In a microscopic world invisible to the naked eye, billions of microorganisms are silently acting as the "purification vanguard" of Earth's water environment. Using nature's nitrogen cycle as a blueprint, they protect our water resources through sophisticated mechanisms evolved over millions of years.
With the rapid development of industry, agriculture, and urbanization, large amounts of nitrogen-containing compounds have entered water environments, causing a series of environmental problems including algal blooms, decreased water transparency, sharply reduced dissolved oxygen, and massive deaths of aquatic organisms.
Reduction in oxygen demand with partial nitrification-denitrification4
Energy savings with anammox technology compared to traditional methods4
Reduction in organic carbon requirement with short-cut nitrogen removal4
While traditional physical and chemical denitrification methods have some effect, they are often costly and can produce secondary pollution. In this context, microbial denitrification technology is becoming a key force in the field of water pollution control due to its low cost, high efficiency, and environmental friendliness4 .
In the microscopic world of microorganisms, a silent nitrogen cycle war takes place every day. Various microorganisms work synergistically to convert nitrogen pollutants in water into harmless nitrogen gas, which is released into the atmosphere.
NH3 → NO2-
Ammonia oxidizers convert ammonia to nitrite
NO2- → NO3-
Nitrite oxidizers convert nitrite to nitrate
NO3- → N2
Denitrifiers reduce nitrate to nitrogen gas
NH4+ + NO2- → N2
Direct conversion under anaerobic conditions
This process not only maintains the ecological balance of water bodies but also provides a natural solution for humanity to address the challenge of nitrogen pollution.
The essence of microbial denitrification is the stepwise conversion of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in water to nitrogen gas through a series of metabolic reactions by specific types of microorganisms. These microorganisms include bacteria, archaea, and even some eukaryotic microorganisms, which together form a complex and sophisticated denitrification network3 .
In the complex network of microbial denitrification, different types of microorganisms have clear divisions of labor and perform their respective duties, forming an efficient "purification alliance." This alliance mainly includes members such as bacteria, archaea, and fungi, which work synergistically to complete the full conversion process from ammonia nitrogen to nitrogen gas.
| Microorganism Type | Function | Living Conditions | Ecological Significance |
|---|---|---|---|
| Ammonia-oxidizing bacteria | Convert ammonia to nitrite | Aerobic | Initiator of nitrification process |
| Ammonia-oxidizing archaea | Convert ammonia to nitrite | Aerobic/Extreme environments | Main nitrification force in special environments |
| Nitrite-oxidizing bacteria | Convert nitrite to nitrate | Aerobic | Completes nitrification process |
| Denitrifying bacteria | Reduce nitrate to nitrogen gas | Anoxic | Key step in denitrification |
| Aerobic denitrifying bacteria | Perform denitrification under aerobic conditions | Aerobic | Simplifies denitrification process |
| Anammox bacteria | Directly convert ammonia and nitrite to nitrogen gas | Anaerobic | Efficient new denitrification method |
These are the "vanguard troops" of the denitrification process, capable of converting ammonia nitrogen to nitrite, initiating the first step of nitrification. Recent research has found that not only traditional ammonia-oxidizing bacteria but also certain ammonia-oxidizing archaea play important roles in this process, especially in extreme environments3 .
These are the "main force" of the denitrification process, reducing nitrate and nitrite to nitrogen gas step by step under anoxic conditions. Interestingly, evolution in nature has also created microorganisms with special denitrification abilities, such as aerobic denitrifiers—they can perform denitrification even under aerobic conditions, breaking traditional understanding4 .
With the development of molecular biology techniques, scientists continue to discover new denitrifying microorganisms and develop corresponding application technologies. Among them, the discovery and application of anammox bacteria is considered a major breakthrough in the field of biological nitrogen removal from wastewater4 .
These microorganisms can directly use ammonia nitrogen as an electron donor and nitrite as an electron acceptor under anaerobic conditions, converting both directly to nitrogen gas. This process requires no organic carbon source and compared to traditional denitrification pathways, can save about 60% of energy consumption, significantly reducing operating costs4 .
Another notable emerging technology is partial nitrification-denitrification, which controls environmental conditions to stop the ammonia oxidation process at the nitrite stage, then directly proceeds to denitrification. This shortcut pathway can reduce aeration requirements by about 40%, lower organic carbon demand by about 30%, and increase denitrification rates, making it a frontier hotspot in biological nitrogen removal4 .
Recent research has also found that certain methanogenic archaea not only participate in the carbon cycle but also play roles in the nitrogen cycle. Through special enzyme systems, they can participate in nitrogen transformation processes. Even under nitrogen-deficient conditions, some methane-oxidizing microorganisms can couple iron reduction with biological nitrogen fixation, achieving nitrogen recycling3 .
To deeply understand microbial denitrification processes and optimize their application conditions, scientists have designed various precise experiments. Below is a research case on iron reduction-dependent anaerobic oxidation of methane coupled with biological nitrogen fixation under nitrogen-deficient conditions, which provides important insights for understanding microbial denitrification in special environments3 .
Researchers selected methane-oxidizing cultures and ferrihydrite as research objects, using various advanced analytical methods to explore how microorganisms perform iron reduction-dependent anaerobic oxidation of methane under nitrogen-deficient conditions and evaluate the possibility of coupling with biological nitrogen fixation.
The experiment yielded several important findings:
| Experimental Condition | Nitrogenase Activity | 15N Assimilation | Ferrihydrite Reduction Degree | Microbial Community Changes |
|---|---|---|---|---|
| N-deficient + Methane | High | Significant | Significant | Methanobacterium (19.32%), Geobacter (6.14%) |
| N-deficient No Methane | Low | Not significant | Not significant | Relatively stable community structure |
| N-rich + Methane | Medium | Medium | Medium | Balanced distribution of different functional groups |
The scientific importance of this experiment lies in:
| Microorganism Type | Relative Abundance (%) | Functional Role | Changes During Experiment |
|---|---|---|---|
| Methanobacterium | 19.32 | Methane metabolism | Significantly increased in methane-added group |
| Geobacter | 6.14 | Iron reduction | Participated in ferrihydrite reduction process |
| Desulfovibrio | 17.52 | Sulfur cycle/Possibly involved in nitrogen cycle | Active during experiment |
Studying microbial denitrification processes requires a series of advanced technical methods and reagent tools. With technological development and research deepening, scientists now have increasingly powerful "toolboxes" to explore the mysteries of the microscopic world.
In the early stages of microbial denitrification research, scientists mainly relied on cultivation methods and physiological and biochemical experiments to understand microbial characteristics.
Traditional reagents and materials include: various nitrogen sources (such as ammonium salts, nitrates, nitrites), carbon sources (such as sodium acetate, glycerol), buffer systems and trace element solutions, which provide basic conditions for microbial growth and denitrification activities2 .
Modern molecular biology development has greatly expanded scientists' ability to study microbial denitrification processes:
| Research Tool Category | Specific Examples | Main Function | Application Value |
|---|---|---|---|
| Sequencing Kits | Microbial amplicon barcode sequencing kit (SQK-MAB114.24)1 | 16S/ITS region full-length sequencing | Microbial community analysis |
| Functional Gene Analysis | Nitrogen cycle functional gene detection (amoA, nirK, nirS, nosZ) | Denitrification potential assessment | Predicting ecosystem function |
| Isotope Tracing | 15N labeled substrates3 | Tracking nitrogen transformation pathways | Revealing reaction mechanisms and rates |
| Electrochemical Analysis | Microbial electrochemical system monitoring | Studying electron transfer processes | Understanding energy metabolism mechanisms |
| Fluorescence Spectroscopy | Three-dimensional fluorescence spectroscopy analysis3 | Dissolved organic matter characterization | Revealing microbial metabolic products |
Microbial denitrification technologies have gradually moved from laboratory research to extensive engineering applications, providing effective solutions for practical environmental problems. These technologies play important roles in multiple scenarios:
Traditional wastewater treatment plants are being upgraded by introducing new microbial denitrification technologies. For example, anammox technology has achieved large-scale application in wastewater treatment in Europe and some Asian countries. Compared to traditional nitrification-denitrification processes, it can significantly reduce energy consumption and operating costs4 .
Partial nitrification-denitrification technology has also been applied in wastewater treatment plants in some cities in China, performing particularly well in treating high-ammonia nitrogen industrial wastewater and landfill leachate, achieving the goal of energy saving and consumption reduction4 .
Nitrogen pollution in agricultural runoff is an important cause of eutrophication in lakes and reservoirs. By constructing constructed wetlands and using plant-microorganism combined systems to treat agricultural drainage, nitrogen entering natural water bodies can be effectively reduced.
Research has found that in constructed wetland systems, microbial communities can form efficient denitrification networks through synergistic effects. Some studies are exploring ways to enhance the denitrification function of wetland systems by adding specific bacterial agents or optimizing carrier materials4 .
In the aquaculture field, microbial denitrification technology is used to maintain water quality in aquaculture systems, preventing the accumulation of ammonia nitrogen and nitrite from harming aquatic organisms. Immobilized microbial technology and biofilm reactors have shown good application effects in this field4 .
For heavily polluted water sediments, scientists have developed remediation technology based on calcium peroxide. Research shows that calcium peroxide, by changing the redox environment of sediments, can significantly affect microbial community structure:
Promoting the proliferation of genera with ammonia oxidation or sulfur oxidation functions (such as Nitrosomonas, Thiobacillus), while inhibiting the growth of anaerobic fermentation or sulfate-reducing genera (such as Clostridium, Desulfovibrio), thus optimizing the functional structure of sediment microbial communities3 .
Microbial denitrification research is moving toward a more refined and systematic new stage. With continuous technological progress and innovation in research methods, scientists will more deeply understand the mechanisms of microbial denitrification at molecular and genetic levels, providing a more solid theoretical foundation for technological applications.
The integration of synthetic biology and genetic engineering may enable the design of synthetic microbial communities with high-efficiency denitrification capabilities, achieving more precise and efficient nitrogen pollution control.
Future research directions may focus on the following aspects:
Combining meta-omics and metabolomics technologies to comprehensively analyze key steps and regulatory mechanisms of microbial denitrification at molecular level3 .
Mining new efficient denitrifying microbial resources from special environments, expanding the diversity of strain libraries3 .
Identifying key rate-limiting steps in denitrification processes, developing precise control strategies to improve denitrification efficiency3 .
Strengthening long-cycle ecological effect research, comprehensively assessing long-term environmental safety impacts of microbial denitrification technologies3 .
The development of microbial denitrification research will not only provide more effective technical means for solving water environmental pollution problems but also deepen our understanding of Earth's nitrogen cycle processes, contributing important strength to building a sustainable future with harmonious coexistence between humans and nature.