflect the conversion process of organic matter. The reaction mechanism of the organic carbon source during the fermentation phase of the heterotrophic denitrification process is similar to that during the composting process (Chen et al., 2003; He et al., 2011). Therefore, in this study, the changes in DOM were used to characterize the change in organic matter during the experiment. Compared to the relatively wellcharacterized toxic effects of high concentrations of organic acids on the activity of microorganisms (Cheung et al., 2010), there is a lack of studies on the effects of K+ during DOM dissolution. The activity and abundance of functional species also play a crucial role in the rate and efficiency of denitrification. Furthermore, the optimal K+ concentration for functional species, including denitrifiers and fermentative bacteria, remains unknown. In this study, the mechanism of nitrate removal enhancement was studied. Banana peel, a typical agricultural waste product, was selected to evaluate the performance of denitrification because it has almost all the characteristics of agricultural waste (further details are provided in the supplementary materials). Therefore, the overarching goal of this study was to examine the
role of K+ on banana peel-driven heterotrophic denitrification and the possible underlying mechanism within a wide range of dissolved K+ concentrations. A series of well-designed batch tests were conducted to: (1) determine the threshold and optimal K+ concentration for denitrification; (2) identify the relationship between K+ concentration and DOM dissolution; (3) evaluate the abundance and structure of microorganisms and dominant functional species under different K+ concentrations; (4) explore the possible mechanism of dissolved K+ during the denitrification process; and (5) perform multivariate statistical analysis and correlation analysis between the denitrification performance, DOM dissolution and functional species relative abundance.
2. Materials and methods 2.1. Microorganism cultivation and bioreactor setup Sludge for the acclimation of denitrification was obtained from the pre-anoxic zone of the Qinghe Wastewater Treatment Plant in Beijing, China. It was then cultured in a 2-L breaker containing culture medium composed of the following mineral substances: 2.7 g/ L CH3COONa, 0.3 g/L NH4Cl, 0.85 g/L NaNO3, 0.15 g/L KH2PO4, 0.1 g/ L MgSO47H2O and 0.005 g/L FeSO47H2O (Yao et al., 2013). The sludge was incubated for one month and the medium was refreshed every 4 days. The initial soluble organics in the inoculum were depleted after cultivation. Five 1-L conical flasks with rubber plugs were employed as the batch reactors. Two holes existed in the rubber plugs for N2 stripping and sampling. Each flask was purged with N2 and sealed with rubber plugs to maintain anaerobic condition. Each bioreactor, with different K+ concentrations, was filled with 1 L of synthetic nitrate wastewater, 10 mL of anaerobic sludge and 1 g of dried banana peel as the fixed carbon source. Nitrate-N was supplied in the form of NaNO3 at a concentration of 50 mg/L and soluble K+ was supplied in the form of KCl. The reactor without additional KCl was designated as control group B-0, while the bioreactors containing different concentrations of additional K+, namely 50, 100, 150 and 200 mg/L, were designed as B-5, B-10, B-15 and B-20, respectively. All bioreactors were provided with 1 g of fixed banana peel (size of 4.75 mm, dried at 105 °C for 24 h in an oven) as the carbon source. In addition, phosphorus used for bacterial growth originated from the dissolution of banana peel during these experiments. The dissolved COD of the banana peel was about 240.83 ± 7.50 mg/g, which meets the C/N ratio required for heterotrophic denitrification for three cycles (the initial nitrate concentration was 50 ± 5 mg-N/L at each cycle). All reactors were operated at 30 ± 2 °C with shaking at 150 rpm. Treatments were performed in duplicate and the mean values of the experimental data are reported. Finally, NO 3 -N reduction with banana peel as the carbon source under different K+ concentrations were individually assessed over three consecutive operating cycles. The end of an operating cycle was defined by the complete reduction of NO 2 -N and only NO3 -N was added, without carbon source, at the beginning of the next cycle. The suitable concentration range of soluble K+ during the denitrification process was also evaluated. Synthetic groundwater was prepared by amending deionized water with NaNO3, maintaining the nitrate concentration at 50 mg-N/L (Zhang et al., 2012). The phosphorus requirement for denitrification was approximately 0.02–0.03 g PO3 4 -P/g NO3 -N. The banana peel provided enough phosphorus to support the requirement of the microorganisms throughout the experiment. Thus, there was no need to add external phosphorus to the synthetic groundwater. In addition, the nitrogen content of the banana
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peel was 0.36 ± 0.02% and no accumulation of NO 3 -N occurred in the dissolution test. 2.2. Chemical and biological analyses Water samples taken from the reactors were immediately filtered through 0.22 lm membranes prior to analysis. The pH was immediately determined with a pH meter (Seven Multi S40, Met tler Toledo, Switzerland). NO 3 -N and NO2 -N were measured using a spectrophotometer (DR6000, HACH, USA) according to the Chinese NEPA standard methods. NH+4-N, PO3 4 -P and CODCr were monitored using phenol-sodium hypochlorite, the water and wastewater monitoring analysis method and, respectively (SEPA, 2002). K+ in the aqueous solution was analyzed by ICP-AES (ICAP 6000 SERIES, Thermo Fisher, Germany). The denitrification performance of the bioreactor was evaluated based on the NO 3 -N removal efficiency (NRE) and NO 3 -N removal rate (NRR), which are defined by Eqs. (1) and (2) (Zhao et al., 2018):
NREð%Þ ¼ ½ðC 0 C t Þ 100 =C 0
ð1Þ
NRRðmg N=ðL hÞÞ ¼ ðC 0 C t Þ =t
ð2Þ
where C0 is the initial NO 3 -N concentration (mg/L); Ct is the NO3 -N concentration at time t (mg/L); and t is the reaction time (h). DOM was characterized by three-dimensional excitationemission matrix (EEM) fluorescence spectroscopy using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) (All samples were diluted 5 times.). The slit width for both the excitation and emission was 10 nm and the scanning speed was set at 40 nm/s. Excitation and emission were simultaneously scanned at wavelengths ranging from 200 to 450 nm. After regulating scattering using interpolation in areas affected by Rayleigh and Raman scattering (He et al., 2011), the fluorescence regional integration (FRI) technique was used for analysis (Wei et al., 2014). Microbial samples from each bioreactor were collected and pretreated ultrasonically. The total genomic DNA was extracted using the FastDNAÒ SPIN Kit (MP Biomedicals, USA) following the manufacturer’s instructions. The extracted DNA was amplified with PCR primers 338F (50-ACTCCTACGGGAGGCAGCAG- 30) and 806R (50GGACTACHVGGGTWTC TAAT-30). A mixture of amplified samples was used for high throughput 16S rRNA gene analysis using the MiSeq platform (Illumina, USA), conducted by Shanghai Majorbio Technology (Shanghai, China). The sequences are available on the NCBI Sequence Read Archive under accession number PRJNA491377.
3
2.3. Statistical analysis + The variation in NO 3 -N, NO2 -N, NH4-N and COD, as well as the area of different DOM, were analyzed using Origin 9.1. Principal component analysis (PCA) was used for data reduction via SPSS version 20.0. In addition, the relationship between bacterial species, DOM dissolution, denitrification performance and K+ concentrations were analyzed.
3. Results and discussion 3.1. Denitrification performance Nitrate-N was gradually removed during the three consecutive operating cycles and microbial NO 3 -N reduction was clearly affected in the presence of different K+ concentrations (Fig. 1). Due to the promotion of microbial activity, NO 3 -N reduction was positively correlated with the K+ concentration up to 229.78 ± 25.80 mg-K/L (Table 1). The observed boundary condition for the K+ concentration was lower than the 1000 mg/L K+ obtained in a previous report (Cyplik et al., 2007). The reasons for this phenomenon are attributed to the different phase of carbon sources and bacterial species because the H. denitrificans used in the previous study is a halotolerant microorganism (Cyplik et al., 2007). The effect of the K+ concentration on banana peel-based heterotrophic denitrification transitioned from stimulation (<229.78 ± 25.80 mg-K/L) to inhibition (>229.78 ± 25.80 mg-K/L). When the K+ concentration exceeded the boundary condition, the B-20 bioreactor only reduced 85.73 ± 0.54 mg NO 3 -N during the three consecutive operating cycles, exhibiting the lowest NO 3 -N reduction rate (1.10 mg/(Lh)) in the first cycle (Table 1). This result clearly confirms that excessive K+ suppresses NO 3 -N reduction. Compared with the NO 3 -N removal efficiencies of B-0 (67.3% ± 0.6%), B-5 (80.7% ± 0.9%) and B-10 (93.9% ± 0.5%), B-15 exhibited the highest NO 3 -N removal efficiency (98.1% ± 0.2%) at 502 h during the three consecutive cycles. This indicates that the K+ concentration of 229.78 ± 25.80 mg-K/L can enhance the continuous denitrification ability with the same amount of solid phase carbon source. The obtained denitrification rates of the banana peel-based system during the first cycle was higher than the denitrification rates obtained in a methanol system (0.98 mg-N/(Lh)) (He et al., 2018) and a rice bran system (0.73–1.05 mg-N/(Lh)) (Hou, 2018). The denitrification rate during the second cycle was higher than the rates obtained in a fish waste system (0.19 mg-N/(Lh))
+ Fig. 1. Variations of NO 3 -N concentration in banana peel-based heterotrophic denitrification system with different K concentrations of three consecutive cycles.
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Table 1 Zero-order kinetic parameters of NO 3 -N removal in bioreactors over typical operating cycles. Reaction system
K1 (mg N/Lh) R2
B-0 B-5 B-10 B-15 B-20
1.9007 1.8931 1.8714 1.8581 1.1044
K2 (mg N/Lh) R2 0.9779 0.9745 0.9720 0.9709 0.9197
0.3268 0.3558 0.4521 0.4515 –
K3 (mg N/Lh) R2 0.9794 0.9866 0.9864 0.9562 –
0.0825 0.1205 0.1290 0.1456 –
0.9447 0.9829 0.9327 0.9011 –
* K1: reduction rate of nitrate in the first cycle; K2: reduction rate of nitrate in the second cycle; K3: Reduction rate of nitrate in the third cycle.
Table 2 Zero-order kinetic equation and parameters of the production and reduction of NO 2 -N in bioreactors over the first typical operating cycle, C0 (mg/L) and Ct (mg/L) are the concentrations of NO 2 -N at initial condition and time t (h), respectively. NO 2 -N production
Reaction system
B-0 B-5 B-10 B-15 B-20
NO 2 -N reduction 2
K0 (mg N/Lh)
R
K (mg N/Lh)
R2
1.1683 1.1797 1.1349 1.0992 0.8584
0.9587 0.9800 0.9550 0.9526 0.9186
0.6676 0.6871 1.0518 0.8604 0.5613
0.9865 0.9801 0.9926 0.9810 0.9371
* K0: the production of NO2-N in bioreactors over the first typical operating cycle; K: the reduction of NO2-N in bioreactors over the first typical operating cycle.
(He et al., 2018) and a eucalyptus mulch system (0.11 mg-N/(Lh)) (He et al., 2018). Due to inhibition by excessive K+, the NO 3 -N reduction capacity of the banana peel in B-20 was significantly lower than the amount that could be reduced in the banana peel-based denitrification system (164.42 ± 1.15 mg-N/g-BP). Excessive K+ may have caused the cell salinity to be so high that it inhibited ion exchange, thereby permeating between intra- and extra-cellular spaces, and the microorganism in our system was not halotolerant. During the first typical operating cycle, 50 ± 2 mg-N/L was completely bioreduced within 24 h in B-0, B-5, B-10 and B-15, which all showed similar NO 3 -N reduction rates (Table 1), while a removal efficiency of 63.4% ± 0.0% was obtained in B-20 (p < 0.05). This reveals that the threshold K+ concentration for NO 3 -N reduction in the banana peel-based denitrification system was around 229.78 ± 25.80 mg-K/L and that the denitrification process would be restrained if this value exceeded the boundary condition. However, in the following operating cycle, both B-10 and B-15 exhibited the same and highest NO 3 -N reduction rate (0.45 mg/(Lh)), followed by B-5 (0.36 mg/(Lh)) and B-0 (0.33 mg/(Lh)), while B-20 could not continue to degrade NO 3 -N after 166 h. During the third operating cycle, B-15 showed the highest NO 3 -N removal rate (0.15 ± 0.03 mg/(Lh)), followed by B-10 (0.13 ± 0.03 mg/(Lh)), B-5 (0.12 ± 0.07 mg/(Lh)) and B-0 (0.08 ± 0.02 mg/(Lh)) (p < 0.05) (Table 1). This further indicates that the K+ concentration of 229.78 ± 25.80 mg-K/L had a lasting promotion effect on denitrification. During the denitrification process, some intermediate reactions may occur synchronously, as follows (Eqs. (3)–(7)) (Li et al., 2016):
3.2. The evolution of dissolved organic matter (DOM)
2NO3 + 4Hþ + 4e !2NO2 + 2H2 O
ð3Þ
2NO2 + 4Hþ + 4e !2NO + 2H2 O
ð4Þ
2NO + 2Hþ + 2e !N2 O + H2 O
ð5Þ
N2 O + 2Hþ + 2e !N2 + H2 O
ð6Þ
2NO3 + C6 H12 O6 + 6Hþ !3NH4 þ + 6CO2 + 3H2 O NO 2 -N
NH+4-N
2002), while little accumulation of NO 2 -N (below the detection limit) and NH+4-N (2.61–3.09 mg-N/L) appeared at the end of the reaction. In addition, B-10 showed the highest NO 2 -N reduction rate (1.05 mg/(Lh)) during the NO 2 -N reduction process, followed by B-15 (0.86 ± 0.03 mg/(Lh)), B-5 (0.69 ± 0.05 mg/(Lh)) and B-0 (0.67 ± 0.04 mg/(Lh)), while B-20 exhibited a rate of 0.56 ± 0.05 mg/(Lh) (p < 0.05). B-10 showed the highest NO 2 -N reduction rate among all the reactors, as excessively high or low K+ concentrations decreased NO 2 -N reduction (Fig. S3). Results demonstrate that the K+ boundary conditions for NO 3 -N and NO 2 -N reduction were 229.78 ± 25.80 and 159.10 ± 24.60 mg-K/L, respectively, in the banana peel-based denitrification system. Furthermore, the banana peel could achieve efficient nitrate removal during long-term operation (398 h; Fig. S6). Variation of pH in five bio-reactors (Fig. S7) showed the same sharply decreasing trend from 0 to 28 h, but then gradually increased and finally stabilized at 7.0–7.5. The optimum pH for nitrate and nitrite reduction ranges from 7 to 8, which is a range that is most suitable for the survival and function of denitrifying microorganisms. During the reaction process, the carbon source was first fermented to produce acid under the action of fermentative microorganisms. The denitrifying bacteria could then use the small molecular acid produced by carbon source fermentation to achieve nitrate reduction. Denitrification is a biological process of alkali production. The OH generated during the denitrification process can neutralize the acid produced by fermentation, thereby increasing the pH during the later stage of the reaction.
ð7Þ
The small amount of and implies the presence of multiple metabolic pathways, including denitrification and dissimilatory nitrate reduction to ammonium (Fig. S2) (Patterson et al.,
The main banana peel components included 12.50% cellulose, 19.90% hemicellulose and 14.10% lignin. To study the changes of these components during the reaction, the DOM was analyzed by EEM. DOM, a heterogeneous mixture of complex organic compounds, includes organic acids, amino acids, humic substances and enzymes (He et al., 2011). It can work as an electron shuttle and mediate electron transfer between reducing agents and electron acceptors (Dawson and Hilton, 2011). These actions could accelerate the composting process by accelerating the degradation of persistent organic matter, such as chloro-organics and nitrophenols (Zhu et al., 2013). Nutrients such as cellulose, hemicellulose and lignin in banana peel can be degraded to glucose, hexose and
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pentose under the action of hydrolases (Jönsson et al., 2013). In addition, these nutrients can dissolve a large amount of DOM; however, the evolutionary pathway of specific DOM components remains to be further clarified. Therefore, the DOM in this study was analyzed by EEM to illustrate the evolution of carbon compounds. Regions I, II and IV were associated with protein-like substances (tyrosine-like, tryptophan-like and soluble microbial byproduct-like substances), whereas regions III and V represented fulvic acid-like and humic acid-like substances, respectively (Chen et al., 2003). A dissolution phenomenon was observed at the beginning of the reaction, with the main peak appearing in region V and an increased integral area (Fig. 2). As the reaction proceeded, the main peaks shifted to regions I and II, and the integral area decreased, meaning that the substances of all regions decreased and the main humic-like components were degraded into low molecular substances (confirmed in Fig. S3). The migration and transformation of the EEM fluorescence spectra reflected the order of DOM utilization (Fig. 2). Protein-like substances (tyrosine-like, tryptophan-like and soluble microbial by-product-like substances) were easily utilized by the denitrifiers due to their easily degradable structures, and the substances of region V were degraded into low molecular weight compounds after being used by the microorganisms (Fig. 2). This result agrees with a previous study, which reported that microorganisms preferentially use labile organic substances in the DOM (e.g. aliphatics, polysaccharides and proteins) during the reaction process, whereas DOM compounds with aromatic structural units were not easily degraded and generally accumulated (Said-Pullicino and Gigliotti, 2007). The change in the amount of DOM during the reaction was measured by the change in COD. The dissolution of organic matter is of positive significance in promoting the heterotrophic denitrification process. However, it is undeniable that residual organic matter could cause secondary
5
COD pollution. At present, physical adsorption methods, such as activated carbon, anthracite and bamboo charcoal, can achieve efficient COD removal (Hata et al., 2016; Mueller et al., 2001). Therefore, in practical engineering applications, COD adsorption could be integrated as a back-end process in denitrification units. Alternatively, COD concentration in wastewater can be reduced by optimizing reactor design including size and packing addition. B-15 exhibited the largest degradation of humic-like substances of 10.59% at the end of the reaction, followed by B-10 (8.17%), B-5 (5.50%), B-0 (4.88%) and B-20 (2.25%) (Fig. S3). Meanwhile, the largest protein-like substance generation occurred in B-15 (7.69%), followed by B-10 (6.78%), B-5 (5.46%), B-0 (3.00%) and B-20 (2.45%) (Fig. 4). The substances of regions I and II were derived from the inherent self-dissolution of the banana peel and the degradation of region V substances. These changes indicate that the utilization degree of DOM was positively correlated with the K+ concentration up to 229.78 ± 25.80 mg/L. Thus, the higher K+ concentration, the more humic-like substances are converted to protein-like compounds within the boundary condition. In addition, correlation analysis was used to evaluate the correlation between the K+ concentration and the change in different regions (regions I + II and region V) (Table S2). There was a significant positive correlation between the change in different regions (the rate of decline in the percentage of region V and increase in regions I + II) and K+ concentration when K+ was below the boundary condition. When the concentration of K+ was above the boundary condition, the degradation of organic matter was suppressed, which had a positive correlation with the denitrification performance under different K+ concentrations, especially during cycles 2 and 3 (confirmed in Table S3). This conclusion indicates that additional K+ enhances the degree of DOM degradation and utilization in the banana peel, and simultaneously improves the denitrification rate. Banana peel fermentation is similar to the composting process,
Fig. 2. EEM spectra of each cycle of different K+ concentration in banana peel-based denitrification system (all samples were diluted 5-fold). (the EEM image represent B-0 (a), B-5 (b), B-10 (c), B-15 (d) and B-20 (e), there were only two cycles in B-20 and then keep stable nitrate concentration without any reaction).
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with both processes achieving humification of waste organic compounds. From this, we can infer that additional K+ can promote composting to some extent. The optimized K+ concentration for the degradation of organic matter was around 229.78 ± 25.80 mg/ L in the banana peel-based denitrification system, and the optimized K+ concentration of DOM degradation was consistent with + the reduction of NO 3 -N. Moreover, the K concentration experiment further confirmed that K+ addition (Fig. 1) accelerated the denitrification rate by promoting the dissolution of organic substances within the appropriate K+ concentration range (Fig. S3). This result is consistent with a previously reported study indicating that K+ can effectively enhance the degradation of organics (Li et al., 2018). Analysis from compound 1 indicates that the concentration of K+ was negatively correlated with the dissolution of organic substances from the banana peel (Fig. 3), which further indicates that addition K+ can improve the dissolution of organic matter from the banana peel. According to the calculation and PCA of different regions in banana peel dissolution, the amount of dissolution change in regions I, II and III had a negative effect with the amount in regions IV and V in compound 2 (Fig. 3). This result demonstrates that the reduction of regions IV and V transferred into regions I, II and III, which were more easily degraded in subsequent reactions. Appropriate addition of K+ could promote the dissolution of organic matter from the banana peel and the increase of organic matter dissolution will increase the amount of degradable organic matter, thereby improving the efficiency of the carbon source. 3.3. Microbial community and mechanisms A total of 46743, 59973, 56112, 69810, 55,341 and 64,675 highquality reads with an average length of 442 bp were recorded from blank sample B-0, B-5, B-10, B-15 and B-20, respectively. Chao 1 and ACE indexes (Table S1) indicate that the abundances in B-0, B-5, B-10 and B-15 were higher than those of B-20 and the blank sample. This implies that the K+ concentration (below the boundary condition) may lead to an increased abundance, while excessive K+ had an inhibitory effect on abundance. The Simpson and Shannon diversity indexes can provide insights into species richness and evenness (Liu et al., 2017). The higher Shannon index and lower Simpson index suggest a higher diversity and evenness in the five samples (B-0, B-5, B-10, B-15 and B-20) compared with blank sample, indicating that K+ can weaken the environmental selectivity of
microorganisms and improve the ability of different functional species to adapt to the same environment (Table S1). It has been previously reported that denitrification can be facilitated by appropriate addition of K+ because it plays an important role in stimulating bacterial activity (Cyplik et al., 2007). Analysis of the 16S rRNA gene sequences further proved that the K+ concentration affected the community structure and relative abundance of dominant functional species. The effect of K+ on the abundance of the microbial community members apparently transitioned from stimulation (<229.78 ± 25.80 mg-K/L) to inhibition (>229.78 ± 25.80 mg-K/L) (Table S1). B-0, B-5, B-10 and B-20 exhibited the higher diversity of microbial species for fermentation and denitrification when compared with the blank sample, (Table 3, Fig. 4(a)), which also corresponded to the denitrification and fermentation performance. These results could be attributed to the interaction between performance and different functional species. A microbial community shift was observed in the five bioreactors at the genus level (Fig. 4(a)). Diaphorobacter, which is closely related to denitrification and can synthesize nitrate and nitrite reductases (Chakravarthy et al., 2011), exhibited relative abundances of 10.05%, 20.07%, 21.84%, 18.29% and 1.85% in B-0, B-5, B-10, B-15 and B-20, respectively. This indicates that the most suitable K+ concentration range for the enrichment of Diaphorobacter is around 114.04 ± 25.38 mg-K/L to 229.78 ± 25.80 mg-K/L. Paludibacter is involved in biodegradation and denitrification processes under anoxic conditions and can utilize various sugars, including glucose, starch and sucrose, while simultaneously producing propionate and acetate (Ueki, 2006; Qiu et al., 2014). The relative abundance of Paludibacter decreased from 15.22% to 1.36% as the K+ concentration increased from 81.88 ± 11.22 mg-K/L to 292.32 ± 28.20 mg-K/L (Table 3). This indicates that the relative abundance is negatively correlated with the K+ concentration. Thauera is capable of degrading isopropanol and acetone under denitrifying conditions (Fida et al., 2017). The relative abundance of Thauera increased from 1.61% (80.88 ± 21.66 mg/L of K+) to 10.15% (229.78 ± 25.80 mg-K/ L) and then decreased to 1.3% (292.32 ± 46.12 mg-K/L). These results significantly confirm that K+ concentrations below 229.78 ± 25.80 mg-K/L are beneficial to Thauera enrichment but concentrations above 229.78 ± 25.80 mg-K/L will inhibit its growth. Furthermore, the relative abundance of Anaerolineaceae, which is associated with the degradation of refractory dissolved organic matter (rDOM) (Li et al., 2018), decreased with increasing K+ concentrations, verifying that a K+ concentration above 80.88 ± 11.22 mg-K/L
Fig. 3. Correlation matrix between K+ concentration and DOM dissolution (a) and PCA of the correlation between K+ concentration and DOM dissolution of different regions (b). * R1: the change between starting and ending in Region I (DRegion I), R2: the change between starting and ending in Region I (DRegion II), R3: the change between starting and ending in Region I (DRegion III), R4: the change between starting and ending in Region I (DRegion IV), R5: the change between starting and ending in Region I (DRegion V), and Ck: different K+ concentration.
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H. Wang et al. / Science of the Total Environment 703 (2020) 134830 Table 3 Relative abundance of functional species at genus level in different reaction systems. Function
Genus
B-0
B-5
B-10
B-15
B-20
Fermentation
Dechlorobacter Paludibacter norank_f_Anaerolineaceae norank_f_Porphyromonadaceae Diaphorobacter Simplicispira Thauera Thiobacillus Pseudomonas Closlridium_sensu_stricto Lentimicrobium norank_o_Ignavibacteriales
– 15.22 10.33 2.93 10.05 8.22 1.61 – – – 8.1 3.18
3.1 11.62 2.75 5.34 20.07 10.03 2.21 – – – 6.22 1.95
8.48 10 1.77 1.78 21.48 3.13 4.44 – – – 6.69 –
8.09 4.3 2.18 1.8 18.29 12.15 10.15 1.52 1.86 1.46 4.16 –
– 1.36 1.32 – 1.85 – 1.3 – – – 1.91 –
Denitrification
Fig. 4. Microbial community structure and multivariate statistical analysis. (a) 1Heatmap to profile the most abundant genera (top 30) of five bioreactors and blank sample (The color bar indicates the range of the percentage of a genus in a sample, based on the color key at the bottom). (b) Correlation matrix between K+ concentration and functional species. (c) PCA of the correlation between K+ concentration and functional species. * K: K+ concentration, De: Dechlorobacter, P: Paludibacter, NA: norank_f_Anaerolineaceae, NP: norank_f_Porphyromonadaceae, Di: Diaphorobacter, T: Thauera, L: Lentimicrobium, S: Simplicispira.
inhibits its activity. Dechlorobacter, which can use nitrate as electron donor in polyhydroxybutyrate valerate (PHBV)-supported systems (Xu et al., 2018), was not only present in the K+ concentration range of 114.04 ± 25.38 to 229.78 ± 25.80 mg-K/L, but also increased from 3.10% to 8.09% with increasing of K+ concentrations. This indicates
that a K+ concentration between 114.04 ± 25.38 and 229.78 ± 25.80 mg-K/L is suitable for its survival and that concentrations outside this range will suppress its enrichment. Simplicispira has the ability to reduce nitrate under anoxic conditions with PBS as carbon source and a biofilm carrier for wastewater treatment (Zhu et al.,
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Fig. 5. Proposed mechanisms of nitrate reduction and DOM degradation of banana peel in B-15.
2015). Interestingly, B-15 exhibited the highest relative abundance of Simplicispira (12.15%), followed by B-5 (10.03%), B-0 (8.22%) and B-10 (3.13%), while it was not found in B-20 (Table 3). Thus, a K+ concentration exceeding the boundary condition is not conducive to the formation of Simplicispira. Lentimicrobium, whose relative abundance decreased with the increasing K+ concentration, might participate in the fermentation of complex organics in food waste (Sun et al., 2016). These enriched denitrifiers and fermentative bacteria may be responsible for the removal of nitrate and the degradation of organic matter. Moreover, Pseudomonas, Closlridium and Thiobacillus were the unique functional species in B-15. Pseudomonas exhibits high resistance to aromatic pollutant toxicity and high degradation efficiencies for a wide variety of aromatic compounds under various environmental conditions. Thiobacillus is an autotrophic denitrifying bacteria that is also is known to simultaneously reduce sulfate and nitrate in the presence of high concentration of organics (Park et al., 2015). These bacteria are beneficial to the degradation of NO 3 -N and complex organic matter (DOM), thereby explaining why the highest denitrification rate was achieved in B-15. In addition, B-15 accounted for the largest proportion of denitrifying functional species (Table 3), which is consistent with the denitrification process shown in Fig. 1. This may be attributed to the fact that denitrifiers can stimulate biodenitrification, thereby accelerating the reaction rate. Moreover, different functional species have their own optimal K+ concentration range, which was indeed demonstrated by our experiments. In addition, the data presented in Fig. 4(b) indicates the correlation between different parameters, namely the K+ concentration, Dechlorobacter, Paludibacter, norank_f_A-naerolineaceae, norank_f_Porphyromonadaceae, Diaphorobacter, Thauera, Lentimicrobium and Simplicispira. These analyses further verify that the K+ concentration was positively cor-
related with Dechlorobacter (fermentor) and Thauera (denitrifier) (p < 0.05) within the boundary condition determined by statistical analysis. The mechanisms of denitrification and fermentation in the B-15 reactor are summarized in Fig. 5. B-15 promoted the degradation of humic-like substances by 217.0%, 192.5% and 129.6% when compared with B-0, B-5 and B-10, respectively, while the excessive K+ in B-20 (above the boundary condition of 229.78 ± 25.80 mg-K/L) suppressed the degradation of humic-like substances, further proving that the K+ boundary condition for promoting fermentation is around 229.78 ± 25.80 mg-K/L. In addition, the denitrification rates of B-15 were 136.4% and 125.0% higher than B-0 and B-5, respectively, during the second cycle, and 187.5%, 125.0% and 115.4% higher than B-0, B-5 and B-10 in the third cycle, respectively, confirming that the K+ boundary condition enhanced the denitrification process in the banana peelbased system. Moreover, the detection of Diaphorobacter, Thauera, Paludibacter, norank_f_Anaerolineaceae and Lentimicrobium verified that both DOM (dissolved from the banana peel) oxidation and nitrate removal can be achieved simultaneously in a banana peel-based system. B-15 had the most abundant microbial community of all the bioreactors, with the presence of Pseudomonas and Thiobacillus being very beneficial for promoting denitrification. Furthermore, this study found that the survival of a large proportion of denitrifiers and fermentative bacteria (e.g. Diaphorobacter and Thauera) was positively correlated with the K+ concentration (below the boundary condition of 229.78 ± 25.80 mg-K/L). 4. Conclusions This study elucidated the stimulatory and inhibitory effects of K+ on denitrification, DOM degradation and microbial community structure in a banana peel-based heterotrophic denitrification sys-
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tem under the given treatment conditions. The main results were as follows: (i) the nitrate removal performance was promoted by K+ up to 229.78 ± 25.80 mg-K/L, but nitrate reduction was significantly inhibited at higher concentrations; (ii) the DOM degradation pathway was demonstrated by showing that protein-like substances are used first, followed by the conversion of humic-like substances into low-molecular weight substances for microorganisms utilization, which leaves macromolecular humic-like substances that cannot be biodegraded; and (iii) microbial community analysis of the five bioreactors showed that the genera Pseudomonas (1.86%) and Thiobacillus (1.52%) are the unique functional species in B-15, which exhibited the best nitrate bioreduction characteristics. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (NSFC) (No. 51578519; No. 21876159) and the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07 202002). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2019.134830. References Chakravarthy, S.S., Pande, S., Kapoor, A., Nerurkar, A.S., 2011. Comparison of Denitrification Between Paracoccus sp. and Diaphorobacter sp. Appl. Biochem. Biotechnol. 165 (1), 260–269. https://doi.org/10.1007/s12010-011-9248-5. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. https://doi. org/10.1021/es034354c. Cheung, H.N.B., Huang, G.H., Yu, H., 2010. Microbial-growth inhibition during composting of food waste: Effects of organic acids. Bioresour. Technol. 101 (15), 5925–5934. https://doi.org/10.1016/j.biortech.2010.02.062. Cyplik, P., Grajek, W., Marecik, R., et al., 2007. Effect of macro/micro nutrients and carbon source over the denitrification rate of Haloferax denitrificans archaeon. Enzyme Microb. Technol. 40 (2), 212–220. Dawson, C.J., Hilton, J., 2011. Fertiliser availability in a resource-limited world: Production and recycling of nitrogen and phosphorus. Food Policy 36, S14–S22. https://doi.org/10.1016/j.foodpol.2010.11.012. Feng, L., Chen, K., Han, D., Zhao, J., Lu, Y., Yang, G., Mu, J., Zhao, X., 2017. Comparison of nitrogen removal and microbial properties in solid-phase denitrification systems for water purification with various pretreated lignocellulosic carriers. Bioresour. Technol. 224, 236–245. https://doi.org/10.1016/j.biortech.2016.11.002. Fida, T.T., Gassara, F., Voordouw, G., 2017. Biodegradation of isopropanol and acetone under denitrifying conditions by Thauera sp. TK001 for nitratemediated microbially enhanced oil recovery. J. Hazard. Mater. 334, 68–75. https://doi.org/10.1016/j.jhazmat. 2017.03.061. Hata, M., Amano, Y., Thiravetyan, P., et al., 2016. Preparation of bamboo chars and bamboo activated carbons to remove color and COD from ink wastewater. Water Environ. Res. 88 (1), 87–96. He, Q., Zhang, D., Main, K., Feng, C., Ergas, S.J., 2018. Biological denitrification in marine aquaculture systems: A multiple electron donor microcosm study. Bioresour. Technol. 263, 340–349. https://doi.org/10.1016/j.biortech.2018.05.018. He, X., Xi, B., Wei, Z., Guo, X., Li, M., An, D., Liu, H., 2011. Spectroscopic characterization of water extractable organic matter during composting of municipal solid waste. Chemosphere 82, 541–548. https://doi.org/10.1016/j. chemosphere.2010.10.057. Hou, T., n.d. Enhancement of rice bran as carbon and microbial sources on the nitrate removal from groundwater. Biochemical Engineering Journal https://doi. org/10.1016/j.bej. 2018.07.010. Jafari, S.J., Moussavi, G., Yaghmaeian, K., 2015. High-rate biological denitrification in the cyclic rotating-bed biological reactor: Effect of COD/NO3, nitrate concentration
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