Heterotrophic Nitrification-aerobic Denitrification Research Articles

Nitrogen removal by heterotrophic nitrification-aerobic denitrification bacteria: A review

The rising urbanization and industrialization are increasing nitrogen pollution in water and wastewaters, calling for advanced remediation treatments. Traditional biological treatments are limited in area and efficiency. Therefore, heterotrophic nitrification-aerobic denitrification (HN-AD) in a single reactor has drawn significant attention and has shown higher efficiency. Here we review HN-AD bacteria with focus on bacterial species, nitrogen metabolism, and factors influencing denitrification. Nitrogen metabolism pathway includes nitrate-nitrite reduction denitrification, hydroxylamine reduction and assimilatory denitrification. Factors influencing denitrification comprise carbon source, C/N, temperature, pH, DO and so on. Compared to published literature reviews on HN-AD, our manuscript comprehensively summary the latest research on its nitrogen remove mechanisms and related enzymes for the first time.

Metabolomic insight into regulatory mechanism of heterotrophic bacteria nitrification-aerobic denitrification bacteria to high-strength ammonium wastewater treatment

This work aimed to elucidate the metabolic mechanism of heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria influenced by varying concentrations of ammonium nitrogen (NH4+-N) in high-strength synthetic wastewater treatment. The results showed that the removal rates of NH4+-N and total nitrogen, along with enzymatic activities related to nitrification and denitrification, increased with rising NH4+-N concentrations (N500:500 mg/L, N1000:1000 mg/L and N2000:2000 mg/L). The relative abundances of HN-AD bacteria were 50 %, 62 % and 82 % in the three groups. In the N2000 group, the cAMP signaling pathway, glycerophospholipid metabolites, purines and pyrimidines related to DNA/RNA synthesis, electron donor NAD+-related energy, the tricarboxylic acid (TCA) cycle and glutamate metabolism were upregulated. Therefore, influent NH4+-N at 2000 mg/L promoted glutamate metabolism to accelerate the TCA cycle, and enhanced cellular energy and advanced denitrification activity of bacteria for HN-AD. This mechanism, in turn, enhanced microbial growth and the carbon and nitrogen metabolism of bacteria for HN-AD.

Response of extracellular polymeric substances to high load shock in an aerobic granular sludge reactor performing simultaneous heterotrophic nitrification-aerobic denitrification

Response of extracellular polymeric substances to high load shock in an aerobic granular sludge reactor performing simultaneous heterotrophic nitrification-aerobic denitrification

Disentangling microbial coupled fillers mechanisms for the permeable layer optimization process in multi-soil-layering systems

The multi-soil-layering (MSL) systems is an emerging solution for environmentally-friendly and cost-effective treatment of decentralized rural domestic wastewater. However, the role of the seemingly simple permeable layer has been overlooked, potentially holding the breakthroughs or directions to addressing suboptimal nitrogen removal performance in MSL systems. In this paper, the mechanism among diverse substrates (zeolite, green zeolite and biological ceramsite) coupled microorganisms in different systems (activated bacterial powder and activated sludge) for rural domestic wastewater purification was investigated. The removal efficiencies performed by zeolite coupled with microorganisms within 3 days were 93.8% for COD, 97.1% for TP, and 98.8% for NH4+-N. Notably, activated sludge showed better nitrification and comprehensive performance than specialized nitrifying bacteria powder. Zeolite attained an impressive 89.4% NH4+-N desorption efficiency, with a substantive fraction of NH4+-N manifesting as exchanged ammonium. High-throughput 16S rRNA gene sequencing revealed that aerobic and parthenogenetic anaerobic bacteria dominated the reactor, with anaerobic bacteria conspicuously absent. And the heterotrophic nitrification-aerobic denitrification (HN-AD) process was significant, with the presence of denitrifying phosphorus-accumulating organisms (DPAOs) for simultaneous nitrogen and phosphorus removal. This study not only raises awareness about the importance of the permeable layer and enhances comprehension of the HN-AD mechanism in MSL systems, but also provides valuable insights for optimizing MSL system construction, operation, and rural domestic wastewater treatment.

Metagenomic analysis revealed the evolution of microbial communities, metabolic pathways, and functional genes in the heterotrophic nitrification-aerobic denitrification process under La3+ stress

Trivalent lanthanum (La3+) exists widely in ammonia nitrogen (NH4+-N) tailing water from ionic rare earth mines; however, its effect on heterotrophic nitrification-aerobic denitrification (HN-AD) is unknown, thereby limiting the application of the HN-AD process in this field. In this study, we conducted an HN-AD process using a sequencing batch reactor (5 L) that was continuously operated to directly treat acidic (NH4)2SO4 wastewater (influent NH4+-N concentration of approximately 110 mg/L and influent pH of 5) containing different La3+ concentrations (0–100 mg/L). The NH4+-N removal efficiency of the reactor reached 98.25 % at a La3+ concentration of 100 mg/L. The reactor was in a neutral-to-alkaline environment, which favored La3+ precipitation and complexation. Metagenomic analysis revealed that the relative abundance of Thauera in the reactor remained high (88.62–92.27 %) under La3+ stress. The relative abundances of Pannonobacter and Hyphomonas significantly increased, whereas that of Azoarcus significantly decreased. Metabolic functions in the reactor were mainly contributed by Thauera, and the abundance of metabolic functions under low La3+ stress (≤5 mg/L) significantly differed from that under high La3+ stress (≥10 mg/L). The relative abundance of ammonia assimilation-related genes in the reactor was high and significantly correlated with ammonia removal. However, traditional ammonia oxidation genes were not annotated, and unknown ammonia oxidation pathways may have been present in the reactor. Moreover, La3+ stimulated amino acid biosynthesis and translocation, the citrate cycle, sulfur metabolism, and oxidative phosphorylation and promoted the overproduction of extracellular polymeric substances, which underwent complexation and adsorbed La3+ to reduce its toxicity. Our results showed that the HN-AD process had a strong tolerance to La3+, stable NH4+-N removal efficiency, the potential to recover La3+, and considerable application prospects in treating NH4+-N tailing water from ionic rare earth mines.

A novel pure biofilm system based on aerobic denitrification for nitrate wastewater treatment: Exploring the feasibility of high total nitrogen removal under low-carbon condition

To improve the poor denitrification efficiency of traditional biological treatment for low-carbon wastewater, a novel pure biofilm moving bed biofilm reactor (PH-MBBR) based on heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria was proposed. The start-up of PH-MBBR inoculated with HN-AD bacteria and the operating performances and microbial community composition under various C/N ratios were studied. The results showed that at C/N ratio of only 4, the total nitrogen (TN) removal rate of 10.02 mg/(L·h) was achieved under aerobic conditions. Microbial analysis indicates that the synergistic effect of oligotrophic bacteria Thauera and Azoarcus was the main reason for the high pollutant removal at C/N ratio of 4. Furthermore, the analysis of functional genes and enzymes reveals that NorB and NosZ played important roles in nitrate metabolism in PH-MBBR, and aerobic denitrification pathway was NO3–-N → NO → N2O → N2. This study provides a practical and theoretical basis for low-carbon nitrate wastewater treatment.

Isolation and characterization of a cold-tolerant heterotrophic nitrification–aerobic denitrification bacterium and evaluation of its nitrogen-removal efficiency

With a view toward addressing the poor efficiency with which nitrogen is removed from wastewater below 10 °C, in this study, we isolated a novel cold-tolerant heterotrophic nitrification–aerobic denitrification (HN-AD) bacterium from a wetland and characterized its nitrogen removal performance and nitrogen metabolic pathway. On the basis of 16S rRNA gene sequencing, this strain was identified as a species of Janthinobacterium, designated J1-1. At 8 °C, strain J1-1 showed excellent removal efficiencies of 89.18% and 68.18% for single-source NH4+-N and NO3−-N, respectively, and removal efficiencies of 96.23% and 79.64% for NH4+-N and NO3−-N, respectively, when supplied with mixed-source nitrogen. Whole-genome sequence analysis and successful amplification of the amoA, napA, and nirK functional genes related to nitrogen metabolism provided further evidence in support of the HN-AD capacity of strain J1-1. The deduced HN-AD metabolic pathway of the strain was NH4+-N→NH2OH→NO2−-N→NO3−-N→NO2−-N→NO→N2O. In addition, assessments of NH4+-N removal under different conditions revealed the following conditions to be optimal for efficient removal: a temperature of 20 °C, pH of 7, shaking speed of 150 rpm, sodium succinate as a carbon source, and a C/N mass ratio of 16. Given its efficient nitrogen removal capacity at 8 °C, the J1-1 strain characterized in this study has considerable application potential in the treatment of low-temperature wastewater.

An examination of the carbon metabolic pathways in Acinetobacter sp. TAC-1 in the context of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) utilization

The present study aimed to unravel the carbon metabolism pathway of Acinetobacter sp. TAC-1, a heterotrophic nitrification-aerobic denitrification (HN-AD) strain that utilizes poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as a carbon source. Sodium acetate was employed as a control to assess the gene expression of carbon metabolic pathways in the TAC-1 strain. The results of genome sequencing demonstrated that the TAC-1 strain possessed various genes encoding carbon metabolic enzymes, such as gltA, icd, sucAB, acs, and pckA. KEGG pathway database analysis further verified the presence of carbon metabolism pathways, including the glycolytic pathway (EMP), pentose phosphate pathway (PPP), glyoxylate cycle (GAC), and tricarboxylic acid (TCA) cycle in the TAC-1 strain. The differential expression of metabolites derived from distinct carbon sources provided further evidence that the carbon metabolism pathway of TAC-1 utilizing PHBV follows the sequential process of PHBV (via the PPP pathway)→gluconate (via the EMP pathway)→acetyl-CoA (entering the TCA cycle)→CO2+H2O (generating electron donors and releasing energy). This study is expected to furnish a theoretical foundation for the advancement and implementation of novel denitrification processes based on HN-AD and solid carbon sources.

Simultaneous aerobic nitrogen and phosphate removal capability of novel salt-tolerant strain, Pseudomonas mendocina A4: Characterization, mechanism and application potential

A salt-tolerant strain, Pseudomonas mendocina A4, was isolated from brackish-water ponds showing simultaneous heterotrophic nitrification-aerobic denitrification and phosphorus removal capability. The optimal conditions for nitrogen and phosphate removal of strain A4 were pH 7–8, carbon/nitrogen ratio 10, phosphorus/nitrogen ratio 0.2, temperature 30 °C, and salinity range of 0–5 % using sodium succinate as the carbon source. The nitrogen and phosphate removal efficiencies were 96–100 % and 88–96 % within 24 h, respectively. The nitrogen and phosphate removal processes were matched with the modified Gompertz model, and the underlying mechanisms were confirmed by the activities of key metabolic enzymes. Under 10 % salinity, the immobilization technology was employed to enhance the nitrogen and phosphate removal efficiencies of strain A4, achieving 87 % and 76 %, respectively. These findings highlight the potential application of strain A4 in both freshwater and marine culture wastewater treatment.

Feast/famine ratio regulates the succession of heterotrophic nitrification-aerobic denitrification and autotrophic ammonia oxidizing bacteria in halophilic aerobic granular sludge treating saline wastewater

Heterotrophic nitrification-aerobic denitrification (HN-AD) shows innovation potential of wastewater treatment process in a single tank. However, how to enrich HN-AD bacteria in activated sludge to enhance their contribution remained unknown. This study explored the impact of the feast/famine (F/F) ratio on the succession of autotrophic ammonia oxidizing bacteria (AOB) and HN-AD bacteria in a halophilic aerobic granular sludge (HAGS) system. As the F/F ratio decreased from 1/9 to 1/15, the total inorganic nitrogen (TIN) removal performance significantly decreased. The proportion of heterotrophic bacteria was dropped from 79.0 % to 33 %. Accordingly, the relative abundance of Paracoccus decreased from 70.8 % to 25.4 %, and the copy number of the napA gene was reduced from 2.2 × 1010 copies/g HAGS to 8.1 × 109 copies/g HAGS. It found the F/F ratio regulated the population succession of autotrophic AOB and HN-AD bacteria, thereby providing a solution to achieve the enrichment of HN-AD bacteria in HAGS.

Indigenous mixotrophic aerobic denitrifiers stimulated by oxygen micro/nanobubble-loaded microporous biochar

The prevalence of hypoxia in surface sediment inhibits the growth of aerobic denitrifiers in natural waters. A novel oxygen micro/nanobubble-loaded microporous biochar (OMB) was developed to activate indigenous aerobic denitrifiers in this study. The results indicate a thin-layer OMB capping mitigates hypoxia effectively. Following a 30-day microcosm-based incubation, a 60 % decrease in total nitrogen concentration was observed, and the oxygen penetration depth in the sediment was increased from <4.0 mm to 38.4 mm. High-throughput sequencing revealed the stimulation of indigenous mixotrophic aerobic denitrifiers, including autotrophic denitrifiers such as Hydrogenophaga and Thiobacillus, heterotrophic denitrifiers like Limnobacter and unclassified_f_Methylophilaceae, and heterotrophic nitrification aerobic denitrification bacteria, including Shinella and Acidovorax, with total relative abundance reaching up to 38.1 %. Further analysis showed OMB enhanced the overall collaborative relationships among microorganisms and promoted the expression of nitrification- and denitrification-related genes. This study introduces an innovative strategy for stimulating indigenous aerobic denitrifiers in aquatic ecosystems.

Microalgae-assisted heterotrophic nitrification-aerobic denitrification process for cost-effective nitrogen and phosphorus removal from high-salinity wastewater: Performance, mechanism, and bacterial community

A microalgae-assisted heterotrophic nitrification-aerobic denitrification (HNAD) system for efficient nutrient removal from high-salinity wastewater was constructed for the first time as a cost-effective process in the present study. Excellent nutrient removal (∼100.0 %) was achieved through the symbiotic system. The biological removal process, biologically induced phosphate precipitation (BIPP), microalgae uptake, and ammonia stripping worked together for nutrient removal. Furthermore, the biological removal process achieved by biofilm contributed to approximately 55.3–71.8 % of nitrogen removal. BIPP undertook approximately 45.6–51.8 % of phosphorus removal. Batch activity tests confirmed that HNAD fulfilled an extremely critical role in nitrogen removal. Microalgal metabolism drove BIPP to achieve efficient phosphorus removal. Moreover, as the main HNAD bacteria, OLB13 and Thauera were enriched. The preliminary energy flow analysis demonstrated that the symbiotic system could achieve energy neutrality, theoretically. The findings provide novel insights into strategies of low-carbon and efficient nutrient removal from high-salinity wastewater.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrifying-aerobic denitrifying strain H1 as a potential candidate in mariculture wastewater treatment.

The nitrogen removal performance and mechanisms of Bacillus subtilis H1 isolated from a mariculture environment were investigated. Strain H1 efficiently removed NH4+-N, NO2--N, and NO3--N in simulated wastewater with removal efficiencies of 85.61%, 90.58%, and 57.82%, respectively. Strain H1 also efficiently degraded mixed nitrogen (NH4+-N mixed with NO2--N and/or NO3--N) and had removal efficiencies ranging from 82.39 to 89.54%. Nitrogen balance analysis revealed that inorganic nitrogen was degraded by heterotrophic nitrification-aerobic denitrification (HN-AD) and assimilation. 15N isotope tracing indicated that N2O was the product of the HN-AD process, while N2 as the final product was only detected during the reduction of 15NO2--N. The nitrogen assimilation and dissimilation pathways by strain H1 were further clarified using complete genome sequencing, nitrification inhibitor addition, and enzymatic activity measurement, and the ammonium oxidation process was speculated as NH4+ → NH2OH → NO → N2O. These results showed the application prospect of B. subtilis H1 in treating mariculture wastewater.

Effect of free ammonia and free nitrous acid on nitrogen removal of heterotrophic nitrification and aerobic denitrification bacteria

Free ammonia (FA) and free nitrous acid (FNA) can inhibit nitrogen removal by nitrifying microorganisms. However, current studies on FA and FNA mainly focus on traditional nitrifying microorganisms, and little is known about the relationship between heterotrophic nitrification-aerobic denitrification (HN-AD) microorganisms and FA and FNA. This study selected Alcaligenes faecalis WT14, an HN-AD strain, as the research object. The effects of different FA and FNA concentrations on the nitrogen removal ability of strain WT14 were investigated under neutral (pH = 7) and alkaline (pH = 9) conditions. The results showed that FA and FNA affected the removal ability of strain WT14 to ammonia nitrogen (NH4+-N) and nitrite nitrogen (NO2−-N). The rapid reduction of NH4+-N removal efficiency was determined at different concentrations of 6.12 mg·L−1 (pH = 7) and 551.18 mg·L−1 (pH = 9) for FA, which was 0.483 mg·L−1 (pH = 7) and 0.005 mg·L−1 (pH = 9) for FNA, respectively. Strain WT14 preferred an alkaline environment for growth; the FA tolerance value was 1134.25 mg·L−1 in such an environment, which was 51.8 times that of a neutral environment. Furthermore, strain WT14 had a higher removal rate of NH4+-N and NO2−-N in an alkaline environment. Its highest removal rates of NH4+-N and NO2−-N were 15.03 mg·L−1·h−1 and 7.16 mg·L−1·h−1, respectively, which was much higher than the reported HN-AD microorganisms.

Response of Nitrogen Removal Performance and Microbial Distribution to Seasonal Shock Nutrients Load in a Lakeshore Multicell Constructed Wetland

Multicell constructed wetlands (MCWs) on lakeshores are a prospective treatment technique. However, the factors affecting the nutrient removal performance of lakeshore MCWs at the field scale are unclear. This study chose a field-scale lakeshore MCW with the highest mass removal efficiency (approximately 49,175.12 mg m−2 day−1) for total nitrogen removal in the wet season to investigate the response of nitrogen removal and microbial distribution to seasonal shock nutrients load. The mass loading rates in the wet season were as high as 43~72 times over those in the dry season. Hence, a storage pond (SP), as a forebay retention cell, was necessary to mitigate the shock loads of the influent, which is beneficial to nitrogen removal of the MCW system. The two major genera in the sediments are heterotrophic nitrification–aerobic denitrification bacteria, and the abundance and species of the nitrogen-related functional genera were higher in the wet season than the dry season. According to the results of redundancy analysis, the hydraulic residence time (29.4%, F = 2.2, p &lt; 0.1) and hydraulic loading rate (85.9, F = 36.5, p &lt; 0.05) were the major factors explaining microbial community variation, instead of environmental factors (temperature, pH, and dissolved oxygen). The shock loads of influent and the periodic saturation in sediments contributed to a complicated oxygen and nitrogen nutrient exchange environment resulting in higher abundance and species of nitrogen-related microbes, which is beneficial to nitrogen removal in lakeshore MCWs. The results provided a scientific basis for the optimal design of constructed wetlands on lakeshores.

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification bacterium Acinetobacter ZQ-A1 and community characteristics analysis of its application in pig farm wastewater.

A heterotrophic nitrifying aerobic denitrifying (HN-AD) strain ZQ-A1 with excellent denitrification performance, identified as Acinetobacter, was isolated from simultaneous nitrification and denitrification (SND) craft. ZQ-A1 was capable of removing NH4+, NO2-, and NO3-; the 21-hour removal rates were 84.84%, 87.13%, and 92.63%. ZQ-A1 has the ability to treat mixed nitrogen sources. In addition, ZQ-A1 can be well applied to actual sewage. According to the analysis of microbial community characteristics, the relative abundance of Acinetobacter in the experimental group increased from 0.06% to 2.38%, which is an important reason for the removal rate of NH4+ exceeding 99% within 30 days. The results of KEGG function prediction showed that with the addition of ZQ-A1, the relative abundance of pathways related to bacterial metabolism, such as tricarboxylic acid cycle metabolism, was higher. The research expanded the thinking of HN-AD bacteria in actual production and laid a foundation for its application in sewage treatment.

Nitrogen Removal Characteristics and Metabolism Mechanism of High-Efficiency Cold-Tolerant Heterotrophic Nitrification-Aerobic Denitrification Bacterium Glutamicibacter sp. WS1 for Various Nitrogen Sources at Low Temperature

For resolving the problems of poor nitrogen removal efficiency and substandard effluent quality in wastewater treatment plants during winter, a cold-tolerant strain Glutamicibacter sp. WS1, with heterotrophic nitrification-aerobic denitrification ability, was isolated from activated sludge. The functional genes for nitrogen conversion of strain WS1 were amplified by PCR, and the nitrogen removal characteristics of the strain were verified under different nitrogen sources at 15℃. In addition, the effects of environmental factors on the aerobic denitrification performance of the strain were explored at low temperature. Finally, a reasonable nitrogen metabolism pathway of strain WS1 was resolved based on functional genes and nitrogen balance analysis. The results showed that strain WS1 contained functional genes related to nitrogen conversion, including amoA, napA, nirS, and nirK genes. Notably, nirS and nirK genes coexisted in the strain. At the low temperature of 15℃, with NH4+-N, NO3--N, NO2--N+NO3--N, and NH4+-N+NO3--N as nitrogen sources, the corresponding removal efficiencies of strain WS1 were 100%, 98.10%, 99.87%+100%, and 100%+94.92%, respectively. The optimal denitrification performance of the strain was achieved with sodium citrate as the carbon source, C/N of 16, pH of 8, DO of 4.5-6.8 mg·L-1, and temperature of 30℃. In addition, the NO3--N removal efficiency of strain WS1 reached 92.50% under low temperature (15℃) and low C/N (10) conditions. Based on the results of PCR amplification and nitrogen balance analysis, heterotrophic nitrification-aerobic denitrification/aerobic denitrification and assimilation were the main pathways for nitrogen substrate removal by strain WS1, in which most of the inorganic nitrogen (47%-56%) was converted to gaseous nitrogen through heterotrophic nitrification-aerobic denitrification/aerobic denitrification. Strain WS1 has broad application prospects in the treatment of low-temperature nitrogenous wastewater.

A review of microbial nitrogen transformations and microbiome engineering for biological nitrogen removal under salinity stress

The osmotic stress caused by salinity exerts severe inhibition on the process of biological nitrogen removal (BNR), leading to the deterioration of biosystems and the discharge of nitrogen with saline wastewater. Feasible strategies to solve the bottleneck in saline wastewater treatment have attracted great attention, but relevant studies to improve nitrogen transformations and enhance the salt-tolerance of biosystems in terms of microbiome engineering have not been systematically reviewed and discussed. This work attempted to provide a more comprehensive explanation of both BNR and microbiome engineering approaches for saline wastewater treatment. The effect of salinity on conventional BNR pathways, nitrification-denitrification and anammox, was summarized at cellular and metabolic levels, including the nitrogen metabolic pathways, the functional microorganisms, and the inhibition threshold of salinity. Promising nitrogen transformations, such as heterotrophic nitrification-aerobic denitrification, ammonium assimilation and the coupling of conventional pathways, were introduced and compared based on advantages and challenges in detail. Strategies to improve the salt tolerance of biosystems were proposed and evaluated from the perspective of microbiome engineering. Finally, prospects of future investigation and applications on halophilic microbiomes in saline wastewater treatment were discussed.

Marine Heterotrophic Nitrification–Aerobic Denitrification Process Treating Mariculture Wastewater: Microbiome and Resistome Responses to Multiantibiotic Stresses

Marine Heterotrophic Nitrification–Aerobic Denitrification Process Treating Mariculture Wastewater: Microbiome and Resistome Responses to Multiantibiotic Stresses

Exploring influence mechanism of small-molecule carbon source on heterotrophic nitrification-aerobic denitrification process from carbon metabolism, nitrogen metabolism and electron transport process

The heterotrophic nitrification-aerobic denitrification (HNAD) process can remove nitrogen and organic carbon under aerobic conditions. To get the in-depth mechanism of the HAND process, a strain named Acinetobacter johnsonii ZHL01 was isolated, and enzyme activity, electron transport, energy production, and gene expression of the strain were studied with small-molecule carbon sources, including sodium citrate, sodium acetate, sodium fumarate, and sodium succinate. The HNAD pathway of ZHL01 was NH4+→NH2OH → NO, and nitrogen balance analysis shows that ZHL01 could assimilate and denitrify 58.29 ± 1.05 % and 16.58 ± 1.07 % of nitrogen, respectively. The assimilation, the nitrification/denitrification, and the respiration processes were regulated by the concentration of reduced nicotinamide adenine dinucleotide (NADH) produced from the different metabolic pathways of small-molecule carbon sources. The HNAD process occurs to reduce intracellular redox levels related to NADH concentrations. This discovery provides a theoretical basis for the practical application of HAND bacteria.