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Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions
Accepted Manuscript Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions Zhen Wang, Yahui Jiang, Mukesh Kumar Awasthi, Jiao Wang, Xinguo Yang, Ali Amjad, Quan Wang, Altaf Hussain Lahori, Zengqiang Zhang PII: DOI: Reference:
S0960-8524(17)32124-7 https://doi.org/10.1016/j.biortech.2017.12.009 BITE 19264
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
1 November 2017 5 December 2017 6 December 2017
Please cite this article as: Wang, Z., Jiang, Y., Awasthi, M.K., Wang, J., Yang, X., Amjad, A., Wang, Q., Lahori, A.H., Zhang, Z., Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.12.009
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Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions Zhen Wang a, Yahui Jiang a, Mukesh Kumar Awasthi a, b, Jiao Wang a, Xinguo Yang c, Ali Amjad a, Quan Wang a, Altaf Hussain Lahori a, Zengqiang Zhang a* a
College of Natural Resources and Environment, Northwest A&F University, Yangling,
Shaanxi 712100, China b
Department of Biotechnology, Amicable Knowledge Solution University, Satna, India
c
Key Laboratory for Restoration and Reconstruction of Degraded Ecosystem in
Northwestern China of Ministry of Education, Ningxia University, Yinchuan 750021, China
*Corresponding author: Prof. Zengqiang Zhang College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi Province 712100, PR China Tel./Fax: +86-013609254113; +86 029-87080055. E-mail address: [email protected] (Z.Q. Zhang). 1
Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions Zhen Wang a, Yahui Jiang a, Mukesh Kumar Awasthi a, b, Jiao Wang a, Xinguo Yang c, Ali Amjad a, Quan Wang a, Altaf Hussain Lahori a, Zengqiang Zhang a* Abstract: In this current study, sawdust and zero-valent iron (Fe0) were used as co-electron donors to evaluate the effects of coexistent ions on the combined heterotrophic and autotrophic denitrification (HAD) processes. The results showed that HCO3- and SO42- drastically enhanced nitrate removal. The promotion effect derived from both biological and chemical process by HCO3- and chemical process by SO42-. However, Ca2+ ions would remarkably increase nitrate removal due to promoting the electron transfer and the metabolic activities of bacteria, whereas the Cu2+ ions inhibited the biological process due to the deleterious effect on bacteria. Meanwhile, Fe2+ and Fe3+ ions exhibited inhibition effect firstly because of their toxicity to bacteria and promotion subsequently due to their enhancement on Fe0 chemical denitrification. Moreover, byproducts such as nitrite, ammonium, dissolved organic carbon (DOC), etc. were also influenced by common ions. Keywords:
Nitrate removal, Cations, Anions, Sawdust, Iron.
2
1. Introduction Excess accumulation of nitrate (NO3-) in drinking water which is harmful to human health due to its contribution to methemoglobinemia, gastric cancer, and non-Hodgkin lymphoma (Sunger and Bose 2009; Wang et al., 2012). The intensive application of especially nitrogenous fertilizers, sewage irrigation, and industrial waste effluent discharge are the major sources of nitrate contamination in ground and surface waters (Zhao et al., 2012; Li et al., 2016). World Health Organization (WHO) stipulated the maximum NO3--N concentration in drinking water is 10 mg N L-1 (WHO, 2008). Among the current remediation technologies, biological denitrification which occurs naturally when denitrifying bacteria use nitrate as a terminal electron acceptor in their respiration process in anaerobic environment, which is considered to be one of the most economical and widely used remediation methods (Schipper et al., 2010; Sahinkaya et al., 2015; Wang and Chu, 2016). During biological denitrification processes nitrate which is finally transformed into nitrogen gas, with formation some nitrogen intermediates, as indicated by Eq. (1): NO3-→ NO2-→ NO → N2O → N2 Biological denitrification includes heterotrophic denitrification (HD) and autotrophic denitrification (AD). Recently, a combined heterotrophic and autotrophic denitrification (HAD) has been proved to be more effective than HD and AD for nitrate removal from groundwater due to the synergetic relationship between HD and AD (Rocca et al., 2007; Huang et al., 2015; Wang et al., 2015).
3
In HAD symbiotic system, Fe0 was used to create an anaerobic environment in water and produce hydrogen. The low DO levels could favor the heterotrophic denitrifiers utilizing cellulose organic carbons as electron donors to degrade nitrate and meanwhile generate CO2. The CO2 can be used as an inorganic carbon source and the H2 as an electron donor by the autotrophic denitrifiers to remove nitrate via autotrophic denitrification process. Although the synergistic and promotive effects mechanisms of the HAD were encouraging, HAD is still not used for nitrate in-situ remediation in groundwater. HAD supported by cellulose materials and Fe0 is adequate for use in permeable reactive barriers (PRBs), because both cellulose materials and Fe0 have been used in-situ nitrate remediation for a long time (Rocca et al., 2007; Schipper et al., 2010; Schmidt and Clark, 2012). The PRB is a very attractive promising technology due to the relative simplicity, low investment and more cost effectiveness in situ groundwater treatment, but it is difficult to control (Wang and Chu 2016). A few reports related to HAD mainly focused on the kinds of electron donors, pollutants and bacteria (Huang et al., 2015; Li et al., 2016),however，it still requires lots of studies about the background circumstance effects on nitrate removal by HAD system, prior to real in situ nitrate remediation application. Common ions such as SO42−, Cl−, HCO3−, Fe2+, Mg2+ and Ca2+ are ubiquitous in groundwater and soil. These ions would pose different effects on the chemical reactivity of Fe0 and the structure or metabolic activities of bacteria. Previous studies have showed that common coexistent ions in soil and groundwater would affect the Fe0 4
reactivity depending on the target pollutants (Liu et al., 2007; Liu and Lo 2011). Some cations (Fe2+, Fe3+ and Cu2+) and anions (SO42−, Cl−, HCO3−) could promote nitrate reduction by Fe0 but there were no distinct effect by Ca2+, Na+ , K+ , and PO43− behaved inhibition effect (Tang et al., 2012). Moreover, the activities of microorganisms will also be influenced by ions because some ions may cause competitive or non-competitive inhibition of microbial enzymes (Zhao 2005). Ting et al. (1994) reported that nitrogen and phosphorus biological removal would be significantly decreased due to the addition of copper. It would strongly inhibit anammox activity, especially in the presence of NO2- (Zhang et al., 2016). In addition, some anions with high concentrations would result in the damage of cell membranes, and caused the leakage of cell inclusion (Wang et al., 2016). Consequently, HAD system includes both bacteria and Fe0, so the presence of common ions may affect the nitrate removal by chemical or biological process. Considering application of the HAD process for nitrate in-situ remediation, the co-exist ions in water needs to be taken care of. And to date, no attempt has been made to describe about the impact of common ions on HAD. In light of the existing work, the main objective of this study was to investigate the effects of co-exist ions on nitrate removal by sawdust and Fe0 supported HAD. To achieve this goal, the effects of 6 common cations and 4 inorganic anions at two different concentration levels on nitrate removal were evaluated. A pseudo-first-order model was introduced to describe the degradation kinetics of nitrate. Furthermore, nitrite, ammonium, DOC and soluble iron which produced during the nitrate removal process were also examined. The observed 5
results may provide more possible guidance to practical application of HAD technology. 2. Materials and methods 2.1. Materials The feed solution was prepared with nitrate (as KNO3) and phosphate (as K2HPO4) at an N/P weight ratio of 22:3, and the pH was about 6.5. Cation and anion stock solutions were prepared with corresponding chloride (NaCl, CaCl2, MgCl2·6H2O, CuCl2·2H2O, FeCl2·4H2O, FeCl3·6H2O) and potassium salts (KH2PO4，K2SO4，KCl， KHCO3), respectively. All the chemicals used in this study were of analytical grade, and all aqueous solutions were prepared with deionized water. Chinese parasol is wide distribution and low cost in China, and it’s frequently used as raw material for manufacturing. In this study, Chinese parasol sawdust was purchased from a wooden door company in Yang Ling city, Shaanxi, China. It was air dried and milled with a lab grinder. The final powder was sieved with a 1-mm screen. Finally, it was sterilized in a vacuum drying oven at 121°C for 60 min and vacuum-stored after cooling for further experiments. We can see the sawdust surface is rough and porous under a scanning electron microscope (SEM), and the C, N content in sawdust were 52.30 ± 0.335%, 0.307± 0.019% respectively. The iron particle (Fe0) (Tianjin Zonghengxing Industrial & Trading Chemical Reagent Co., China), was of 98% analytical grade, and irregular in shape with smooth and scale surface. The sludge was obtained from a sewage treatment plant in Xi Ning city, Qinghai, China. The C, N and P content in sludge were 27.3%, 6.24% and 7.63% as measured by an elemental analyzer. 6
2.2. Inoculums Ten grams of wet sludge was added as primary bacterial source into a flask which contained 5.00 g of sawdust and 5.00 g of Fe0. The flask was filled with 1000.0 mL of nitrate solution spiked with 50 mg L-1 of NO3--N, and the C/N/P weight ratio of approximately 1000/22/3 (Rocca et al., 2005). The flask was purged with Ar gas for 20 min to remove dissolved oxygen, then sealed and incubated at 25 °C in dark without shaking until nitrate and the intermediate product of nitrite were completely removed. Nitrate and nitrite concentration was measured every 2 d. 2.3. Experiments The effects of common ions on the denitrification activity of HAD were studied in 1000 mL tapered flasks. Five grams of Fe0, 5.00 g of sawdust and 100.0 mL of bacteria solution were added into each reactor, followed by adding nitrate and specific ion stock solutions. The initial pH value of nitrate solution was about 6.5, and the nitrate concentration was 50 mg N L-1. Cations concentration were Ca2+ (200, 400 mg L-1), Mg2+ (200, 400 mg L-1), Cu 2+ (100, 200 mg L-1), Fe2+ (50, 100 mg L-1) and Fe3+ (50, 100 mg L-1). The anions concentration were Cl- (30, 120 mg L-1), SO42- (50, 200 mg L-1), PO43- (5, 20 mg L-1), HCO3- (100, 400 mg L-1) separately. All the ion concentrations were according to their typical concentrations in natural groundwater (Dou et al., 2010). After the Ar gas bubbling, all the reactors were sealed tightly and mixed well before transferring to a constant temperature incubator (HPS-250; China) for static incubation at 25 °C under a dark condition. Aliquots of the samples were taken with a10-mL disposable syringe at certain time 7
intervals, and analyzed immediately after being filtered through a 0.45µm filter membrane. The filtrate was collected for pH, nitrate, nitrite, ammonium, ions and dissolved iron and organic carbon measurement, and all tests were conducted in duplicate. 2.4 Analytical methods The surface morphology of iron powder and sawdust was observed by a JSM-6360LV scanning electron microscope (JEOL, Japan). Nitrate was measured using a UV spectophotometric screening method at 220 and 275 nm (UVmini-1240, Shimadzu). Nitrite and ammonium ions were analyzed by the colorimetric method at 540 nm and 636 nm respectively (Tang et al., 2012). The initial pH value of each reactor was measured with a Mettler Toledo 320-S pH-meter. Dissolved organic carbon (DOC) was measured using a TOC analyzer (HACH, IL530 TOC-TN). Atomic absorption spectrophotometer was used to determine the concentration of dissolved iron (Hitachi ZA3000). Anions were analyzed using an ion chromatograph (861 advanced compact IC, Metrohm) and cations were measured with an inductively coupled plasma mass spectrometry (ICP-MASS, Agilent 7800) (SEPA, 2002). 2.5 Nitrate degradation rate constants The pseudo-first-order model as follow was applied to describe the removal of nitrate by HAD system (Su et al., 2012): C = (C0-Cf)·e-kt + Cf
(2)
In this expression, C is the nitrate concentration at any time, C0 is the initial concentration, Cf is the final concentration, k expresses the pseudo-first-order rate 8
constant (h-1), and t is the reaction time expressed in hours. 3. Results and discussion 3.1. The effect of anions Effects of various anions at two different concentration levels on nitrate removal, nitrite/ammonium acummulation by HAD system was shown in Fig. 1. In control system, nitrate decreased sharply during the first 48 h then slowed down and completely removed within 240 hours (Fig.1a). As shown in Table 1, all anions except Cl- enhanced nitrate reduction in HAD. Compared to other anions, Cl- exhibited non-significant influence on nitrate degradation, nitrite/ammonium accumulation (Fig. 1b and Fig. 1c) and the total nitrogen removal (Fig. 2a). In previous studies, Cl- performed enhancement for nitrate removal (Tang et al., 2012), and 4-chloronitrobenzene degradation (Devlin and Allin 2005) by Fe0, because it can promote iron pitting corrosion and increase ionic strength. However, Cl- could cause the cell wall broken and the protoplast disintegrated. More importantly, chloride could remarkably inhibit the activity of nitrate reductase (Wang et al., 2016). In HAD system, the enhancement of Cl- on Fe0 corrosion may just offset the inhibition on denitrification of denitrify bacteria. From Fig. 1a, it could be seen that SO42- promoted nitrate removal by HAD after 72 h reaction, especially when it was at a higher concentration (200 mg L-1). As the SO42- concentration drastically increased from 50 to 200 mg L-1, the rate constant increased from 1.01e-2 to 1.41e-2 h-1. In the reaction of 72 h, SO42- inhibited nitrate reduction and the higher concentration of SO42- the stronger inhibitory of the nitrate reduction. This phenomenon was also found by Nguyen et al. (2016). Since SO42- could 9
act as an electron acceptor to compete with NO3- during the biological denitrification process, the nitrate removal rate was reduced, and the concentration of nitrite detected was as high as 16 mg L-1. However, lots of studies suggested that sulfate can remove iron oxides and hydroxides from the iron surface, thereby hindering the formation of the passivation film on ZVI (Simpson and Melendres, 1996) and thus increasing the reactivity of the iron (Devlin and Allin, 2005; Tang et al., 2012). Moreover, SO42− also contributed to the production of green rust, which was also favorable for nitrate reduction (Devlin and Allin, 2005). In HAD system, after 72 h reaction, there was a sharp decrease of nitrate concentration and a high concentration of ammonium (17.5 mg L-1) presented. The results illustrated that the promotion effect of SO42- on chemical denitrification by Fe0 was greater than the inhibit effect of biological denitrification from bacteria. Although the nitrate degradation was accelerated by SO42-, the total nitrogen removal rate was 24% lower than in control system (Fig. 2a). This phenomenon was primarily due to the high transformation rate of ammonia nitrogen which produced during the nitrate reduction by Fe0. Phosphorus is one of the essential nutrients for microbial, and the demand is about 10 -4-10-3 mol L-1. As shown in Fig. 1a and Table 1, the nitrate removal rate constants were slightly higher in the presence of PO43- than that in control. Research has shown that phosphorus could increase the number of dominant microorganism and change the biological community structure，thereby increase the nitrate removal rate (Liu et al., 2015). When P concentration is lower than 0.034 mg L-1，the nitrate removal rate is higher with increasing phosphorus concentration. However, when it’s higher than that concentration, the impact of phosphorus on nitrate removal turns out to be small (Niu 10
and Li, 2010). Nonetheless, PO43-could inhibit nitrate reduction by Fe0 and decrease the translation of nitrate to ammonium (Tang et al., 2012). In this study，the concentration of phosphorus far exceeded 0.034 mg L-1，thereby the nitrate removal profiles were close to each other. However, the decrease of ammonium accumulation (Fig. 1c) indicated that nitrate reduction by Fe0 was inhibited. Furthermore, the increase of total nitrogen removal rate (Fig. 2a) revealed that biological denitrification must be promoted. It is noteworthy that the promotion effect of HCO32- on nitrate removal was very extraordinary. Nitrate concentration was reduced to 10 mg N L-1 within 72 h (Fig. 1a), and the rate constant increased from 1.69e-2 h-1 to 2.08e-2 h-1 with the increase of HCO3- (Table 1). By contrast, the control system took almost168 h to get the level of 10 mg N L-1. The promotion of HCO32- can be interpreted in two aspects. In biological process HCO3- could be used as inorganic carbon by autotrophic denitrification bacteria and help control the pH (Niu and Li, 2010). In chemical process, similar to GRSO42−, the GR
HCO3
−
also had potential for nitrate reduction (Su and Puls, 2004). Moreover, the
hydrolysis of H2CO3/HCO3- could promote iron corrosion (Su et al., 2012) and thus enhance nitrate chemical reduction. At the end of the reaction, high ammonium accumulation (Fig. 1c) indicated that HCO3- promoted the nitrate reduction by Fe0, or dissimilatory nitrate reduction to ammonium (DNRA), or both. It is why that the total nitrogen removal rate in HCO3- system reduced about 8% compared with the control system (Fig. 2a). When HCO3- present in HAD, especially the concentration is as high as 400 mg L-1, NO2--N sharply accumulated with a peak of 19 mg L-1 (Fig. 1b). The reason may be
that during the competition of nitrate reductase (NR) and
nitrite reductase (NIR), HCO3- could cause inhibition of the later thus impeding the further transformation of nitrite. 11
3.2 The effect of anions on byproduct Previous studies indicated that one of the shortcomings is organic compounds release when using solid cellulose substrates as organic carbon source for denitrification (Rocca et al., 2007; Schipper et al., 2010). It also can be seen from the results (Fig. 2b), the concentration of the DOC was a little high in HAD systems. Most of the anions tested in this study promoted the increase of DOC, particularly the Cl- and SO42-. As previously mentioned, this phenomenon was due to the inhibition of Cl- and SO42- on denitrify bacterial. Except DOC, other by-product, such as dissolved iron should be considered in application. The results indicated that except PO43- all the anions mentioned in the experiment could reduce the dissolved iron (Fig. 2c). This decrease probably attributed to the formation of iron green rust compounds (GR), which is formed in water especially with high Cl− and SO42− concentrations (Rocca et al., 2007). At the end of experiment, a green color was observed on the iron surface in the system with externally adding Cl−, SO42− and HCO3− and this coincide with the promotion of nitrate removal. Phosphate could inhibit the corrosion of Fe0 (Harms et al., 2003), so it was not the reason that the concentration of dissolved iron increased, but maybe the microbe activities was. 3.3 The effect of cations A variety of cations co-exist in the nitrate contaminated groundwater such as Na+,Ca2+, Mg2+, Cu2+, Fe2+and Fe3+. These cations not merely affect nitrate reduction by Fe0 but also affect microbial growth and metabolism. As illustrated in Fig. 3a and Table 2, the results were different depending on the specific ions. As a common ion, Na+ 12
exhibited non-significantly influence on nitrate degradation and byproducts in our measure range. The hindrance effect of Cu 2+ was very extraordinary. When the Cu 2+ concentration was 100 mg L-1, the rate constant was only 4.650e-3 h-1, and only 52.04% nitrate removal efficiency achieved after 240 h. However, the rate constant was 1.010e-2 h-1 and the nitrate removal efficiency was nearly 100% in control system. Although the nitrate removal rate increased with the increase of Cu 2+ concentration, it is still far lower than in control. On the contrary, previous studies have shown that Cu 2+ performed promotion effect during the process of various pollutants reduction by Fe0 (Su et al., 2012; Tang et al., 2012). The enhancement effect of Cu 2+ ion towards the pollutants reduction may result from the bimetallic system promoting electron transfer, and the catalytic effect of Cu(0) (Hosseini et al., 2011; Shih et al., 2011). The inverse results in this study suggested that in HAD the microbe played a leading role in nitrate removal process. As a heavy metal, Cu 2+ has a deleterious effect on microorganism by irreversible inhibiting the extracellular or intracellular enzymes and decreases biological denitrification potential (Li et al., 2015). A study showed that the addition of Cu inhibited denitrification and increased ammonium accumulation in the sediment-water environment (Sakadevan et al., 1999). Similar role of Cu2+ occurred in this study. Interestingly, lower Cu2+ dosage showed higher inhibition effect, possible because higher Cu2+ dosage promoted nitrate chemical reduction by ZVI. This inference was proved by the result that more ammonium was detected in the presence of more Cu 2+ loading (Fig. 3c).
Moreover, 180.5 µg L-1 and 450.45 µg L-1 of Cu2+ residue
in solution were observed after 240 h in the presence of 100 mg L-1 and 200 mg L-1 Cu2+, 13
respectively, which demonstrated that Cu2+ was involved in chemical reaction and enhanced nitrate reduction by Fe0. Previous studies reported that Cu2+ could accelerate nitrite accumulation during nitrate removal by ZVI, and more Cu2+ addition favored nitrite accumulation (Hao et al., 2005; Tang et al., 2012). But in HAD process no more than 1.8 mg L-1 (Fig. 3b) nitrite was detected which once again proved that the microbe played a leading role in HAD system. Because of the high conversion rate of ammonium, the total nitrogen removal rate was no more than 20% (Fig. 4a). Results showed that the Fe2+ demonstrated inhibition firstly and promotion subsequently (Fig. 3a). Studies have shown that denitrification with iron as electron donor can take place anaerobically under abiotic, biotic or both conditions. Stoichiometric equations of denitrification are as follws (Ottley et al., 1997): 10Fe2+ + 2NO3− + 14H2O → N2 + 10FeOOH + 18H+
(3)
15Fe2+ + 2NO3− + 14H2O → N2 + 5Fe3O4 + 28H+
(4)
Moreover, some researchers suggested that the equations mentioned above only proceed in the Fe2+ concentration more than 76 mg L-1, and the oxidation of ferrous iron could as
well
be
coupled
to
the
reduction of
nitrate
to ammonium (Straub et
al., 2001). Besides, Fe2+ could accelerate the nitrate reduction by Fe0 in chemical process (Huang and Zhang 2006; Tang et al., 2012). At the inhibition phase, we speculated that the inhibition caused by Fe2+ in HAD was due to its toxicity to the denitrifying bacteria , thereby decreased the nitrate removal and caused a little nitrite accumulation(Fig. 3b). Mélanie et al. (2008) also found that Fe2+ was toxic for Escherichia coli. After the buffer phase, the promotion on one hand maybe because Fe2+ promoted nitrate removal by Fe0, for another, Eq. (3) and (4) may take place in reactor with Fe2+ of high concentration. Moreover, the high ammonium accumulation (Fig. 3c) 14
also indicated that Fe2+ accelerated chemical denitrification and produced ammonium. Ferric iron is one of the biogenic elements. It is a part of growth media recommended for bacterial cells. Results indicated that Fe3+ at lower concentration demonstrated inhibition firstly and promotion subsequently on nitrate degradation, but when it at higher concentration the nitrate removal was restrained during the whole process (Fig. 3a). Bacteria growth will be enhanced when the Fe3+ at a low level but the dehydrogenase activity decreased with the Fe3+ concentration increase. Blažková et al. (2017) explored that nitrite accumulated during the ferric-iron-influenced autotrophic denitrification. The same phenomenon was also found in HAD, the nitrite as high as 11.35 mg L-1 was detected when the Fe3+ concentration was at 50 mg L-1 (Fig. 3b). Consequently, we speculated that Fe3+ at a high concentration could restrain the activity of NIR and the bacteria would be wrapped by Fe(OH)3 which produced by the precipitation of Fe3+, so that the activity of bacteria was also restrained. From Fig.3c we can see that Fe3+ caused a high ammonium accumulation and this was due to Fe2+ which supplied by redox reaction (Eq.5) promoted nitrate reduction by Fe0. Fe + 2Fe3+ → 3Fe2+
(5)
For this reason, the total nitrogen removal rate was no more than 70% at the end of the experiment (Fig. 4a). Results revealed that Ca2+and Mg2+inhibited nitrate degradation at the beginning of 48 h, but Ca2+started promotion effect after 48 h and Mg2+ after 72 h (Fig. 3a). Ca2+demonstrated extraordinary promotion effect among all the tested cations. It was no more than 96 h for the nitrate degradation to 10 mg L-1, while the control system took about 144 h. There was no obvious difference of promotion effect between two tested concentrations of Ca2+, and so did Mg2+. Studies reported that Ca2+ could form a 15
complex with surface sites of iron (oxy)hydroxides and then provide a bridge between iron and pollutants, such as arsenic (Tanboonchuy et al., 2012) and Cr(VI) (Liu et al., 2009). However, there was no significant effect on nitrate reduction by zero-valent iron (Huang et al., 2003). In HAD system, the presence of Ca2+ significantly promoted nitrate removal with small increments increase of ammonium (Fig. 3c) which indicated that promotion was mainly because of the enhancement on biodegradation not the iron reduction. A study showed that Ca2+ may have a direct/indirect effect on the structure or metabolic activities of bacteria (Ren et al., 2008). Moreover, the enhancement on biodenitrification maybe due to Ca2+ favourable of the electron transfer between NO3and H2, meanwhile increased the close contact between bactieria and H2 (Liang et al., 2015). Lai and Lo (2008) reported that the Mg2+ precipitates on the Fe0 may deteriorate Fe0 reactivity. Conversely, there were studies showed the presence of Mg2+ had no impact
on
the
degradation of
hexachlorobenzene
(Su
et
al.,
2012)
and
para-chloronitrobenzene (Le et al., 2011) by NZVI particles. Similar to Ca2+ the promotion of Mg2+ on nitrate removal in HAD system was mainly due to it enhancement on biodegradation. Magnesium is essential for the normal functions of organisms. It may be responsible for nucleotide synthesis, and promote cell growth (Chen et al., 2016). As an activator, Mg2+ could enhance the degradation of cellulose and
supply
more organic carbon
source
for
microorganisms,
thereby
to
promote the growth of microorganisms (Schmidt and Clark 2012). 3.4 The effect of cations on by product Except the reactors adding Fe2+ and Fe3+ ions, dissolved iron in most of the 16
reactors rose first and then fell before 144 h (Fig. 4b). There was a sharp decrease of dissolved iron in Fe2+, Fe3+ adding reactors in 24 h which caused by redox reaction , hydrolysis reaction (Tang et al., 2012) and GR formation (Rocca et al., 2007). In Cu 2+ adding reactors, as high as 18.2 mg L-1 dissolved iron was detected because of the substitution reaction between Fe0 and Cu2+ and it decreased to below 4 mg L-1 at the end of reaction due to consumption of Fe2+ for nitrate reduction by ZVI (Hosseini et al., 2011). As the reaction going on, the pH value increased and CO32- was produced (Rocca et al., 2007). In Ca2+, Mg2+presented reactors, CaCO3, MgCO3, Ca(OH)2, Mg(OH)2 may produce, thereby hindering the activity of Fe0. Except Ca2+ and Cu2+, most of the cations had little impact on DOC release in HAD system. Minimum DOC concentration (55.98 mg L-1) was observed in the presence of Ca2+ (200 mg L-1) and maximum DOC concentration (102.4 mg L-1) in the presence of Cu2+ (200 mg L-1), large variation compared to the concentration (77.9 mg L-1) obtained without any other co-existing anion. The lower DOC in Ca2+ adding reactors was not only because the benefit of Ca2+on the growth of bacteria (Ren et al., 2008), but also Ca2+ would react with some organic acid coming from sawdust and microbial metabolism. On the contrary, due to the restrain of Cu 2+ on bacteria growth (Li et al., 2015), the consumption of DOC decreased, thereby exhibiting an increase of DOC concentration. 4. Conclusion Most of the anions enhanced nitrate removal, and the strongest promotion came from HCO3- and SO42-. The most remarkable enhancement of cations on nitrate removal 17
was Ca2+, while the strongest inhibition came from Cu 2+. Cl- and Na+ had no obvious effect on nitrate remediation. Byproducts such as nitrite, ammonium, DOC, etc. were also influenced by common ions, especially in the presence of HCO3- , SO42-, Cu2+ and Fe3+. Overall, results obtained in this study indicated that sawdust-iron based HAD can retain its high removal efficiency in more complex environments with some significantly influence ions taking into consideration in the design. Acknowledgments This Study was supported by the Open Project Program of Breeding Base for State Key Laboratory of Land Degradation and Ecological Restoration of North-western China/ Key Laboratory for Restoration and Reconstruction of Degraded Ecosystem in North-western China of Ministry of Education (Grant No. 2017KF011). References 1.
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Denitrifying bioreactors-an approach for reducing nitrate loads to receiving waters. Ecol. Eng. 36 (11), 1532-1543. 29. Schmidt, C.A., Clark, M.W., 2012. Efficacy of a denitrification wall to treat continuously high nitrate loads. Ecol. Eng. 42, 203-211. 30. SEPA, 2002. Water and Wastewater Monitoring Analysis Method, fourth ed. China Environmental Science Press, Beijing. 31. Shih, Y. H., Chen, M., Su, Y., 2011. Pentachlorophenol reduction by pd/fe bimetallic nanoparticles: effects of copper, nickel, and ferric cations. Appl. Catal., B Environ. 105 (1), 24-29. 32. Simpson,
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addenda to third edition, vol. 1. WHO Press, Geneva. Recommendations. 46. Zhang, Z., Zhang, Q., Xu, J., Deng, R., Ji, Z., Wu, Y., Jin, R., 2016. Evaluation of the inhibitory effects of heavy metals on anammox activity: a batch test study. Bioresour. Technol. 200, 208-216. 47. Zhao, H., 2005. Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids. J. Mol. Catal. B: Enzym. 37 (1), 16-25. 48. Zhao, Y., Zhang, B., Feng, C., Huang, F., Zhang, P., Zhang, Z., Yang, Y., Sugiura, N., 2012. Behavior of autotrophic denitrification and heterotrophic denitrification in an intensified biofilm-electrode reactor for nitrate-contaminated drinking water treatment. Bioresour. Technol. 107 (4), 159-165.
24
Figure captions: Fig.1. Effects of anions on (a) nitrate removal, (b) nitrite and (c) ammonium accumulation in HAD system. Results are the mean of three replicates and error bars indicates standard deviation. Fig.2. Effects of anions on (a) TN removal, (b) DOC and (c) iron release in HAD system. Results are the mean of three replicates and error bars indicates standard deviation. Fig.3. Effects of cations on (a) nitrate removal, (b) nitrite and (c) ammonium accumulation in HAD system. Results are the mean of three replicates and error bars indicates standard deviation. Fig.4. Effects of cations on (a) TN removal and (b) iron dissolution
25
Fig.1.
26
Fig.2.
27
Fig.3.
28
Fig.4.
29
Tables. Table 1 Nitrate removal rate constants in HAD containing various anions. Table 2 Nitrate removal rate constants in HAD containing various cations
30
Anion species
PO43HCO3SO42ClControl
Anions concentration (mg L-1)
First-order kinetics equation: C = (C0-Cf)·e-kt + Cf
k (h-1)
R2
5
1.010e-2
0.9879
20
9.950e-3
0.9836
100
1.690e-2
0.9763
400
2.080e-2
0.9458
50
1.010e-2
0.9909
200
1.410e-2
0.9451
30
8.960e-3
0.9903
120
8.830e-3
0.9904
----
8.860e-3
0.9880
Table 1.
31
Cation species
Na+ Mg2+ Ca2+ Cu 2+ Fe2+ Fe3+
Control
Cations concentration -1
(mg L )
First-order kinetics equation: C = (C0-Cf)·e-kt + Cf k (h-1)
R2
100
1.010e-2
0.9907
200
1.010e-2
0.9935
200
1.180e-2
0.9902
400
1.100e-2
0.9786
200
1.440e-2
0.9751
400
1.430e-2
0.9830
100
4.650e-3
0.9586
200
4.240e-3
0.9802
50
8.880e-3
0.9750
100
9.990e-3
0.9727
50
7.880e-3
0.9453
100
2.216e-5
0.9677
----
1.010e-2
0.9918
Table 2.
32
Highlights: ·The removal of nitrate by HAD is not affected by Na+ and Cl-. ·HCO3- and Ca2+ significantly enhance nitrate removal. ·Cu
2+
inhibited nitrate removal due to the deleterious effect on bacteria.
·Cu2+, Fe2+ and Fe3+ ions cause high ammonium accumulation. ·HCO3-and SO42- promote nitrite and ammonium production.
33
Graphical abstract.
34
S0960-8524(17)32124-7 https://doi.org/10.1016/j.biortech.2017.12.009 BITE 19264
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
1 November 2017 5 December 2017 6 December 2017
Please cite this article as: Wang, Z., Jiang, Y., Awasthi, M.K., Wang, J., Yang, X., Amjad, A., Wang, Q., Lahori, A.H., Zhang, Z., Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.12.009
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Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions Zhen Wang a, Yahui Jiang a, Mukesh Kumar Awasthi a, b, Jiao Wang a, Xinguo Yang c, Ali Amjad a, Quan Wang a, Altaf Hussain Lahori a, Zengqiang Zhang a* a
College of Natural Resources and Environment, Northwest A&F University, Yangling,
Shaanxi 712100, China b
Department of Biotechnology, Amicable Knowledge Solution University, Satna, India
c
Key Laboratory for Restoration and Reconstruction of Degraded Ecosystem in
Northwestern China of Ministry of Education, Ningxia University, Yinchuan 750021, China
*Corresponding author: Prof. Zengqiang Zhang College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi Province 712100, PR China Tel./Fax: +86-013609254113; +86 029-87080055. E-mail address: [email protected] (Z.Q. Zhang). 1
Nitrate removal by combined heterotrophic and autotrophic denitrification processes: Impact of coexistent ions Zhen Wang a, Yahui Jiang a, Mukesh Kumar Awasthi a, b, Jiao Wang a, Xinguo Yang c, Ali Amjad a, Quan Wang a, Altaf Hussain Lahori a, Zengqiang Zhang a* Abstract: In this current study, sawdust and zero-valent iron (Fe0) were used as co-electron donors to evaluate the effects of coexistent ions on the combined heterotrophic and autotrophic denitrification (HAD) processes. The results showed that HCO3- and SO42- drastically enhanced nitrate removal. The promotion effect derived from both biological and chemical process by HCO3- and chemical process by SO42-. However, Ca2+ ions would remarkably increase nitrate removal due to promoting the electron transfer and the metabolic activities of bacteria, whereas the Cu2+ ions inhibited the biological process due to the deleterious effect on bacteria. Meanwhile, Fe2+ and Fe3+ ions exhibited inhibition effect firstly because of their toxicity to bacteria and promotion subsequently due to their enhancement on Fe0 chemical denitrification. Moreover, byproducts such as nitrite, ammonium, dissolved organic carbon (DOC), etc. were also influenced by common ions. Keywords:
Nitrate removal, Cations, Anions, Sawdust, Iron.
2
1. Introduction Excess accumulation of nitrate (NO3-) in drinking water which is harmful to human health due to its contribution to methemoglobinemia, gastric cancer, and non-Hodgkin lymphoma (Sunger and Bose 2009; Wang et al., 2012). The intensive application of especially nitrogenous fertilizers, sewage irrigation, and industrial waste effluent discharge are the major sources of nitrate contamination in ground and surface waters (Zhao et al., 2012; Li et al., 2016). World Health Organization (WHO) stipulated the maximum NO3--N concentration in drinking water is 10 mg N L-1 (WHO, 2008). Among the current remediation technologies, biological denitrification which occurs naturally when denitrifying bacteria use nitrate as a terminal electron acceptor in their respiration process in anaerobic environment, which is considered to be one of the most economical and widely used remediation methods (Schipper et al., 2010; Sahinkaya et al., 2015; Wang and Chu, 2016). During biological denitrification processes nitrate which is finally transformed into nitrogen gas, with formation some nitrogen intermediates, as indicated by Eq. (1): NO3-→ NO2-→ NO → N2O → N2 Biological denitrification includes heterotrophic denitrification (HD) and autotrophic denitrification (AD). Recently, a combined heterotrophic and autotrophic denitrification (HAD) has been proved to be more effective than HD and AD for nitrate removal from groundwater due to the synergetic relationship between HD and AD (Rocca et al., 2007; Huang et al., 2015; Wang et al., 2015).
3
In HAD symbiotic system, Fe0 was used to create an anaerobic environment in water and produce hydrogen. The low DO levels could favor the heterotrophic denitrifiers utilizing cellulose organic carbons as electron donors to degrade nitrate and meanwhile generate CO2. The CO2 can be used as an inorganic carbon source and the H2 as an electron donor by the autotrophic denitrifiers to remove nitrate via autotrophic denitrification process. Although the synergistic and promotive effects mechanisms of the HAD were encouraging, HAD is still not used for nitrate in-situ remediation in groundwater. HAD supported by cellulose materials and Fe0 is adequate for use in permeable reactive barriers (PRBs), because both cellulose materials and Fe0 have been used in-situ nitrate remediation for a long time (Rocca et al., 2007; Schipper et al., 2010; Schmidt and Clark, 2012). The PRB is a very attractive promising technology due to the relative simplicity, low investment and more cost effectiveness in situ groundwater treatment, but it is difficult to control (Wang and Chu 2016). A few reports related to HAD mainly focused on the kinds of electron donors, pollutants and bacteria (Huang et al., 2015; Li et al., 2016),however，it still requires lots of studies about the background circumstance effects on nitrate removal by HAD system, prior to real in situ nitrate remediation application. Common ions such as SO42−, Cl−, HCO3−, Fe2+, Mg2+ and Ca2+ are ubiquitous in groundwater and soil. These ions would pose different effects on the chemical reactivity of Fe0 and the structure or metabolic activities of bacteria. Previous studies have showed that common coexistent ions in soil and groundwater would affect the Fe0 4
reactivity depending on the target pollutants (Liu et al., 2007; Liu and Lo 2011). Some cations (Fe2+, Fe3+ and Cu2+) and anions (SO42−, Cl−, HCO3−) could promote nitrate reduction by Fe0 but there were no distinct effect by Ca2+, Na+ , K+ , and PO43− behaved inhibition effect (Tang et al., 2012). Moreover, the activities of microorganisms will also be influenced by ions because some ions may cause competitive or non-competitive inhibition of microbial enzymes (Zhao 2005). Ting et al. (1994) reported that nitrogen and phosphorus biological removal would be significantly decreased due to the addition of copper. It would strongly inhibit anammox activity, especially in the presence of NO2- (Zhang et al., 2016). In addition, some anions with high concentrations would result in the damage of cell membranes, and caused the leakage of cell inclusion (Wang et al., 2016). Consequently, HAD system includes both bacteria and Fe0, so the presence of common ions may affect the nitrate removal by chemical or biological process. Considering application of the HAD process for nitrate in-situ remediation, the co-exist ions in water needs to be taken care of. And to date, no attempt has been made to describe about the impact of common ions on HAD. In light of the existing work, the main objective of this study was to investigate the effects of co-exist ions on nitrate removal by sawdust and Fe0 supported HAD. To achieve this goal, the effects of 6 common cations and 4 inorganic anions at two different concentration levels on nitrate removal were evaluated. A pseudo-first-order model was introduced to describe the degradation kinetics of nitrate. Furthermore, nitrite, ammonium, DOC and soluble iron which produced during the nitrate removal process were also examined. The observed 5
results may provide more possible guidance to practical application of HAD technology. 2. Materials and methods 2.1. Materials The feed solution was prepared with nitrate (as KNO3) and phosphate (as K2HPO4) at an N/P weight ratio of 22:3, and the pH was about 6.5. Cation and anion stock solutions were prepared with corresponding chloride (NaCl, CaCl2, MgCl2·6H2O, CuCl2·2H2O, FeCl2·4H2O, FeCl3·6H2O) and potassium salts (KH2PO4，K2SO4，KCl， KHCO3), respectively. All the chemicals used in this study were of analytical grade, and all aqueous solutions were prepared with deionized water. Chinese parasol is wide distribution and low cost in China, and it’s frequently used as raw material for manufacturing. In this study, Chinese parasol sawdust was purchased from a wooden door company in Yang Ling city, Shaanxi, China. It was air dried and milled with a lab grinder. The final powder was sieved with a 1-mm screen. Finally, it was sterilized in a vacuum drying oven at 121°C for 60 min and vacuum-stored after cooling for further experiments. We can see the sawdust surface is rough and porous under a scanning electron microscope (SEM), and the C, N content in sawdust were 52.30 ± 0.335%, 0.307± 0.019% respectively. The iron particle (Fe0) (Tianjin Zonghengxing Industrial & Trading Chemical Reagent Co., China), was of 98% analytical grade, and irregular in shape with smooth and scale surface. The sludge was obtained from a sewage treatment plant in Xi Ning city, Qinghai, China. The C, N and P content in sludge were 27.3%, 6.24% and 7.63% as measured by an elemental analyzer. 6
2.2. Inoculums Ten grams of wet sludge was added as primary bacterial source into a flask which contained 5.00 g of sawdust and 5.00 g of Fe0. The flask was filled with 1000.0 mL of nitrate solution spiked with 50 mg L-1 of NO3--N, and the C/N/P weight ratio of approximately 1000/22/3 (Rocca et al., 2005). The flask was purged with Ar gas for 20 min to remove dissolved oxygen, then sealed and incubated at 25 °C in dark without shaking until nitrate and the intermediate product of nitrite were completely removed. Nitrate and nitrite concentration was measured every 2 d. 2.3. Experiments The effects of common ions on the denitrification activity of HAD were studied in 1000 mL tapered flasks. Five grams of Fe0, 5.00 g of sawdust and 100.0 mL of bacteria solution were added into each reactor, followed by adding nitrate and specific ion stock solutions. The initial pH value of nitrate solution was about 6.5, and the nitrate concentration was 50 mg N L-1. Cations concentration were Ca2+ (200, 400 mg L-1), Mg2+ (200, 400 mg L-1), Cu 2+ (100, 200 mg L-1), Fe2+ (50, 100 mg L-1) and Fe3+ (50, 100 mg L-1). The anions concentration were Cl- (30, 120 mg L-1), SO42- (50, 200 mg L-1), PO43- (5, 20 mg L-1), HCO3- (100, 400 mg L-1) separately. All the ion concentrations were according to their typical concentrations in natural groundwater (Dou et al., 2010). After the Ar gas bubbling, all the reactors were sealed tightly and mixed well before transferring to a constant temperature incubator (HPS-250; China) for static incubation at 25 °C under a dark condition. Aliquots of the samples were taken with a10-mL disposable syringe at certain time 7
intervals, and analyzed immediately after being filtered through a 0.45µm filter membrane. The filtrate was collected for pH, nitrate, nitrite, ammonium, ions and dissolved iron and organic carbon measurement, and all tests were conducted in duplicate. 2.4 Analytical methods The surface morphology of iron powder and sawdust was observed by a JSM-6360LV scanning electron microscope (JEOL, Japan). Nitrate was measured using a UV spectophotometric screening method at 220 and 275 nm (UVmini-1240, Shimadzu). Nitrite and ammonium ions were analyzed by the colorimetric method at 540 nm and 636 nm respectively (Tang et al., 2012). The initial pH value of each reactor was measured with a Mettler Toledo 320-S pH-meter. Dissolved organic carbon (DOC) was measured using a TOC analyzer (HACH, IL530 TOC-TN). Atomic absorption spectrophotometer was used to determine the concentration of dissolved iron (Hitachi ZA3000). Anions were analyzed using an ion chromatograph (861 advanced compact IC, Metrohm) and cations were measured with an inductively coupled plasma mass spectrometry (ICP-MASS, Agilent 7800) (SEPA, 2002). 2.5 Nitrate degradation rate constants The pseudo-first-order model as follow was applied to describe the removal of nitrate by HAD system (Su et al., 2012): C = (C0-Cf)·e-kt + Cf
(2)
In this expression, C is the nitrate concentration at any time, C0 is the initial concentration, Cf is the final concentration, k expresses the pseudo-first-order rate 8
constant (h-1), and t is the reaction time expressed in hours. 3. Results and discussion 3.1. The effect of anions Effects of various anions at two different concentration levels on nitrate removal, nitrite/ammonium acummulation by HAD system was shown in Fig. 1. In control system, nitrate decreased sharply during the first 48 h then slowed down and completely removed within 240 hours (Fig.1a). As shown in Table 1, all anions except Cl- enhanced nitrate reduction in HAD. Compared to other anions, Cl- exhibited non-significant influence on nitrate degradation, nitrite/ammonium accumulation (Fig. 1b and Fig. 1c) and the total nitrogen removal (Fig. 2a). In previous studies, Cl- performed enhancement for nitrate removal (Tang et al., 2012), and 4-chloronitrobenzene degradation (Devlin and Allin 2005) by Fe0, because it can promote iron pitting corrosion and increase ionic strength. However, Cl- could cause the cell wall broken and the protoplast disintegrated. More importantly, chloride could remarkably inhibit the activity of nitrate reductase (Wang et al., 2016). In HAD system, the enhancement of Cl- on Fe0 corrosion may just offset the inhibition on denitrification of denitrify bacteria. From Fig. 1a, it could be seen that SO42- promoted nitrate removal by HAD after 72 h reaction, especially when it was at a higher concentration (200 mg L-1). As the SO42- concentration drastically increased from 50 to 200 mg L-1, the rate constant increased from 1.01e-2 to 1.41e-2 h-1. In the reaction of 72 h, SO42- inhibited nitrate reduction and the higher concentration of SO42- the stronger inhibitory of the nitrate reduction. This phenomenon was also found by Nguyen et al. (2016). Since SO42- could 9
act as an electron acceptor to compete with NO3- during the biological denitrification process, the nitrate removal rate was reduced, and the concentration of nitrite detected was as high as 16 mg L-1. However, lots of studies suggested that sulfate can remove iron oxides and hydroxides from the iron surface, thereby hindering the formation of the passivation film on ZVI (Simpson and Melendres, 1996) and thus increasing the reactivity of the iron (Devlin and Allin, 2005; Tang et al., 2012). Moreover, SO42− also contributed to the production of green rust, which was also favorable for nitrate reduction (Devlin and Allin, 2005). In HAD system, after 72 h reaction, there was a sharp decrease of nitrate concentration and a high concentration of ammonium (17.5 mg L-1) presented. The results illustrated that the promotion effect of SO42- on chemical denitrification by Fe0 was greater than the inhibit effect of biological denitrification from bacteria. Although the nitrate degradation was accelerated by SO42-, the total nitrogen removal rate was 24% lower than in control system (Fig. 2a). This phenomenon was primarily due to the high transformation rate of ammonia nitrogen which produced during the nitrate reduction by Fe0. Phosphorus is one of the essential nutrients for microbial, and the demand is about 10 -4-10-3 mol L-1. As shown in Fig. 1a and Table 1, the nitrate removal rate constants were slightly higher in the presence of PO43- than that in control. Research has shown that phosphorus could increase the number of dominant microorganism and change the biological community structure，thereby increase the nitrate removal rate (Liu et al., 2015). When P concentration is lower than 0.034 mg L-1，the nitrate removal rate is higher with increasing phosphorus concentration. However, when it’s higher than that concentration, the impact of phosphorus on nitrate removal turns out to be small (Niu 10
and Li, 2010). Nonetheless, PO43-could inhibit nitrate reduction by Fe0 and decrease the translation of nitrate to ammonium (Tang et al., 2012). In this study，the concentration of phosphorus far exceeded 0.034 mg L-1，thereby the nitrate removal profiles were close to each other. However, the decrease of ammonium accumulation (Fig. 1c) indicated that nitrate reduction by Fe0 was inhibited. Furthermore, the increase of total nitrogen removal rate (Fig. 2a) revealed that biological denitrification must be promoted. It is noteworthy that the promotion effect of HCO32- on nitrate removal was very extraordinary. Nitrate concentration was reduced to 10 mg N L-1 within 72 h (Fig. 1a), and the rate constant increased from 1.69e-2 h-1 to 2.08e-2 h-1 with the increase of HCO3- (Table 1). By contrast, the control system took almost168 h to get the level of 10 mg N L-1. The promotion of HCO32- can be interpreted in two aspects. In biological process HCO3- could be used as inorganic carbon by autotrophic denitrification bacteria and help control the pH (Niu and Li, 2010). In chemical process, similar to GRSO42−, the GR
HCO3
−
also had potential for nitrate reduction (Su and Puls, 2004). Moreover, the
hydrolysis of H2CO3/HCO3- could promote iron corrosion (Su et al., 2012) and thus enhance nitrate chemical reduction. At the end of the reaction, high ammonium accumulation (Fig. 1c) indicated that HCO3- promoted the nitrate reduction by Fe0, or dissimilatory nitrate reduction to ammonium (DNRA), or both. It is why that the total nitrogen removal rate in HCO3- system reduced about 8% compared with the control system (Fig. 2a). When HCO3- present in HAD, especially the concentration is as high as 400 mg L-1, NO2--N sharply accumulated with a peak of 19 mg L-1 (Fig. 1b). The reason may be
that during the competition of nitrate reductase (NR) and
nitrite reductase (NIR), HCO3- could cause inhibition of the later thus impeding the further transformation of nitrite. 11
3.2 The effect of anions on byproduct Previous studies indicated that one of the shortcomings is organic compounds release when using solid cellulose substrates as organic carbon source for denitrification (Rocca et al., 2007; Schipper et al., 2010). It also can be seen from the results (Fig. 2b), the concentration of the DOC was a little high in HAD systems. Most of the anions tested in this study promoted the increase of DOC, particularly the Cl- and SO42-. As previously mentioned, this phenomenon was due to the inhibition of Cl- and SO42- on denitrify bacterial. Except DOC, other by-product, such as dissolved iron should be considered in application. The results indicated that except PO43- all the anions mentioned in the experiment could reduce the dissolved iron (Fig. 2c). This decrease probably attributed to the formation of iron green rust compounds (GR), which is formed in water especially with high Cl− and SO42− concentrations (Rocca et al., 2007). At the end of experiment, a green color was observed on the iron surface in the system with externally adding Cl−, SO42− and HCO3− and this coincide with the promotion of nitrate removal. Phosphate could inhibit the corrosion of Fe0 (Harms et al., 2003), so it was not the reason that the concentration of dissolved iron increased, but maybe the microbe activities was. 3.3 The effect of cations A variety of cations co-exist in the nitrate contaminated groundwater such as Na+,Ca2+, Mg2+, Cu2+, Fe2+and Fe3+. These cations not merely affect nitrate reduction by Fe0 but also affect microbial growth and metabolism. As illustrated in Fig. 3a and Table 2, the results were different depending on the specific ions. As a common ion, Na+ 12
exhibited non-significantly influence on nitrate degradation and byproducts in our measure range. The hindrance effect of Cu 2+ was very extraordinary. When the Cu 2+ concentration was 100 mg L-1, the rate constant was only 4.650e-3 h-1, and only 52.04% nitrate removal efficiency achieved after 240 h. However, the rate constant was 1.010e-2 h-1 and the nitrate removal efficiency was nearly 100% in control system. Although the nitrate removal rate increased with the increase of Cu 2+ concentration, it is still far lower than in control. On the contrary, previous studies have shown that Cu 2+ performed promotion effect during the process of various pollutants reduction by Fe0 (Su et al., 2012; Tang et al., 2012). The enhancement effect of Cu 2+ ion towards the pollutants reduction may result from the bimetallic system promoting electron transfer, and the catalytic effect of Cu(0) (Hosseini et al., 2011; Shih et al., 2011). The inverse results in this study suggested that in HAD the microbe played a leading role in nitrate removal process. As a heavy metal, Cu 2+ has a deleterious effect on microorganism by irreversible inhibiting the extracellular or intracellular enzymes and decreases biological denitrification potential (Li et al., 2015). A study showed that the addition of Cu inhibited denitrification and increased ammonium accumulation in the sediment-water environment (Sakadevan et al., 1999). Similar role of Cu2+ occurred in this study. Interestingly, lower Cu2+ dosage showed higher inhibition effect, possible because higher Cu2+ dosage promoted nitrate chemical reduction by ZVI. This inference was proved by the result that more ammonium was detected in the presence of more Cu 2+ loading (Fig. 3c).
Moreover, 180.5 µg L-1 and 450.45 µg L-1 of Cu2+ residue
in solution were observed after 240 h in the presence of 100 mg L-1 and 200 mg L-1 Cu2+, 13
respectively, which demonstrated that Cu2+ was involved in chemical reaction and enhanced nitrate reduction by Fe0. Previous studies reported that Cu2+ could accelerate nitrite accumulation during nitrate removal by ZVI, and more Cu2+ addition favored nitrite accumulation (Hao et al., 2005; Tang et al., 2012). But in HAD process no more than 1.8 mg L-1 (Fig. 3b) nitrite was detected which once again proved that the microbe played a leading role in HAD system. Because of the high conversion rate of ammonium, the total nitrogen removal rate was no more than 20% (Fig. 4a). Results showed that the Fe2+ demonstrated inhibition firstly and promotion subsequently (Fig. 3a). Studies have shown that denitrification with iron as electron donor can take place anaerobically under abiotic, biotic or both conditions. Stoichiometric equations of denitrification are as follws (Ottley et al., 1997): 10Fe2+ + 2NO3− + 14H2O → N2 + 10FeOOH + 18H+
(3)
15Fe2+ + 2NO3− + 14H2O → N2 + 5Fe3O4 + 28H+
(4)
Moreover, some researchers suggested that the equations mentioned above only proceed in the Fe2+ concentration more than 76 mg L-1, and the oxidation of ferrous iron could as
well
be
coupled
to
the
reduction of
nitrate
to ammonium (Straub et
al., 2001). Besides, Fe2+ could accelerate the nitrate reduction by Fe0 in chemical process (Huang and Zhang 2006; Tang et al., 2012). At the inhibition phase, we speculated that the inhibition caused by Fe2+ in HAD was due to its toxicity to the denitrifying bacteria , thereby decreased the nitrate removal and caused a little nitrite accumulation(Fig. 3b). Mélanie et al. (2008) also found that Fe2+ was toxic for Escherichia coli. After the buffer phase, the promotion on one hand maybe because Fe2+ promoted nitrate removal by Fe0, for another, Eq. (3) and (4) may take place in reactor with Fe2+ of high concentration. Moreover, the high ammonium accumulation (Fig. 3c) 14
also indicated that Fe2+ accelerated chemical denitrification and produced ammonium. Ferric iron is one of the biogenic elements. It is a part of growth media recommended for bacterial cells. Results indicated that Fe3+ at lower concentration demonstrated inhibition firstly and promotion subsequently on nitrate degradation, but when it at higher concentration the nitrate removal was restrained during the whole process (Fig. 3a). Bacteria growth will be enhanced when the Fe3+ at a low level but the dehydrogenase activity decreased with the Fe3+ concentration increase. Blažková et al. (2017) explored that nitrite accumulated during the ferric-iron-influenced autotrophic denitrification. The same phenomenon was also found in HAD, the nitrite as high as 11.35 mg L-1 was detected when the Fe3+ concentration was at 50 mg L-1 (Fig. 3b). Consequently, we speculated that Fe3+ at a high concentration could restrain the activity of NIR and the bacteria would be wrapped by Fe(OH)3 which produced by the precipitation of Fe3+, so that the activity of bacteria was also restrained. From Fig.3c we can see that Fe3+ caused a high ammonium accumulation and this was due to Fe2+ which supplied by redox reaction (Eq.5) promoted nitrate reduction by Fe0. Fe + 2Fe3+ → 3Fe2+
(5)
For this reason, the total nitrogen removal rate was no more than 70% at the end of the experiment (Fig. 4a). Results revealed that Ca2+and Mg2+inhibited nitrate degradation at the beginning of 48 h, but Ca2+started promotion effect after 48 h and Mg2+ after 72 h (Fig. 3a). Ca2+demonstrated extraordinary promotion effect among all the tested cations. It was no more than 96 h for the nitrate degradation to 10 mg L-1, while the control system took about 144 h. There was no obvious difference of promotion effect between two tested concentrations of Ca2+, and so did Mg2+. Studies reported that Ca2+ could form a 15
complex with surface sites of iron (oxy)hydroxides and then provide a bridge between iron and pollutants, such as arsenic (Tanboonchuy et al., 2012) and Cr(VI) (Liu et al., 2009). However, there was no significant effect on nitrate reduction by zero-valent iron (Huang et al., 2003). In HAD system, the presence of Ca2+ significantly promoted nitrate removal with small increments increase of ammonium (Fig. 3c) which indicated that promotion was mainly because of the enhancement on biodegradation not the iron reduction. A study showed that Ca2+ may have a direct/indirect effect on the structure or metabolic activities of bacteria (Ren et al., 2008). Moreover, the enhancement on biodenitrification maybe due to Ca2+ favourable of the electron transfer between NO3and H2, meanwhile increased the close contact between bactieria and H2 (Liang et al., 2015). Lai and Lo (2008) reported that the Mg2+ precipitates on the Fe0 may deteriorate Fe0 reactivity. Conversely, there were studies showed the presence of Mg2+ had no impact
on
the
degradation of
hexachlorobenzene
(Su
et
al.,
2012)
and
para-chloronitrobenzene (Le et al., 2011) by NZVI particles. Similar to Ca2+ the promotion of Mg2+ on nitrate removal in HAD system was mainly due to it enhancement on biodegradation. Magnesium is essential for the normal functions of organisms. It may be responsible for nucleotide synthesis, and promote cell growth (Chen et al., 2016). As an activator, Mg2+ could enhance the degradation of cellulose and
supply
more organic carbon
source
for
microorganisms,
thereby
to
promote the growth of microorganisms (Schmidt and Clark 2012). 3.4 The effect of cations on by product Except the reactors adding Fe2+ and Fe3+ ions, dissolved iron in most of the 16
reactors rose first and then fell before 144 h (Fig. 4b). There was a sharp decrease of dissolved iron in Fe2+, Fe3+ adding reactors in 24 h which caused by redox reaction , hydrolysis reaction (Tang et al., 2012) and GR formation (Rocca et al., 2007). In Cu 2+ adding reactors, as high as 18.2 mg L-1 dissolved iron was detected because of the substitution reaction between Fe0 and Cu2+ and it decreased to below 4 mg L-1 at the end of reaction due to consumption of Fe2+ for nitrate reduction by ZVI (Hosseini et al., 2011). As the reaction going on, the pH value increased and CO32- was produced (Rocca et al., 2007). In Ca2+, Mg2+presented reactors, CaCO3, MgCO3, Ca(OH)2, Mg(OH)2 may produce, thereby hindering the activity of Fe0. Except Ca2+ and Cu2+, most of the cations had little impact on DOC release in HAD system. Minimum DOC concentration (55.98 mg L-1) was observed in the presence of Ca2+ (200 mg L-1) and maximum DOC concentration (102.4 mg L-1) in the presence of Cu2+ (200 mg L-1), large variation compared to the concentration (77.9 mg L-1) obtained without any other co-existing anion. The lower DOC in Ca2+ adding reactors was not only because the benefit of Ca2+on the growth of bacteria (Ren et al., 2008), but also Ca2+ would react with some organic acid coming from sawdust and microbial metabolism. On the contrary, due to the restrain of Cu 2+ on bacteria growth (Li et al., 2015), the consumption of DOC decreased, thereby exhibiting an increase of DOC concentration. 4. Conclusion Most of the anions enhanced nitrate removal, and the strongest promotion came from HCO3- and SO42-. The most remarkable enhancement of cations on nitrate removal 17
was Ca2+, while the strongest inhibition came from Cu 2+. Cl- and Na+ had no obvious effect on nitrate remediation. Byproducts such as nitrite, ammonium, DOC, etc. were also influenced by common ions, especially in the presence of HCO3- , SO42-, Cu2+ and Fe3+. Overall, results obtained in this study indicated that sawdust-iron based HAD can retain its high removal efficiency in more complex environments with some significantly influence ions taking into consideration in the design. Acknowledgments This Study was supported by the Open Project Program of Breeding Base for State Key Laboratory of Land Degradation and Ecological Restoration of North-western China/ Key Laboratory for Restoration and Reconstruction of Degraded Ecosystem in North-western China of Ministry of Education (Grant No. 2017KF011). References 1.
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24
Figure captions: Fig.1. Effects of anions on (a) nitrate removal, (b) nitrite and (c) ammonium accumulation in HAD system. Results are the mean of three replicates and error bars indicates standard deviation. Fig.2. Effects of anions on (a) TN removal, (b) DOC and (c) iron release in HAD system. Results are the mean of three replicates and error bars indicates standard deviation. Fig.3. Effects of cations on (a) nitrate removal, (b) nitrite and (c) ammonium accumulation in HAD system. Results are the mean of three replicates and error bars indicates standard deviation. Fig.4. Effects of cations on (a) TN removal and (b) iron dissolution
25
Fig.1.
26
Fig.2.
27
Fig.3.
28
Fig.4.
29
Tables. Table 1 Nitrate removal rate constants in HAD containing various anions. Table 2 Nitrate removal rate constants in HAD containing various cations
30
Anion species
PO43HCO3SO42ClControl
Anions concentration (mg L-1)
First-order kinetics equation: C = (C0-Cf)·e-kt + Cf
k (h-1)
R2
5
1.010e-2
0.9879
20
9.950e-3
0.9836
100
1.690e-2
0.9763
400
2.080e-2
0.9458
50
1.010e-2
0.9909
200
1.410e-2
0.9451
30
8.960e-3
0.9903
120
8.830e-3
0.9904
----
8.860e-3
0.9880
Table 1.
31
Cation species
Na+ Mg2+ Ca2+ Cu 2+ Fe2+ Fe3+
Control
Cations concentration -1
(mg L )
First-order kinetics equation: C = (C0-Cf)·e-kt + Cf k (h-1)
R2
100
1.010e-2
0.9907
200
1.010e-2
0.9935
200
1.180e-2
0.9902
400
1.100e-2
0.9786
200
1.440e-2
0.9751
400
1.430e-2
0.9830
100
4.650e-3
0.9586
200
4.240e-3
0.9802
50
8.880e-3
0.9750
100
9.990e-3
0.9727
50
7.880e-3
0.9453
100
2.216e-5
0.9677
----
1.010e-2
0.9918
Table 2.
32
Highlights: ·The removal of nitrate by HAD is not affected by Na+ and Cl-. ·HCO3- and Ca2+ significantly enhance nitrate removal. ·Cu
2+
inhibited nitrate removal due to the deleterious effect on bacteria.
·Cu2+, Fe2+ and Fe3+ ions cause high ammonium accumulation. ·HCO3-and SO42- promote nitrite and ammonium production.
33
Graphical abstract.
34