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Article

Accelerating Microbial Activity of Soil Aquifer Treatment by Hydrogen Peroxide

by
Liron Friedman
1,2,
Kartik Chandran
1,*,
Dror Avisar
3,
Edris Taher
1,
Amanda Kirchmaier-Hurpia
1 and
Hadas Mamane
2
1
Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA
2
Environmental Engineering Program, School of Mechanical Engineering, Tel Aviv University, Tel Aviv 69978, Israel
3
Hydrochemistry Lab, Water Research Center, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel
*
Author to whom correspondence should be addressed.
Energies 2022, 15(11), 3852; https://doi.org/10.3390/en15113852
Submission received: 11 April 2022 / Revised: 14 May 2022 / Accepted: 17 May 2022 / Published: 24 May 2022
(This article belongs to the Special Issue Advanced Wastewater Treatment Processes and Their Integration in Water Reuse Systems)
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Versions Notes

Abstract

:
Soil aquifer treatment (SAT), as a gravity-based wastewater reuse process, is limited by oxygen availability to the microbial community in the soil. Using oxygen from enzymatic degradation of H2O2 to generate hyper-oxygen conditions can exceed solubility limitations associated with aeration, but little is known about the effect of hyper-oxygen conditions on the microbial community and the dominant bio-reactions. This study examined the impact of H2O2 addition on the community structure and process performance, along with SAT depth. Overall, two soil columns were incrementally fed synthetic secondary effluents to simulate infiltration through SAT. The experimental column received 14 mg/L hydrogen peroxide to double the level of natural oxygen available. The microbial kinetics of nitrifiers and heterotrophs were evaluated. We found that all of the H2O2 was degraded within the top 10 cm of the column, accompanied by a higher removal of COD (23 ± 0.25%) and ammonia (31 ± 3%) in comparison to the reference column. Higher nitrogen removal (23 ± 0.04%) was obtained for the whole process using H2O2. Analysis of nitrifiers indicated that ammonia-oxidizing bacteria were most influenced, obtaining higher concentration and abundance when exposed to H2O2. DNA sequencing analysis of samples exposed to H2O2 revealed significant community structure and diversity differences among heterotrophs. This study shows that not only aerobic, but also anoxic, microbial activity and process performance in a SAT system could be accelerated in existing infrastructure with H2O2, which could significantly decrease the associated environmental footprint.

Graphical Abstract

1. Introduction

Wastewater treatment for reuse is one of the leading practices to address water scarcity due to climate instability and population growth in water-stressed regions. Tertiary treatments of secondary wastewater effluent are used to remove residual concentrations of contaminants, improve water quality, and allow a broad spectrum of reuse [1,2,3]. Several technologies have been developed for water remediation as natural materials, such as cellulose fibers and lignocellulose for contaminant biosorption [4,5]. Tertiary membrane-based filtration, such as microfiltration (MF) and ultrafiltration (UF), is commonly used, and is efficient in removing organic matter and reducing oxygen demand [6]. In addition, membrane bioreactors that combine microbial activity are widely used for removing nitrogen. However, membrane-based processes require considerable maintenance, intense chemical usage, and energy. In contrast, gravity-driven processes, such as soil infiltration, present a substantially lower associated environmental impact than membrane-based processes [7]. Soil aquifer treatment (SAT) is a chemical-free tertiary treatment and seasonal storage of the treated wastewater which combines infiltration, bioactivity, and aquifer recharge. The SAT process involves cycles of incremental aeration and the flooding of soil basins. While percolation along the soil depth in the vadose zone occurs, aeration periods provide oxygen availability for the fixed biomass on the soil grains, which efficiently removes the residual organic material and nitrogen from the secondary effluents [8]. The sequential flow creates air pockets in the soil due to preferential flow paths that can provide an equivalent of up to 42 mg/L of dissolved oxygen to support microbial aerobic activity [9]. In terms of drawbacks, SAT is a relatively slow process, and requires intensive land use. Consequently, a pretreatment could be considered to reduce loads [10,11]. More extended aeration periods can achieve higher oxygen availability and better performance, but significantly decrease the rate [12]. To meet the oxygen demand in fixed biomass systems, several studies have examined using pure oxygen or hydrogen peroxide (H2O2) [13,14].
Although many studies have shown the critical role of aeration and oxygen limitations in SAT in terms of biotransformation, the direct influence of oxygen on biokinetics has not been well understood. Specifically, the potential rates of microbial activity in SAT are affected by the carbon-to-nitrogen (C/N) ratio [15], biodegradability [16], aggregate size, and durations between flooding [17]. Biological carbon and nitrogen removal are often dominated by a few microbial functional groups (all affected by oxygen availability) as follows: carbon oxidation and denitrification by ordinary heterotrophic organisms (OHO) [18], and nitrification by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) [19]. Among NOB, the genus Nitrobacter spp. Prefer high nitrite levels characterized by a faster growth rate, whereas Nitrospira spp. Thrive, despite limited nitrite and oxygen availability [20]. Studies have revealed wider metabolic versatility among Nitrospira spp., such as complete ammonia oxidation (CMX) [21]. In comparison to nitrifiers, heterotrophs are more diverse in their potential functions when denitrification activity is limited by oxygen inhibition [22]. Studies and calculative models noted that the competition between microbial functional groups in the soil is governed by mass transfer; while nitrite and nitrate are actively transported through the bacterial membrane, oxygen is transported passively by diffusion. The different transport mechanisms enable nitrogen removal via simultaneous nitrification and denitrification (SND) activity [23]. While limited aeration periods in SAT resulted in poor performance, longer aeration periods did achieve higher oxygen availability and better performance, but significantly reduced infiltration [12,24]. To overcome these limitations, pure oxygen or hydrogen peroxide (H2O2) degradation in fixed biomass systems can be used [13,25]. Wang et al. (2017) [26] investigated the dynamics of microbial communities in soil treatment systems when applied with H2O2, and found a significant sensitivity of anaerobic groups. H2O2 is widely used as an oxidative agent that can be coupled with a catalyst for the removal of organic materials from water and for control of biofilm [27,28], or as an oxidant in advanced oxidation processes (AOP) for the degradation of trace organic compounds (TrOc) [29,30,31].
In relatively low concentrations, microbes can degrade H2O2 into oxygen and water (Equation (1)), using the enzyme catalase, without introducing toxic or polluting substances into the environment [32].
1 H2O2 => 0.53 H2O + 0.47 O2
Equation (1). The formula for bacterial degradation of H2O2 stoichiometry is by weight (g) [33].
The generated oxygen is an oxygen source that can substitute aeration [34], accelerate performance in a biofilter system [35], and act as a SAT pretreatment to reduce oxygen demand [36]. However, to the best of our knowledge, none of the studies assessed the possible acceleration of SAT performance using H2O2 directly without releasing any byproducts into the environment, as with AOP treatments. In this paper, we characterized carbon and nitrogen transformations and related microbial community dynamics to assess the performance of SAT. This paper presents the use of H2O2 for accelerating the microbial activity driving SAT performance. The introduction of H2O2 to SAT has the potential to reduce land demand through a cleaner, more resilient, and more sustainable production process for reused wastewater.

2. Materials and Methods

2.1. Accelerating Aerobic Microbial Activity by H2O2 Addition

The laboratory-scale setup was designed to simulate the vadose zone of SAT, which was fed with constant secondary effluent quality, similar to the one at the Shafdan wastewater treatment plant in Israel (Figure 1). In brief, two 100-cm depth soil columns, 10-mm in diameter, were fed with synthetic secondary effluent (SE). The columns were operated in a 48-h cycle of 12-h flooding and 36-h of soil aeration (unsaturated mode).
The residence time, determined by tracer measurements, was 5.8-h for the reference column (RC), and 6.0-h for the H2O2 column (named HPC herein). While the control column was fed only with the SE, the H2O2 column was also supplemented with 14 mg/L H2O2 (from a 30% stock solution, Fisher Scientific). When all of the H2O2 was fully degraded, it contributed ~7 mg/L of dissolved oxygen into the solution, in addition to the 7 mg/L from naturally dissolved oxygen when saturated, which is twice as much as the natural limitations of oxygen solubility at room temperature. The H2O2 concentration added was intended to double the available oxygen for bacterial activity in the column (in comparison to naturally dissolved oxygen from the air) to meet the oxygen demand for nitrification of the secondary effluent. Thus, the total available oxygen following a full degradation of the H2O2 would be 14 mg/L (Equation (1)). A hydrogen peroxide test kit (Millipore Corporation) was used and calibrated with a detection range of 1 to 6 mg/L and a measurement error of 0.1 mg/L. Samples were diluted at a 1:3 ratio to adjust to the detection range. The column saturation rate was calculated as the percentage of the delta volume of the measured inlet and the outlet as a fraction of the void volume inside the columns (Supplemental S4 Figure S2).

2.2. Synthetic Secondary Effluent Column Feed

The synthetic secondary effluent (SE) feed content for both columns was similar (Table 1). To simulate the effluent quality of the Shafdan Wastewater Treatment Plant (WWTP), the columns contained higher nitrogen content than is common in such plants.

2.3. Verifying Microbial Only H2O2-Based Activity

The degradation rates of H2O2 were examined in a mixed-batch reactor with soil (see S5.3), and were found to be 7–10 min for full degradation. Considering the 30-min residence time for each 10-cm of the column, it was assumed that the H2O2 would be fully degraded within the top 3 cm. No significant degradation of H2O2 was detected with autoclaved soil soaked in SE solution. Moreover, no significant chemical removal of NH4+ or COD was observed in the presence of H2O2 within 7 h of incubation (Supplemental Table S4).

2.4. Sampling and Analysis of Solute Quality and Soil Bacteria along Depth

The sampling and analysis of solute quality and soil bacteria were performed as in Friedman et al. (2018), where solute samples were taken and analyzed in duplicates. Calibration and system characterization was conducted for 5 months until a stable removal of COD and ammonia was achieved, indicating a steady state.

2.5. Bacterial Population by qPCR and 16S rRNA Gene Analysis

The microbial functional groups of the soil’s biofilm concentrations throughout the column were determined by targeted qPCR analysis of DNA extracted from soil samples. Soil samples of 10 g each were taken along the column’s depth. DNA was extracted in duplicates using PowerSoil® DNA Isolation Kit (Mobio, Carlsbad, CA, USA). The qPCR analysis of the extracted DNA samples was conducted in triplicate for each sample, focusing on primer-sets related to the following known functional groups: amoA for AOB [37], NSPRA 16S for Nitrospira, Nitro 16S for Nitrobacter, Pla46F for Anammox [38], and universal bacteria Eub 16S for total bacteria [39], as detailed in Park et al. (2017). NOB in this study refers to the sum of the concentrations (number of DNA copies per mg soil) of the genera Nitrospira and Nitrobacter

2.6. Topsoil 16S rRNA Gene Sequencing, bacterial Population Analysis

To determine the effect of H2O2 addition on the structure of the microbial community at the location of exposure, especially among heterotrophs, the topsoil samples of both columns were sequenced based on the 16S rRNA gene. A detailed description of the sequencing method and data analysis is provided in Supplementary S7 and elsewhere [40,41,42]. Briefly, the extracted DNA of every sample was analyzed by next-generation sequencing of the amplicon library of the Ribosomal Database Project (RDP), and classifier software was used to classify the 16S rRNA gene sequences and assign them to the closest known genus in the NCBI database with a 95% threshold. In addition, to focus on the dominant genus in the samples, all genera with an abundance lower than 2% were excluded.

3. Results

3.1. Evaluation of Soil Column Performance along with the Depth

Along with the depth of the column, no H2O2 was detected, indicating that it was fully degraded within the top 10 cm (or prior). A slight and similar decrease in dissolved oxygen concentration was observed for both columns, with 6.8 ± 0.2 mg/L and 6.1 ± 0.3 mg/L at the top and bottom, respectively, indicating aerobic conditions along the column, as expected in the vadose zone of SAT systems. Similar trends were observed for the pH, with values of 8.1 ± 0.2 and 7.6 ± 0.2 at the top and bottom, respectively, suggesting nitrification activity.
The highest COD-removal rates were obtained in the first 10 cm for both columns when, with the H2O2 column (HPC), 20% higher removal was obtained than in the reference column (RC) (Table S5, Figure 2a). The removal of COD exceeded the concentration of the rbCOD (glucose) in the feed, suggesting that this fraction of organic material was the first to be utilized, although other organic fractions were also used. This indicates that the addition of H2O2 accelerated the removal of the more degradable COD. Almost all of the COD was used at the 70 cm depth, indicating the removal of sbCOD further down the column.
In the first 10 cm, the ammonia removal was 30% higher in HPC than RC (1.64 mg/l-N vs. 1.25 mg/l-N). In both columns, an increase in ammonia concentrations was observed between −10 cm to −30 cm (Figure 2b), which is suggested to occur due to the ammonification of organic nitrogen [43]. Simultaneous reactions could explain the lower ammonia values in the first 10 cm of HPC: accelerated COD degradation, accelerated ammonification, and higher ammonia oxidation rates. The accelerated COD degradation and ammonification rates were equally balanced, with higher ammonia oxidation rates. Interestingly, at −70 cm, ammonia was fully removed in both columns (Figure 2b).
Nitrite was fully removed in the first 10 cm of HPC and was not detected at a deeper depth. For RC, although there was a significant drop in nitrite concentration at the top, it was not completely removed until reaching −70 cm (Figure 2b). In contrast to RC, nitrate was detected only below 40 cm in HPC, with a gradual increase by the bottom of the column (Figure 2c). The absence of nitrite in HPC with the introduction of nitrate only 40 cm deep indicates good SND conditions, suggesting a competitive role of nitrite in the process. Deeper in the RC (<−40 cm), the parallel removal of ammonia and introduction of nitrate reflects nitrification, while the presence of nitrite, although minor, suggests that nitrification is not complete [20] (Figure 2b,c).
The higher rates of ammonia and COD removal at the top of the HPC reflect accelerated aerobic activity due to H2O2 addition. This was also indicated by a stronger correlation between the profiles of ammonia and nitrate in the HPC compared to the RC (−0.94 and −0.87, respectively). Unexpectedly, in the case of accelerated aerobic activity, the nitrogen balance of the whole column revealed better nitrogen removal (Table S5). It is suggested that the higher availability of oxygen also allows for the activity of nitrifiers and the introduction of NOx species in parallel to COD oxidation. It was already demonstrated that, when H2O2 was added to WWTP secondary effluents, genes related to nitrification and, simultaneously, denitrification were significant and dominant in the microbial community structure [35]. Here, we suggest that the addition of H2O2 increased oxygen availability and accelerated the introduction of NOx via nitrification, which subsequently enhanced higher denitrification rates in the top 10 cm, followed by higher nitrogen removal in total.
In addition, the calculated consumption of ammonia for assimilation was slightly higher in the RC than in the HPC (34% and 28%, respectively) (Table S5). This difference suggests that enhanced nitrogen removal in the HPC did not necessarily occur where the H2O2 was degraded. As expected, the addition of H2O2 accelerated aerobic activity at the location of exposure, but it also resulted in a significant increase in the performance of nitrogen removal further along soil depth. This result suggests that the accelerated removal of COD by adding H2O2 at the top of the column reduced the available substrate for heterotrophs, thereby creating advantageous conditions further along the column for nitrifiers to compete for oxygen. In a previous study, we showed SND activity in a similar setup, and suggested the effect of COD availability and degradability levels on the microbial dynamics of N removal [15].

3.2. The Difference in Community Structure of Topsoil Samples Due to Exposure to H2O2

Considering the difference in oxygen availability between the top of both columns, the microbial community structure in the topsoil of HPC was less diverse than that in the topsoil of RC (Figure 3). The richness of the microbial species in both samples was similar, while the main difference between the two communities was found for the genera Rhodobacter (2.1% vs. 21.6%), Dechloromonas (9.1% vs. 1.7%), and Arthrobacter (0.0% vs. 5.9%). In addition, the genera Novosphingobium and Pseudocardia, both considered aerobic, were insignificant in the HPC, but relatively abundant (~6%) in the RC, indicating more obligate aerobes in the latter. The genus Rhodobacter are highly diverse in regards to function, but some were shown to denitrify or be active in limited oxygen conditions [44,45].
Dechloromonas species are characterized as facultative anaerobes, capable of utilizing humic acids [46]. Arthrobacter species are characterized as facultative aerobes, but can denitrify and utilize complex carbon compounds that might be more advantageous under the high availability of oxygen and various carbon sources [47]. The difference in the structures between the two communities suggests that H2O2 had a role in supporting the domination of the genera Rhodobacter and Arthrobacter (Figure 3).

3.3. Nitrogen Functional Group Dynamics

The profiles of nitrogen-functional groups along the columns were analyzed to reveal the functional potential of the biomass and relate it to the observed performance. The concentration of ammonia oxidizers (amoA) was significantly higher in the HPC than the RC in the topsoil (1.1 ± 0.04 × 109 vs. 8.5 ± 0.035 × 109 DNA copies/mg soil) and continued to be dominant at a depth of −10 cm (Figure 4a). This finding could be related to the higher removal rates of ammonia in the top 10 cm of the HPC. In both columns, the profiles of total bacteria (Eub) and NOB showed similar trends and values. Interestingly, the profiles of ammonia oxidizers and NOB overlapped all along with the RC, which resembles an equal portion between AOB and NOB, among other nitrifiers.
The total bacteria concentrations at the top of the RC and HPC were similar, and gradually dropped by two orders of magnitude by the bottom of the columns. The largest drop in total bacteria along the columns, corresponding with the drop in COD, was observed at −10 cm in both columns (Figure 4b).
In general, the microbial profiles paralleled the chemical ones. These findings indicate that the main acceleration in ammonia consumption with the addition of H2O2 resulted from AOB, presumably due to higher oxygen availability. This can be explained by the higher degradation rate of H2O2 or AOB’s lower affinity to oxygen compared to heterotrophs and NOB, which can be expressed in terms of the oxygen half-saturation rate of the Monod kinetic equation [41].
The concentrations of anammox-related bacteria (AMX) at the top of both columns were identical, around 2.4 × 108 DNA copies/mg soil. At a depth of −10 cm, a drastic decrease in concentration was observed in the RC. In the HPC, a similar drop was observed at a deeper level, at −20 cm (Figure 4d), although ammonia was still sufficient. This difference could indicate a higher availability of NO2− at −10 cm depth in the HPC, consistent with the higher presence of AOB in this location. In contrast to the other functional groups, the profile of AMX showed a gradual recovery of 3–4 logs until 70 cm depth. The observed trend of the AMX reflects competition for nitrite reduction with OHO. AMX-related bacteria might over-compete for the available nitrite when the COD availability drops.

3.4. Influence of H2O2 on the Community Structure of NOB

To understand the competitive relationships among NOB, the differentiation of Nitrospira and Nitrobacter species was investigated. A significant abundance of Nitrobacter species (~25%) among NOB was observed in the top samples of both columns (Figure 5). At all other depths, for both columns, Nitrospira was the most abundant genus by far (>90%), compared to Nitrobacter. The dominance of Nitrospira species’ concentration and abundance in the top 40 cm of the RC, if ammonia was available (>1.5 mg N/L), suggests a possible role of comammox (CMX) activity in ammonia oxidation [48]. A similar dynamic was seen for the HPC (−10 cm and −20 cm) in both columns (Figure 5a,b).
Nitrobacter species are found in environments with relatively high concentrations of substrate (KsNO2−,Nitrobacter > KsNO2−,Nitrospira, 0.69–7.6 mg N/L vs. 0.13–0.97 mg N/L, respectively). In addition, some species of Nitrospira were found to have a higher affinity to oxygen than the known Nitrobacter species (KO2,Nitrobacter > KO2,Nitrospira) [41]. It is suggested that the higher abundance of Nitrobacter species at the top of both columns, relative to other depths, reflects the higher availability of nitrite, introduced from the feed solution rather than produced by nitritation.
Previous studies have shown that SND could occur in soil systems and is related to oxygen availability [49], and that the addition of H2O2 results in significant expression of genes related to denitrification activity [35]. We suggest that the mechanism of accelerating oxidation via microbial activity when H2O2 is added is rooted in the rapid degradation of the H2O2, which releases oxygen close to the cell membrane, thereby generating conditions of higher oxygen availability than present in standard diffusion from air. When there is a significant oxygen demand and the biofilm community is characterized with oxygen-competitive nitrifiers, nitrification is generated, and NOx species are introduced, although not all of the COD is oxidized. Thus, when COD is still available, denitrification and nitrogen removal are also accelerated.
With regard to SAT, the impact of adding H2O2 to the stream of the secondary effluent occurs within the first few cm of the soil depth and provides a prominent source of oxygen to accelerate bacterial activity beyond the limitations of oxygen solubility. In this study, although SND was observed at the location of exposure to H2O2, the main enhancement in anoxic activity occurred further down the column (Figure 2). Furthermore, the oxidative “signature”, resulting from the addition of H2O2 at the top of the columns, affected the total removal of nitrogen and the profiles of related functional groups, along with the depth (Figure 3).
We suggest that the change in the C/N ratio and the reduction in the potential oxygen demand of the solute, along with the columns due to the enhanced activity, is affected downstream in the process, which was similarly suggested by Moshe et al. (2020) [12]. With relatively high ammonia in the treated effluents, as in this study, the use of H2O2 may offer a wider role than aerobic microbial activity in biofilm-based processes, leading not only to faster and better utilization, but also enhancing nitrogen removal.

4. Discussion

Potential for Application and Enviro-Economic Evaluation

Applying H2O2 to SAT has several advantages compared to conventional SAT, which solely relies on gaseous oxygen, and to other tertiary processes. Since the bacteria degrade H2O2, the oxygen produced is highly available with minimal oxygen transport, and can provide oxygen availability beyond the natural constraints of solubility, such as temperature and pressure. Additionally, since H2O2-degrading enzymes are ubiquitous among microbial organisms, H2O2 can be considered a bio-specific oxygen source for aerobic activity [50].
Applying H2O2 is relatively easy, requiring minimal maintenance, space, and capital costs [51,52]. Considering a cost of 400 USD/m3 for a 35% H2O2 industrial-grade solution, the operational cost of improving SAT performance by treating similar secondary effluents would save ~0.0215 USD/m3 on the operational cost of SAT, estimated to be 0.13–0.17 USD/m3 [53,54]. Other solutions to treat secondary effluents may have much higher costs: microfiltration (MF) + reverse osmosis (RO) + UV/H2O2 are estimated to be 0.28 USD/m3, while MF + ultra-filtration (UF) followed by RO are estimated to be 0.4 USD/m3 [1,55,56]. Developments in on-site production of H2O2 may eliminate the cost of delivery and storage [57,58].
In terms of efficiency, a gravitational-based process like SAT has several limitations compared to intensive membrane processes such as MF, UF, and RO. These processes have higher and more intensive removal rates, the ability to remove a broad spectrum of contaminants, and a relatively small land demand. Although SAT presents relatively slower rates of contaminant removal, it is a robust process. Membrane-based processes have a significant environmental footprint due to relatively higher energy demand (0.5–0.6kWh/m3) [59] and continuous production of permeate.
AOPs are highly effcient in removal of TrOC, but are not capable of removing nutrients. SAT is used to address the potential risks of water scarcity through the reuse of wastewater for unrestricted irrigation, and has a significant advantage in meeting the required water quality with minimal energy demand (0.05–0.07 kWh/m3) and minimal climate footprint [10,60].

5. Conclusions

In this study, we demonstrated that the acceleration of bioactivity via H2O2 addition to SAT creates an oxidative “signature”, resulting in enhanced aerobic microbial activity and better total nitrogen removal, along with column depth at a rate greater than that which is predicted by theoretical calculations of oxygen demand. The degradation rate of H2O2 was relatively quick and is suggested to lead to higher oxygen availability at the location of exposure, the top of the column, where it was fully utilized for aerobic activity. Accelerating microbial activity by H2O2 addition affected the microbial community structure and diversity, favoring AOB, between nitrifiers, at the location of exposure, which suggests that AOB mostly utilized the H2O2-generated oxygen. Combining H2O2 with SAT offers a potential path toward significantly decreasing the environmental footprint of filtration systems, as well as the production of treated wastewater for reuse. This study presents wastewater reuse designers with an evaluation of a gravity-based and relatively low energy tertiary solution, which can be used for designing new systems or intensifying the use of existing SAT infrastructure.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/en15113852/s1, Figure S1: Experiment setup to measure saturation rate. The outlet is drained to a bucket which continuously weights on a balance; Figure S2: Volume (mL) of inlet, outlet, and delta (ml) of Column CC (a) and PC (b) (peroxide treatment) over time (min); Figure S3: Ammonia and COD values measured over 9 hours at the control (a) and peroxide (b) column. Figure S4: Ammonia and COD values measured from control (a) and peroxide (b) column at the top, −20 cm and −120 cm; Scheme S1: The test bottles and content; Table S1: Specification of secondary effluent anions and ions; Table S2: Concentration, biodegradability and nitrogen content (by weight) of COD and ammonia concentration at the feed. * Measured nitrogen content of LB (Table S4); Table S3: Concentrations of ammonia, COD, H2O2, at start and 7 hours in the sealed vi-als. Number of the treatment is referred in brackets; Table S4: Calculations of N assimilation. * Calculated based on total nitrogen measurements (data not shown); Table S5. Comparison between RC and HPC for the removal of COD, NH4+, and N.

Author Contributions

Conceptualization, L.F. and H.M.; methodology, A.K.-H. and E.T.; validation, L.F., A.K.-H. and E.T.; formal analysis, L.F. and E.T.; resources, H.M., K.C. and D.A.; writing—original draft preparation, L.F.; writing—review and editing, L.F., H.M. and K.C.; visualization, A.K.-H.; supervision, H.M., K.C. and D.A.; All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Informed Consent Statement

Not applicable.

Acknowledgments

The authors would like to thank M. R. Park and Z. Li for their help and inspiration, S. Dagan for the sequencing analysis and innovative thinking, S. Katz for the analytical and graphical help, and T. C. Siegel for her professional artistic touch to express the science in graphics. The first author sincerely appreciates the support of The Manna Center for Food Safety and Security fellowship of Tel Aviv University. This work is dedicated to the memory of Allan Witztum, a passionate botanist, an inspirational teacher, and a kind and humble man.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Soil column setup. Control column (RC; (left)) and H2O2 column (HPC; (right)) fed with synthetic secondary effluent (tanks in the middle) and H2O2 (tank on the right). All pumps were controlled by a timer.
Figure 1. Soil column setup. Control column (RC; (left)) and H2O2 column (HPC; (right)) fed with synthetic secondary effluent (tanks in the middle) and H2O2 (tank on the right). All pumps were controlled by a timer.
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Figure 2. Comparison between the control (RC) and peroxide column (HPC) of the chemical concentrations’ profiles along depths. (a) Comparison of the COD profile (mg/L); (b) comparison of the NH4+, NO2 (mg/l-N); and (c) NO3 (mg/l-N) profiles. Standard errors of averaged values are represented by error bars. The suggested dominant nitrogen reactions are noted in highlighted boxes next to the plot lines or highlighted in dotted red circles.
Figure 2. Comparison between the control (RC) and peroxide column (HPC) of the chemical concentrations’ profiles along depths. (a) Comparison of the COD profile (mg/L); (b) comparison of the NH4+, NO2 (mg/l-N); and (c) NO3 (mg/l-N) profiles. Standard errors of averaged values are represented by error bars. The suggested dominant nitrogen reactions are noted in highlighted boxes next to the plot lines or highlighted in dotted red circles.
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Figure 3. Comparison of the bacterial community structure (16S rRNA gene) of only the dominant genus (>2%) of the top of the two columns.
Figure 3. Comparison of the bacterial community structure (16S rRNA gene) of only the dominant genus (>2%) of the top of the two columns.
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Figure 4. Comparison between control (RC) and peroxide columns (HPC) profiles of the main microbial functional groups along depths. (a) Profile concentrations of amoA (AOB); (b) NOB (sum of Nitrospira spp. and Nitrobacter spp.); (c) total bacteria (universal bacteria, Eub); and (d) anammox. Units are in log DNA copy number/mg soil. Columns were fed with synthetic effluent with no H2O2 (top) and 14 mg/L H2O2 (bottom).
Figure 4. Comparison between control (RC) and peroxide columns (HPC) profiles of the main microbial functional groups along depths. (a) Profile concentrations of amoA (AOB); (b) NOB (sum of Nitrospira spp. and Nitrobacter spp.); (c) total bacteria (universal bacteria, Eub); and (d) anammox. Units are in log DNA copy number/mg soil. Columns were fed with synthetic effluent with no H2O2 (top) and 14 mg/L H2O2 (bottom).
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Figure 5. The fraction of Nitrospira spp. and Nitrobacter spp. out of NOB (sum of Nitrospira spp. and Nitrobacter spp.) along column depth, for (a) RC and (b) HPC. Dotted circle emphasizes the location of significant differences.
Figure 5. The fraction of Nitrospira spp. and Nitrobacter spp. out of NOB (sum of Nitrospira spp. and Nitrobacter spp.) along column depth, for (a) RC and (b) HPC. Dotted circle emphasizes the location of significant differences.
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Table
Table 1. The measured concentrations of ammonia (NH4+), nitrite (NO2−), nitrate (NO3−), organic nitrogen, and COD in the SE. COD concentration was composed of materials with three levels of biodegradability: readily–rbCOD (glucose), moderate–mbCOD (LB broth), and slow–sbCOD (humic acids). Further details are provided in Supplemental Tables S2 and S3.
Table 1. The measured concentrations of ammonia (NH4+), nitrite (NO2−), nitrate (NO3−), organic nitrogen, and COD in the SE. COD concentration was composed of materials with three levels of biodegradability: readily–rbCOD (glucose), moderate–mbCOD (LB broth), and slow–sbCOD (humic acids). Further details are provided in Supplemental Tables S2 and S3.
CompoundConcentrationUnit
NH4+4.1 ± 0.2mg N/L
NO2−1.8 ± 0.05mg N/L
NO3−0.22 ± 0.01mg N/L
Organic nitrogen4.5 ± 0.1mg N/L
COD63.2 ± 1.1mg/L
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Friedman, L.; Chandran, K.; Avisar, D.; Taher, E.; Kirchmaier-Hurpia, A.; Mamane, H. Accelerating Microbial Activity of Soil Aquifer Treatment by Hydrogen Peroxide. Energies 2022, 15, 3852. https://doi.org/10.3390/en15113852

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Friedman L, Chandran K, Avisar D, Taher E, Kirchmaier-Hurpia A, Mamane H. Accelerating Microbial Activity of Soil Aquifer Treatment by Hydrogen Peroxide. Energies. 2022; 15(11):3852. https://doi.org/10.3390/en15113852

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Friedman, Liron, Kartik Chandran, Dror Avisar, Edris Taher, Amanda Kirchmaier-Hurpia, and Hadas Mamane. 2022. "Accelerating Microbial Activity of Soil Aquifer Treatment by Hydrogen Peroxide" Energies 15, no. 11: 3852. https://doi.org/10.3390/en15113852

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