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Article

Thauera sp. in Hydrogen-Based Denitrification: Effects of Plentiful Bicarbonate Supplementation on Powerful Nitrite Reducer

by
Suphatchai Rujakom
1,*,
Tatsuru Kamei
2 and
Futaba Kazama
2
1
School of Interdisciplinary Studies, Mahidol University Kanchanaburi Campus, No. 199, Moo 9, Lumsum Sub-District, Saiyok District, Kanchanaburi 71150, Thailand
2
Interdisciplinary Research Centre for River Basin Environment, University of Yamanashi, 4-4-37 Takeda, Kofu 400-0016, Yamanashi, Japan
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(1), 277; https://doi.org/10.3390/su15010277
Submission received: 15 November 2022 / Revised: 12 December 2022 / Accepted: 21 December 2022 / Published: 24 December 2022
(This article belongs to the Special Issue Water Sustainability: River Basin Management, Water Quality and Quantity Monitoring)
Browse Figures
Versions Notes

Abstract

:
Nitrite accumulation in hydrogen-based denitrification (HD) has been reported as a difficulty for achieving complete denitrification. Thauera sp. has been found as the dominant bacterial species in HD previously when using a plentiful amount of HCO3−. This present study was successful in isolating Pseudomonas sp., Dietzia sp., Pannonibacter sp., Halomonas sp., Bacillus sp., and Thauera sp. These isolated strains were selected for investigating the nitrogen removal performance under the plentiful HCO3− condition. Only Pseudomonas sp. and Thauera sp. were capable of removing NO2− where the specific NO2− removal rate of Thauera sp. (36.02 ± 5.66 mgN gVSS−1 day−1) was 9 times quicker than that of Pseudomonas sp. (3.94 ± 0.80 mgN gVSS−1 day−1). The Thauera sp. strain was then tested at different HCO3− amounts. As a result, Thauera sp. had no ability to function both NO3− and NO2− removals under HCO3− deficit condition. This study provided evidence on the role of Thauera sp. and the necessity of bicarbonate in the hydrogen-based denitrification process to enhance its efficiency and to simultaneously reduce the operational cost especially for hydrogen.

1. Introduction

Autotrophic denitrification is an alternative process for the abatement of NO3− and NO2− contaminated water [1,2,3]. It can overcome the high sludge formation problem requiring post-treatment in heterotrophic denitrification since autotrophic denitrification provides lower sludge yields [4,5]. Additionally, several non-toxic inorganic elements can be used as an electron donor for autotrophs, such as ferrous iron, sulphur and H2 gas, which could lead the denitrification system to a satisfactory performance [1,2,3]. Since H2 can be considered as an environmentally friendly substance due to its non-toxicity and non-harmfulness [6,7], using H2 gas is a favorable option as it provides decent efficiency compared to other known electron donors for the autotrophic denitrification system [8]. H2-based denitrification is also well-known as hydrogenotrophic denitrification, H2-based autotrophic denitrification, autohydrogenotrophic denitrification or hydrogen-based autotrophic denitrification (HD) [3,6,9,10,11,12]. Decent efficiency of the HD system has been observed in several studies [3,9,13,14,15,16,17]. Still, several studies experienced the accumulation of NO2−, which hindered the complete denitrification [3,9,10,11,12,15,16,17].
Considering the inorganic carbon source for HD, bicarbonate (HCO3−) was found to be an important inorganic carbon source for autotrophic H2-oxidizing denitrifiers, which provided higher potential in acclimatizing the HD bacteria enhancing the HD efficiency compared to utilizing CO2 [3,11,12]. Moreover, as presented in Equations (1) and (2), HCO3− of only 0.05 moles is required to remove 1 mole of NO3− while 0.122 moles HCO3− is required to convert 1 mole of NO2− to N2 gas [18]. This implicitly suggests the importance of HCO3− on NO2− reduction and that providing a larger amount of HCO3− to the HD system could overcome the NO2− accumulation issue leading to complete denitrification.
NO3− + 1.13 H2 + 0.05 HCO3− → 0.99 NO2− + 0.01 C5H7O2N + 1.09 H2O + 0.06 OH−
NO2− + 1.78 H2 + 0.122 HCO3− → 0.488 N2 + 0.0244 C5H7O2N + 1.19 H2O + 1.122 OH−
Furthermore, information on denitrification behavior, dose of supplementary HCO3− amount, the expression of denitrification-functional genes, and the abundances of bacteria dominating to the HD system of our former study [9] was gathered to comprehend their relationships through canonical correspondence analysis (CCA) as illustrated in Figure 1. The results implied that bacteria belonging to the genus Thauera might be the major players having roles on NO2− reduction, leading the system operated with the high C/N ratio to the high HD efficiency. An explicitly high abundance of nirS gene, indicating the greater rate of the NO2− removal activity, was also observed, as well as an exceptionally high abundance of the genus Thauera in the sludge under the plentiful HCO3− condition where the great HD efficiencies were achieved. In this regard, HD can be considered as eco-friendly due to the non-toxic properties of both H2 and HCO3− to the environment.
Moreover, the genus Thauera was reported as gram-negative bacteria having intracellular poly-β-hydroxybutyrate (PHB). It possesses a cellular respiration system to extract carbon sources as chemical energy for its cell processes. It can grow in both aerobic and anaerobic conditions, where heterotrophic and autotrophic growth was observed depending on its utilization of carbon sources and electron donors [19,20]. Hence, Thauera can be referred to as facultatively mixotrophic bacteria. The genus Thauera has been found to be a dominant bacterial species in the HD system [6,9,18]. Our previous study [9] found that inorganic HCO3- was influential in acclimatizing Thauera under an H2-based anoxic condition, leading to a high nitrite reduction rate achieving complete denitrification. Thus, the isolation of Thauera sp. from the mixed-culture sludge used as an inoculant is necessary to comprehend the denitrifying behaviors of Thauera sp. in the H2-based anoxic condition with HCO3−. It was also interesting to comprehend that HCO3− has a powerful impact on Thauera sp. in performing denitrifying activities, especially NO2− reduction, independent from the cooperative functions of other bacteria due to a lack of information on the effect of HCO3− on the Thauera strain in denitrification. To explore the possible way to overcome the NO2− accumulation in the HD system, Thauera sp. was isolated in this study to investigate the important role of HCO3− as an inorganic carbon source on the ability of NO3− and NO2− removal. This could suggest the strategy of acclimatizing the Thauera-rich sludge using HCO3− for operation of the HD system in possessing the powerful NO2− reducer, which can aid in solving the NO2− accumulation issue and enhancing the complete denitrification.

2. Materials and Methods

2.1. Bacterial Isolation

This study attempted to isolate Thauera sp. from the mix-culture sludge enriched on the HD reactor. As the bacterial sludge had been cultivated and enriched in the previous study [9] where the HD performance was found satisfying and bacteria in the genus Thauera were found highly abundant, the sludge was subsequently obtained to be used for the bacterial isolation. First, a serial-dilution (from 10−1 to 10−5) was performed for the obtained sludge prior to spreading onto an agar plate for which the dilution was using 0.9 % NaCl (w/v). The agar was prepared following the instructions provided by the manufacturer, as mentioned in a previous study [21], as a selective medium for cultivating NO3−-reducing bacteria or denitrifiers (Table S1). Furthermore, it is necessary to also prepare three solutions (I, II, and III). These solutions were autoclaved to provide the aseptic condition and were cooled down before mixing in the ratios of 98:1:1 for solution I, II, and III, respectively, to be used as the media in this experiment. As the spread-plate technique was performed, 100 µL of each dilution of the sludge was separately spread on the prepared agar plate in triplicate. The agar plates were then incubated at 35 °C aerobically until the colonies were observed (around three weeks for this study). Bacterial cultivation was performed under aerobic condition in this study as Thauera sp. was found to be facultatively mixotrophic bacteria [19,20,22]. The different-morphology colonies were randomly picked from several plates, which contained around 30 to 50 colonies, and were then streaked onto a new agar plate for each colony to purify the colonies. The plates were incubated at the identical conditions until the growth of the colony was observed. The purified colony was taken and cultivated in Luria–Bertani (LB) broth until the bacterial growth was observed by the optical density measured at 600 nm (data not shown).

2.2. Identification of Bacterial Strain

The DNA sequencing was performed for each isolated bacterial strain to identify its taxonomical information. The preserved bacterial glycerol stocks were used to directly amplify bacterial 16S rRNA gene through Polymerase Chain Reaction (PCR) using primers Univ-8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and Univ-1512R (5′-ACG GYT ACC TTG TTA CGA CTT-3′) [23,24]. A TaKaRa PCR Thermal Cycler Dice® Gradient (TAKARA, Shiga, Japan) was used to perform the amplification in which the reaction mixtures for PCR contained 5 µL bacterial glycerol stock; 0.25 µM forward and reverse primers (Eurofins Genomics Inc., Tokyo, Japan); 25 µL Sapphire Amp® Fast PCR Master Mix (TAKARA, Shiga, Japan); and 19.5 µL sterile DNAse-free water. The thermal cycling conditions of PCR for 16S rRNA were 94 °C for 60 s; 35 cycles at 98 °C for 5 s; 55 °C for 5 s, and 72 °C for 15 s; and the final extension step at 72 °C for 10 s. After that, the PCR products were purified using Cica Geneus® PCR & Gel Prep Kit (Kanto Chemical Co., Inc., Tokyo, Japan) to obtain the purified DNA for the commercial DNA sequencing service (Eurofins Genomics Co., Ltd., Tokyo, Japan). The sequencing was then analyzed using the clone library method with primer Univ-1512R. Afterwards, the obtained sequences were aligned using MUSCLE [25]. The comparison between the obtained sequences and the sequences available on the database of GeneBank was simultaneously conducted in order to identify the most similarly identified bacterial strain sequences to the sequences obtained in this study through the BLAST search (blastn suite). The distance estimation and the phylogenetic tree generation was performed using the Maximum composite Likelihood model [26] and Neighbor-Joining method [27], respectively, by clustering together in the bootstrap test for 1000 replicates [28] using MEGA software version 11 [29]. The sequences obtained in this study are available on GeneBank with accession numbers OP676119 to OP676134 as presented in Table 1.

2.3. Inoculants Preparation

Single strains of bacteria were prepared as inocula following the method adapted from a previous study [30] where all the apparatuses used in this experiment were autoclaved to perform the aseptic technique. The grown bacterial strain in LB broth of 20 mL was inoculated to an autoclaved Erlenmeyer flask containing 250 mL LB broth and was then placed in a shaking incubator for 48 h at 35 °C and 150 rpm. The bacterial cells were then aseptically harvested by centrifugation at 4000 rpm for 10 min. The collected cells were washed with sterile 0.9% NaCl (w/v) prior to the inoculation. The procedures of the bacterial cell preparation are illustrated in Figure 2.

2.4. Effects of HCO3− on Bacterial Behavior in Performing Nitrogen Removal

In this study, several experiments were performed with different amounts of HCO3−: inadequate, sufficient, and plentiful amounts. An inadequate HCO3− amount was prepared with no addition of HCO3− to simulate the inorganic carbon source deficit condition because the bacteria as low HCO3− amount is already available in tap water [9]. Moreover, a 275 mg HCO3− L−1 was supplemented to provide a sufficient carbon source to remove 20 mg N L−1 that was spiked as an initial concentration in this study based on the theoretical equations in Equations (1) and (2) and the previous report [3]. For the plentiful HCO3− amount, 6886 mg HCO3− L−1 was added to the synthetic medium to provide the suitable condition for Thauera sp. as reported in our previous finding [9]. The effect of HCO3− on several selected bacterial strains isolated from the mixed H2-dependent denitrifiers in NO3− and NO2− removals under the plentiful HCO3− amount supplementary was investigated to comprehend and to compare the behaviors of each strain on nitrogen removal. A 300 mL-working volume Erlenmeyer flask was used as an experimental unit (Figure 3). The prepared inoculant of each bacterial strain was individually put into an autoclaved flask containing 300 mL of tap water-based synthetic medium prepared with NaHCO3 and either NaNO3 (20 mg NO3−-N L−1) or NaNO2 (20 mg NO2−-N L−1). As the target strain of this study was Thauera sp., the different HCO3− amounts, inadequate, sufficient, and plentiful, were varied. Details of all experiments are summarized in Table S2. The flask was then tightened with a rubber stopper equipped with a sampling channel, H2 inlet channel and a vent in which the whole part of this rubber stopper was also sterile. Experimental units were sparged with H2 gas continuously through a 0.2 µm filter from a gas cylinder and were operated in an incubator at 35 °C throughout the study. The experiment was carried out in the duplicate for each condition of synthetic medium. The water sample was taken frequently for the immediate measurements of the pH and dissolved H2 (DH) in water prior to the preservation in −4 °C for further analyses of NO3− and NO2− concentrations. Additionally, there were experimental units performed as controls of this experiment by operating with no bacterial inoculation.

2.5. Analytical Methods for Water Samples and Necessary Calculations

As the water samples were taken from several experiments in this study, all samples were preserved at −4 °C for the analyses of NO3−-N and NO2−-N concentrations, which were determined through colorimetric methods, according to the standard methods for the examination of water and wastewater [31] using a UV-1800 spectrophotometer (SHIMADZU, Tokyo, Japan). Volatile suspended solid (VSS) in each experimental unit was measured to identify the specific rates of NO3−-N and NO2−-N removals. DH and pH were measured using a pH meter (HORIBA Scientific, Kyoto, Japan) and a DH meter (TRUSTLEX Inc., Osaka, Japan), respectively. The calculations of the specific rates of NO3− and NO2− removals and denitrification [mgN gVSS−1 day−1] were performed using Equation (3) where C0 and Ct were the initial and final concentration of either NO3− or NO2− [mgN L−1] at a time (t), respectively; t is an operation time [day]; and VSS is a concentration of the volatile suspended solid [gVSS L−1]. To calculate the denitrification rate, concentration of total nitrogen was used for C0 and Ct.
Specific rate of removals = (C0 − Ct)/(t × VSS)

3. Results and Discussion

3.1. Isolation of Single Bacterial Strains from the Mixed-Culture Sludge

From the isolation of bacteria from the mixed-culture sludge of the HD system, 16 isolates of bacterial strain were obtained. The strains were named as Is1 to Is16 prior to the DNA sequencing. The sequences of the 16S rRNA gene of each isolated strain were compared with the sequences available on the NCBI database as presented in Figure 4 and Table 1. It was found that the bacterial strains isolated in this study belonged to different classes, which were α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Actinobacteria, and Bacilli, as illustrated in Figure 4. Among these isolates, there were six clusters of isolated strains for which the results in Table 1 revealed that the strains were found belonging to six different families: Pseudomonadaceae, Dietziaceae, Rhodobacteraceae, Halomonadaceae, Zoogloeaceae, and Bacillaceae. The exceptionally high similarity of the isolates in this study to the available sequences on the database were also found ranging from 99.34 to 100.00%. At the genus level, it showed that the bacterial isolations in this study were able to isolate bacterial strains from six genera: Pseudomonas, Dietzia, Pannonibacter, Halomonas, Thauera, and Bacillus. Is1, Is2, Is3, and Is4 exhibited the closest match to Pseudomonas spp. with similarities of 100%. Is5 showed a 100% similarity to Dietzia sp. Similarly, Pannonibacter sp. was found to be the closest match for Is6, Is8, Is9, Is10, Is11, Is12, and Is14 with similarities ranging from 99.88% to 99.89%. Is7 had a closest match to Halomonas sp. with a similarity of 99.89%. Is13 was found to have a 99.89% similarity to Thauera sp. while Is15 and Is16 had the closest match to Bacillus spp. with a 99.34% to 99.83% similarity.
According to previous studies, it was determined that these 16 isolates obtained in this study had the capability of denitrification. Pseudomonas spp. were capable of aerobically and anaerobically removing nitrate and achieving complete denitrification [32,33]. Dietzia spp. had abilities in utilizing petroleum hydrocarbons as a carbon source and reducing NO3− [34,35]. Pannonibacter spp. were found capable of performing denitrification in aerobic and anaerobic conditions with a remarkable potential in removing NO3− and NO2− in saline water [36,37]. Moreover, the complete denitrification was achieved aerobically by Halomonas spp. in which denitrification-functional genes (i.e., napA, narG, nirS, norB, and norZ) were found to coexist in the Halomonas spp. group [38,39]. Thauera spp. were dominant in both autotrophic and heterotrophic denitrification processes, where Thauera spp. exhibited a great potential in denitrifying activities achieving complete denitrification as the expression of napA, narG, nirK, nirS, norB, and nosZ genes were determined [9,40,41,42]. Bacillus spp. was found as an efficient aerobic denitrifier in which the complete denitrification was confirmed by detecting the nirS gene indicating the NO2− reducing activity [43,44,45]. In this study, however, the Thauera sp. strain was successfully isolated from the mixed-culture sludge of the HD reactor and shared a high similarity to Thauera phenylacetica strain B4P, as presented in Table 1.

3.2. Behavior of Single Isolated Strains in HD under Plentiful HCO3− Supplementary Conditions

As several bacterial strains were obtained, the strains belonging to genera Pseudomonas, Pannonibacter, and Bacillus were found as the often-observed strains in this study. The bacterial strains Is1, Is6, and Is15 were used as inoculants to represent the three aforementioned bacterial strains. Thauera sp. strain Is13 was also used as an inoculant to compare its behaviors to the other strains. The strains were separately inoculated into each reactor containing liquid medium spiked with either NO3− or NO2− under H2-sparging anoxic condition to simulate the HD system, where the amount of HCO3− varied from inadequate to plentiful amounts in each individual reactor. The reactors were incubated at 35 °C and continuously sparged with H2 gas at a stable rate to maintain the concentration of dissolved H2 to be higher than 0.2 mg L−1, which is excess and does not limit the system during the experimental studies. The control units without bacterial inoculation showed neither NO3− nor NO2− removal activities (data not shown) indicating that the experiments were performed aseptically. The results showed that Pseudomonas sp. strain Is1 was capable of completely removing NO3− and NO2− in about 70 h, whereas the accumulation of intermediate NO2− was observed during the NO3− reduction when the reactors under the H2-based anoxic condition supplemented with the plentiful HCO3− amount, as illustrated in Figure S1. Moreover, the results obtained from the reactors inoculated with Pannonibacter sp. strain Is6 implied that complete reduction of NO3− was achieved within 35 h, where NO2− accumulated as high as the initial NO3− concentration (Figure S2a), in which no further removal of NO2− was seen. Figure S2b also confirmed that Pannonibacter sp. was incapable of NO2− reduction in the plentiful HCO3− condition. For Bacillus sp. strain Is15, the results showed that no removal of both NO3− and NO2− was observed despite operating for over 150 h, as presented in Figure S3, indicating that Bacillus sp. might not be a main player in the HD system supplemented with plentiful HCO3−.
However, Thauera sp. was capable of performing rapid NO3− and NO2− removal, achieving a faster complete denitrification compared to the other strains, as illustrated in Figure 5e. Considering the NO2− removal enhancing denitrification, it was additionally found that the specific NO2− removal rate of Thauera sp. strain Is13 (36.02 ± 5.66 mgN gVSS−1 day−1) was about 9 times quicker than that of Pseudomonas sp. strain Is1 (3.94 ± 0.80 mgN gVSS−1 day−1), as described in Table 2. This suggested that the plentiful HCO3− condition may be favorable for Thauera sp. to perform outstandingly satisfactory NO2− removal to induce faster complete denitrification. The specific removal rates of NO3− and NO2− performed by Thauera sp. strain Is13 were also explicitly higher than those performed by the other strains. The results also implied that each bacterial strain had its suitable and favorable conditions to perform denitrifying activities (NO3− and NO2− removals), as reported in previous studies [32,33,36,37,43,44,45]. Moreover, the initial and final pH of the synthetic medium supplemented with plentiful HCO3− of the overall experiments were found to be 9.19 ± 0.05 and 9.47 ± 0.11, respectively, suggesting that HCO3− could also be utilized as a buffer for the solution to avoid the pH shift in the HD system, as also reported previously [9,14,46,47].

3.3. Effects of HCO3− on Thauera sp. in Performing Nitrogen Removal

As mentioned previously, HCO3− could be a significant factor inducing the performance of Thauera sp. in denitrification. Several concentrations of HCO3−: inadequate, sufficient, and plentiful, were varied. Figure 5e illustrates that Thauera sp. strain Is13 was capable of achieving complete denitrification within 45 h when plentiful HCO3− amount was supplemented to the reactor. Moreover, there was no intermediate NO2− observed during the operation. Furthermore, Thauera sp. strain Is13 in the reactor with the sufficient HCO3− amount was able to completely remove NO3− by 34 h. Figure 5c shows that Thauera sp. strain Is13 simultaneously accumulated NO2− as a transient intermediate, which was 22.9% of the initial NO3− concentration and the intermediate was subsequently reduced until complete denitrification by 65 h was achieved. However, there were no reductions in both NO3− and NO2− when the inadequate HCO3− amount was supplemented in spite of the reactor being operated until 65 h (Figure 5a).
In addition, Thauera sp. strain Is13 was also inoculated to the reactors spiked with NO2−. Figure 5 also shows the profiles of the NO2− concentration and the pH. A rapidly complete NO2− reduction was observed at 20 h when the plentiful HCO3− amount was supplemented (Figure 5b) while the complete NO2− reduction was observed in the reactor with the sufficient HCO3− amount at about 34 h (Figure 5d). In the inadequate HCO3− amount reactor, no reduction of NO2− was seen even though the reactor was operated up to 60 h (Figure 5f). This implied that Thauera sp. required at least the sufficient HCO3− amount to enhance the ability of the removal of NO2−. The results suggested that Thauera sp. might be a powerful NO2− reducer to overcome the NO2− accumulation problem occurring in the HD system. This could be evidence that Thauera sp. required sufficient HCO3− to perform the NO3− and NO2− reductions in which removing NO2− was the key to achieving complete denitrification.
Additionally, considering the changes in the pH in this study, it was found that the reactors supplemented with plentiful HCO3− had a capacity in buffering the shift of the pH, as presented in Figure 5e,f as previously mentioned. However, in the synthetic water with sufficient HCO3−, as illustrated in Figure 5c,d, the initial and final pH were approximately 9.29 ± 0.13 and 10.34 ± 0.04, respectively. This could affirm that a lower HCO3− amount can reduce the buffering capacity of the solution as the pH is increased from the complete NO2− conversion step producing a high OH− concentration, as described in Equation (2). Moreover, the inadequate HCO3− supplemented synthetic medium should have the weakest buffering capacity as no additional HCO3− was supplied. In contrast, the initial and final pH were 8.47 ± 0.25 and 9.23 ± 0.21, respectively, which were not as different as observed in the sufficient HCO3− condition. This implied that neither the NO3− nor NO2− reductions were observed in this condition.
After all the experiments were carried out using Thauera sp. strain Is13, which was isolated from the mixed HD culture sludge, different behaviors on N removal under the different supplementary HCO3− amounts were observed, as mentioned earlier. The specific rates of NO3− and NO2− removals and denitrification were determined, as presented in Table 2. It is evident that the rates of NO3− and NO2− removals were found as high as the larger amount of HCO3− was supplemented to the system resulting in the denitrification rate being induced despite the bacterium having insufficient potential to remove NO3− or NO2− when the inadequate amount of HCO3− was supplemented. This finding is consistent with a previous study that showed that the removal rates of NO3− and NO3− were as low as the smaller amount of HCO3− was supplemented [3,9]. It is apparent that Thauera sp. provided the more powerful potential on nitrogen removal as a larger supplementary HCO3− amount was provided.

3.4. Contribution of Thauera sp. on Nitrogen Removal in Actual HD Reactor

To consider the powerful potential of Thauera sp. in the actual HD reactor, which was supplemented with the plentiful amount of HCO3− and inoculated with the mixed HD culture sludge as performed in the actual HD system in the former study [9], the specific rates of NO3− and NO2− removals and denitrification of the actual HD reactor were compared with the specific rates obtained in this experiment, using Thauera sp. strain Is13. As illustrated in Figure 6, when the inadequate HCO3− amount was supplemented to the system inoculated with Thauera sp. strain Is13, no activities of NO3− and NO2− removals were seen. This was consistent with a former study [9] that Thauera sp. was not a main player in the HD system supplemented with inadequate HCO3− and that the satisfactory performances were seen while a sufficient retention time was provided to the system. This implied that other bacteria were responsible for nitrogen removal under the HCO3− deficit condition with slower reducing activities instead of Thauera sp.
Additionally, the rates found in the reactor inoculated with Thauera sp. strain Is13 and supplemented with the sufficient HCO3− amount were, however, lower than the rates observed in the actual HD reactor. This revealed that other dominant bacteria were responsible for NO3− and NO2− removal as it was discussed in a former study [9] that the decent denitrification efficiency was achieved through the different structures of bacterial community where Thauera sp. was not found dominant in the system.
Lastly, Thauera sp. strain Is13 had a powerful potential in removing both NO3− and NO2− in the plentiful HCO3− condition, resulting in providing high specific denitrification rate. Those rates almost reached the rates observed in the actual HD reactors at the identical supplementary HCO3− amount. This implied that Thauera sp. was the most truly dominant bacteria functioning on nitrogen removal, especially NO2− removal when supplemented with plentiful HCO3−.

4. Conclusions

The aim of this present study was to explore possible ways to overcome the NO2− accumulation in HD system following the findings found in previous studies that Thauera sp. and HCO3− would be the key to solve the issue. Several bacterial strains were isolated from the mixed-culture sludge from the HD system using random selection of colony approach. The phylogenetic analysis found that the isolated strains belonged to six genera in which, fortunately, Thauera sp. was also successfully isolated. This Thauera sp. strain was about 100% similar to Thauera phenylacetica strain B4P (NR027224) as compared to the sequences available in GeneBank. Thauera sp. and the other three strains were enriched and individually inoculated to the system operated under the H2-based anoxic condition, which was individually spiked with NO3− and NO2− to compare the performance of Thauera sp. and the others. It was found that only Thauera sp. was able to outstandingly perform rapid nitrogen removal, especially NO2− removal in the plentiful HCO3− condition. The removal rate of NO3− and NO3− by Thauera sp. decreased as the supplementary amount of HCO3− was reduced. It was evident that the acclimatizing Thauera sp. strain required the sufficient HCO3− amount. As a consequence, the Thauera sp. strain was capable of providing the satisfying performance on denitrification when the plentiful HCO3− was supplemented to the system. The results obtained in this study also revealed that Thauera sp. might be a predominant H2-oxidizing denitrifier that can fully function on the NO3− and NO2− removal in the plentiful-HCO3−-supplemented HD system. Considering the overall conclusion of this study, it is advantageous to use HCO3− as a strategic factor for the rapid acclimatization of HD bacteria responsible for NO2− removal in which Thauera sp. could be one among those. This strategy can be used to overcome the NO2− accumulation problem usually occurring in the HD process, leading to the incomplete denitrification, and to shorten the operation time. In other words, it is possible to develop Thauera-rich sludge using the plentiful HCO3− amount to be used as an inoculum for the HD system to enhance the HD efficiency.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su15010277/s1. Table S1 Recipe of the minimal medium used to prepare an agar for isolating the denitrifiers. Table S2 Details of experiments carried out in this study. Figure S1 Profiles of NO3- and NO2- concentrations and pH in a system inoculated with Pseudomonas sp. strain Is1 when spiked with (a) NO3- and (b) NO2- at plentifully supplementary HCO3- amount. Figure S2 Profiles of NO3- and NO2- concentrations and pH in a system inoculated with Pannonibacter sp. strain Is6 when spiked with (a) NO3- and (b) NO2- at plentifully supplementary HCO3- amount. Figure S3 Profiles of NO3- and NO2- concentrations and pH in a system inoculated with Bacillus sp. strain Is15 when spiked with (a) NO3- and (b) NO2- at plentifully supplementary HCO3- amount.

Author Contributions

Planning, conceptualization, formal analyses, investigation, visualization, writing—original draft preparation, S.R.; methodology, writing—review and editing, S.R. and T.K.; supervision, F.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by the Science and Technology Research Partnership for Sustainable Development (SATREPS) program of Japan Science and Technology Agency (JST) and Japan International Cooperation Agency (JICA).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors acknowledge the Interdisciplinary Centre for River Basin Environment, University of Yamanashi, Japan for facilitating this research.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Canonical Correspondence Analysis (CCA) representing the correlation between operational and affected parameters and abundances of denitrification-functional genes and dominant bacteria. The data from previous studies were utilized to interpret the results [9].
Figure 1. Canonical Correspondence Analysis (CCA) representing the correlation between operational and affected parameters and abundances of denitrification-functional genes and dominant bacteria. The data from previous studies were utilized to interpret the results [9].
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Figure 2. Preparation of the experimental unit for the observation of isolated strain on nitrogen removal.
Figure 2. Preparation of the experimental unit for the observation of isolated strain on nitrogen removal.
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Figure 3. Schematic diagram of the experimental unit used for observing the pure bacterial strain ability on N removal consisting of (a) a 300 mL-working volume reactor, (b) a rubber stopper, (c) a H2 gas inlet, (d) a 0.2-µm filter, (e) a sampling channel, (f) a vent, (g) a magnetic bar, and (h) a magnetic stirrer.
Figure 3. Schematic diagram of the experimental unit used for observing the pure bacterial strain ability on N removal consisting of (a) a 300 mL-working volume reactor, (b) a rubber stopper, (c) a H2 gas inlet, (d) a 0.2-µm filter, (e) a sampling channel, (f) a vent, (g) a magnetic bar, and (h) a magnetic stirrer.
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Figure 4. 16S rRNA gene-based phylogenetic tree showing the relationship of several bacterial strains isolated from the mixed-culture HD bacterial sludge obtained from the former study [9] (isolate Is1—16) inferred using the Neighbor-Joining method. The values next to the branches indicate the percentage of the bootstrap values of 1000 replications above 50%. Length of branches represents 2% distances in the units of base substitution number per site. Paracoccus denitrificans strain DSM 413 was used as an outgroup sequence to root the tree.
Figure 4. 16S rRNA gene-based phylogenetic tree showing the relationship of several bacterial strains isolated from the mixed-culture HD bacterial sludge obtained from the former study [9] (isolate Is1—16) inferred using the Neighbor-Joining method. The values next to the branches indicate the percentage of the bootstrap values of 1000 replications above 50%. Length of branches represents 2% distances in the units of base substitution number per site. Paracoccus denitrificans strain DSM 413 was used as an outgroup sequence to root the tree.
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Figure 5. Profiles of NO3− and NO2− concentrations and pH in a system inoculated with Thauera sp. strain Is13 when spiked with NO3− or NO2− at different supplementary HCO3− amounts as individually described in each figure (a–f). Inadequate, sufficient, and plentiful HCO3− amounts are no addition, 275, and 6886 mg HCO3− L−1, respectively.
Figure 5. Profiles of NO3− and NO2− concentrations and pH in a system inoculated with Thauera sp. strain Is13 when spiked with NO3− or NO2− at different supplementary HCO3− amounts as individually described in each figure (a–f). Inadequate, sufficient, and plentiful HCO3− amounts are no addition, 275, and 6886 mg HCO3− L−1, respectively.
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Figure 6. Specific rates of (a) NO3− removal, (b) NO2− removal, and (c) denitrification comparing between reactors inoculated with mixed-culture sludge of the former study [9] and with isolated Thauera sp. stain Is13 of this study at different amounts of HCO3− supplement.
Figure 6. Specific rates of (a) NO3− removal, (b) NO2− removal, and (c) denitrification comparing between reactors inoculated with mixed-culture sludge of the former study [9] and with isolated Thauera sp. stain Is13 of this study at different amounts of HCO3− supplement.
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Table
Table 1. Identification of bacterial strains isolated in this study.
Table 1. Identification of bacterial strains isolated in this study.
IsolateAccession No.% SimilarityClosest MatchPhylogenetic Group (Family)
Is1OP676119100.00Pseudomonas mendocina strain HDB-3Pseudomonadaceae
Is2OP676120100.00Pseudomonas mendocina strain HDB-3Pseudomonadaceae
Is3OP676121100.00Pseudomonas mendocina strain HDB-3Pseudomonadaceae
Is4OP676122100.00Pseudomonas mendocina strain HDB-3Pseudomonadaceae
Is5OP676123100.00Dietzia natronolimnaea strain NF047Dietziaceae
Is6OP67612499.89Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is7OP67612599.89Halomonas desiderata strain FB2 16SHalomonadaceae
Is8OP67612699.88Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is9OP67612799.88Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is10OP67612899.88Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is11OP67612999.88Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is12OP67613099.88Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is13OP67613199.89Thauera phenylacetica strain B4PZoogloeaceae
Is14OP67613299.88Pannonibacter phragmitetus strain PB-Rt1Rhodobacteraceae
Is15OP67613399.34Bacillus flexus strain HDB-2Bacillaceae
Is16OP67613499.83Bacillus aryabhattai strain B8W22T.44Bacillaceae
Table
Table 2. Averaged rates of NO3− and NO3− removals and denitrification performed by several strains isolated in study.
Table 2. Averaged rates of NO3− and NO3− removals and denitrification performed by several strains isolated in study.
Bacterial InoculaHCO3− AmountSpecific Rate (mgN gVSS−1 day−1)
NO3− RemovalNO2− RemovalDenitrification
Pseudomonas sp. strain Is1Plentiful4.15 ± 0.843.94 ± 0.804.14 ± 0.84
Pannonibacter sp. strain Is615.94 ± 4.19n.d. *n.d. *
Bacillus sp. strain Is15n.d. *n.d. *n.d.
Thauera sp. strain Is13Plentiful22.59 ± 2.6636.02 ± 5.6622.61 ± 2.66
Sufficient14.32 ± 5.1618.72 ± 2.147.66 ± 2.76
Inadequaten.d. *n.d. *n.d. *
* n.d. = Not detected.
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Rujakom, S.; Kamei, T.; Kazama, F. Thauera sp. in Hydrogen-Based Denitrification: Effects of Plentiful Bicarbonate Supplementation on Powerful Nitrite Reducer. Sustainability 2023, 15, 277. https://doi.org/10.3390/su15010277

AMA Style

Rujakom S, Kamei T, Kazama F. Thauera sp. in Hydrogen-Based Denitrification: Effects of Plentiful Bicarbonate Supplementation on Powerful Nitrite Reducer. Sustainability. 2023; 15(1):277. https://doi.org/10.3390/su15010277

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Rujakom, Suphatchai, Tatsuru Kamei, and Futaba Kazama. 2023. "Thauera sp. in Hydrogen-Based Denitrification: Effects of Plentiful Bicarbonate Supplementation on Powerful Nitrite Reducer" Sustainability 15, no. 1: 277. https://doi.org/10.3390/su15010277

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