Improving the Water Intake Mode

The influent mode can affect the nitrification by affecting the concentration of dissolved oxygen, such as tidal influent and drop influent, which can increase the concentration of dissolved oxygen, thus strengthening the nitrification.

Tidal operation is the use of the suction generated in the alternating process of water inlet and drainage to absorb oxygen in the atmosphere in the constructed

Fig. 1.

Various enhanced nitrogen removal measures in constructed wetlands.

1. Tidal inlet; 2. Water drop influent; 3. Substrate combinations; 4. Plant mixed planting;

7. Add carbon source; 8. Add Fe/MnOx; 9. Immobilized microorganism; 10. Aeration. wetland, and the operation cycle is “influent-flood-effluent-drain” [48]. In the water inlet stage, the constructed wetland is gradually flooded, and the air in the substrate is continually depleted. During the drainage phase, the air re-enters the constructed wetland, and the substrate is re-oxygenated, which improves the efficiency of oxygen transport [49]. As wastewater and air circulate, oxygen supply and consumption in the wetland are greatly improved, and nitrification and denitrification are gradually intensified, which is conducive to maximizing TN removal efficiency [50]. Thus, wastewater can act as a timed air pump, providing alternate aerobic and anaerobic environments for nitrification and denitrification. Tidal influent allows maximum wastewater contact with the biofilm, enhancing oxygen transfer by drawing air into the wetland substrate [51]. A study claimed that the reoxygenation capacity of tidal stream-constructed wetlands was 350 gm^-2d^-1, which was much higher than that of traditional-constructed wetlands (<100 gm^-2d^-1) [52]. Li et al. [53] used tidal influent to treat low-pollution synthetic sewage. It was shown that the removal efficiency of TN and NO₃^--N in tidal influent-constructed wetland was higher than that of intermittently aerated constructed wetland, with the removal rate of NH₄⁺-N above 90% and COD above 87%. Compared to aeration, tidal flow wetlands can save half of the energy and area for treating the same volume of wastewater [54]. However, tidal operation is more complex and requires skilled operation mode in order to give full play to the best nitrogen removal efficiency of constructed wetlands.

The principle of falling into the water is that the sewage is falling from a high place; when the water is falling into the air, the oxygen in the air is in full contact with the sewage, which can effectively promote the process of transferring the air-oxygen to the water. In addition, the water falling from a high place can produce waves on the surface of the water, and at the same time, it is conducive to the diffusion of air [55]. Therefore, the concentration of dissolved oxygen in wastewater increases with the increase of the drop. The research by Li et al. [56] showed that the greater the height difference, the greater the reoxygenation amount, and the best reoxygenation effect was achieved when the drop height reached 2.5 m. Zheng et al. [57] also found that the dissolved oxygen concentration increased by 5.20±0.17 mg/L when the height difference was 50 cm, and the removal of COD and NH₄⁺-N by treating rural wastewater with falling water reoxygenation and tidal operation was 99.50±0.21% and 87.16%±1.76%, respectively. Falling into the water can take advantage of the potential energy present in the terrain difference to reduce the output of energy or use a pump to raise the water level to a higher level, but it requires a portion of the energy consumption [58]. In addition, the oxygenation efficiency of multi-stage drop inlet water is higher. Zou et al. [59] used a vertical flow-constructed wetland to treat rural sewage and showed that, compared with the direct fall-off water, the multi-stage, two-layer fall-off water increased the dissolved oxygen per meter by 2-6 mg/L.

Optimization of Substrate Combinations

Complementary effects and synergistic effects resulting from the combination of substrates can increase the efficiency of nitrogen removal. For instance, the combination of manganese ore and walnut shell, whose shell is rich in organic matter, can improve denitrification, and manganese ore can provide a better redox potential environment. The combination of both can simultaneously strengthen the nitrification and denitrification processes [60]. In recent years, the application of pyrite in constructed wetlands has become more and more common, and the efficient purification of wastewater has been realized. The pyrite surface has many active sites and provides nutrients for allochthonous Fe (III)-reducing microorganisms. Pyrite-based constructed wetland systems can promote denitrification using sulfur and ferrous iron as electron donors for denitrification [61]. Uniform mixing of pyrite with high porosity substrates (e.g., activated carbon, volcanic stone, etc.) can enhance microorganisms and their effective adhesion to the contact area of pyrite, promote the growth of autotrophic and heterotrophic denitrifying bacteria in the constructed wetland system, and ultimately improve the denitrification capacity of the constructed wetland [62]. Jiang et al. [63] selected pyrite, alkali-modified rice husk, quartz sand, and gravel as substrates for vertical flow-constructed wetlands and found that pyrite could enhance autotrophic denitrification and alkali-modified rice husk could enhance heterotrophic denitrification, and the combination of the two could effectively remove nitrogen from low C/N wastewater.

The combination of substrates in the appropriate proportions can provide a favorable environment for the growth and multiplication of microorganisms and plants. The addition of special porous substrates and organic substrates can provide carbon sources for denitrification and promote the removal of nitrogen. When the substrate is combined, different substrates have different abilities in different parts. When the removal ability of the upper substrate to nitrogen is gradually consumed, the removal site of nitrogen will move to the lower layer, coupled with the different properties and nitrogen removal capacity of each layer of substrate, so that the removal mechanism of nitrogen under the synergy of substrate, plants, and microorganisms is different [64]. When selecting substrate combinations, choosing substrate combinations with different particle sizes can alter the dissolution oxygen distribution in constructed wetlands. As Fig. 2, a and b show, a vertical flow-constructed wetland and a horizontal flow-constructed wetland using a combination of sand, fine gravel, and coarse gravel, with the three substrates filled to different depths. According to the porosity of the substrate and particle size, the combination of filling from small to large can form an upper aerobic, middle anaerobic, and lower hypoxic environment, produce a rich microbial community structure, and remove different forms of nitrogen by aerobic nitrification, aerobic denitrification, anaerobic denitrification, and anaerobic ammonia oxidation. On the contrary, according to the matrix porosity and particle size, from large to small order combination filling can improve the overall dissolved oxygen level, and form a high dissolved oxygen environment in the upper layer, conducive to the survival of aerobic microorganisms and various aerobic animals, produce different dissolved oxygen gradients, and promote nitrogen removal [65]. Li et al. [66] found in the subsurface flow-constructed wetland that when treating high-intensity nitrogenous wastewater, the substrate combination sequence has a great impact on the removal of pollutants, and the removal rates of COD, NH₄⁺-N, and TN can be as high as 97%, 95%, and 94%, respectively, by combining gravel, slag, and wood dust in sequence.

Fig. 2.

Differences in the filling of the different substrate combinations.

Optimize the Plant Species Configuration

Plant species configuration is an essential factor influencing the nitrogen removal efficacy of constructed wetlands. Plants with well-developed roots can provide a larger adhesion surface for microorganisms, thus enhancing rhizosphere purification capacity and improving wetland performance [67]. However, different plants can absorb different nutrients, and the degree of absorption is also different. Therefore, constructed wetlands with monoculture and mixed planting have different removal efficiency of pollutants. Selecting the appropriate mix of plants can have complementary effects, but inappropriate selection can produce competition between species, which can affect the efficiency of nitrogen removal from constructed wetlands.

It has been found that increased plant abundance can improve biomass yield and nitrogen uptake capacity [68]. Therefore, mixed planting is one of the ways to increase the nitrogen removal effect of constructed wetlands. Tu et al. [69] showed that when free surface flow constructed wetland treated domestic sewage, the pollutant removal effect of mixed planting was significantly higher than that of single planting.

Due to plant species competition and stubble growth, monoculture and mixed planted constructed wetlands have significant differences in plant growth, biome structure, and nutrient removal. In addition, monoculture is also thought to be more susceptible to the death of plants caused by predation or disease; compared to monoculture, mixed cultivation is more resilient [70]. Relative to the monoculture wetlands, the presence of different plants in mixed-planting wetlands may provide more efficient root structures and diverse microbial habitats [71]. Therefore, the selection of suitable plant mixture planting has a good spatiotemporal compensation effect on plant growth and microbial community development, which can boost the nitrogen removal effect of wetlands [72]. As shown in Fig. 3, a and b are shown in the mixed planting and monoculture, respectively, in which the root structure of the mixed planting is richer, the nitrogen uptake is greater, and the oxygen secretion and growth status are also different. Wang et al. [73] found that mixed plant culture improved the diversity and nutrient removal efficiency of constructed wetlands, and the removal rates of COD and TN were 74.7% and 94.2%, respectively, when mixed cultivation was used with Iris pseudacorus, Iris sibirica, Juncus effusus, and Hydrocotyle vulgaris.

However, in mixed-planted wetlands, plant competition, such as competition for space, light, and nutrients, can lead to the dominance or inhibition of certain species, which can have an influence on the purification of wastewater in constructed wetlands [74]. Zhang et al. [75] found that in vertical flow-constructed wetlands, due to competition, the removal of pollutants by mixed planting is not greater than that by single planting, but mixed planting can provide other conditions conducive to plant growth, such as dissolved oxygen and pH. Therefore, when selecting plants, the competition between plant species, the growth, reproduction, regeneration, adaptation, and resource utilization capacity of plants should be fully considered, and the appropriate plant planting density should be allocated to reduce the negative impact of competition. In addition, species of the same family or genus should be avoided, as there may be additional competition between them [76].

Novel-Constructed Wetland Coupling Process

The combined use of constructed wetlands with other processes can maximize the performance of a wastewater treatment system, overcome the shortcomings of a single treatment unit, boost the effectiveness of wastewater treatment, and have a better removal efficiency of nitrogen and other pollutants [77]. In order to improve the wastewater treatment capacity of constructed wetlands, several novel wastewater treatment technologies (for example, microbial fuel cells, and biofilm electrode reactors) coupled with constructed wetlands have been proposed. Tables 1 and 2 summarize in detail the nitrogen removal effects of the two coupled processes.

Constructed wetlands coupled with microbial fuel cells are a clean energy technology for wastewater treatment and biopower generation, which can efficiently remove nitrogen from domestic wastewater [78]. Microbial fuel cells typically consist of a reaction chamber, electrodes, and external circuitry, with electrochemically active bacteria being the main biocatalysts in power generation. Electrode materials and microorganisms determine the number of electrons in the circuit and the rate of transport. Electricity production and wastewater treatment occur primarily at the anode, so microbial diversity is greater at the anode than at the cathode. The addition of conductive materials can extend the range of electroactivity of the anode to adjacent filler materials and contribute to the formation of electroactive bacterial communities and biofilms [79]. It has been found that the main dominant phyla in the treatment of synthetic wastewater by microbial fuel cells in constructed wetlands are Proteobacteria, Actinobacteria, Chloroflexi, and Bacteroidetes, with relative abundances of 76.08%, 2.02%, 8.33%, and 2.89%, respectively, and most of the electroactive bacteria belong to Proteobacteria [80]. In the anaerobic zone, electrochemically active bacteria generate electrons and transport them to the anode. The anode acts as an interim electron acceptor, and then the electrons are transported to the cathode through an external circuit. The aerobic region receives electrons and protons from the anaerobic region, and the final electron acceptor, such as O_2, receives electrons from the cathode [81]. Externally supplied anodes or conducting materials can act as artificial electron acceptors in constructed wetland anaerobic zones, facilitating the oxidation of pollutants such as NH₄⁺-N and organic matter. Another aspect is that externally supplied cathodes can act as electron donors, reducing contaminants such as NO₂^--N and NO₃^- -N, and electroactive microorganisms remove TN more effectively than ordinary microorganisms even at low population numbers [82]. According to Araneda et al. [83], when the microbial fuel cell combination system of a constructed wetland treats gray water, the removal rate of sCOD and nitrate can reach 91.7%±5.1%, 86.5±7.1%, respectively, and the maximum power density can reach 719.57±67.67 mWm^-3, which can effectively treat ash water and energy recovery.

Fig. 3.

Mixed and monoculture of plants.

The combination of a biofilm electrode reactor and constructed wetland can form an efficient heterotrophic/ autotrophic denitrification system to promote denitrification. Nitrifying bacteria can use carbon dioxide, carbonate, and bicarbonate as carbon sources to oxidize ammonia nitrogen to nitrate and do not need to add additional organic carbon sources. Hydrogen created by water electrolysis can be utilized as an electron donor for the autotrophic denitrification process, and the coupling system can strengthen the removal of TN and NO₃^--N, and the enhanced removal rate can reach 23.26%, respectively, and 24.20% [84, 85]. In a coupled system of biofilm electrode reactors and constructed wetlands, the appropriate current intensity can provide the right amount of H₂ to facilitate the rate of autotrophic denitrification. The anode electrode material (graphite) can produce an inorganic carbon source (CO₂) to act as a pH buffer and also provide an inorganic carbon source for denitrifying bacteria [86, 87]. Wang et al. [88] found that the biofilm electrode reactor-coupled system with constructed wetland could promote about 20.8% denitrification when I = 10 mA, pH = 7.5. Furthermore, the growth of autotrophic denitrifying bacteria can accelerate the action of the microelectric field, thereby improving the removal rate of NH₃-N and NO₃-N by a biofilm electrode reactor-coupled system with a constructed wetland.

Add Carbon Source

The denitrification process requires sufficient organic carbon sources to effectively remove nitrate [89]. At present, the main type of carbon source added to the constructed wetland is agricultural waste. Such as corn cobs, palmetto bark, and wood chips, which are easily accessible, low-cost, and have a stable release of carbon sources, can enhance denitrification and promote nitrogen removal [90, 91]. The addition of plant carbon sources can provide more attachment sites for nitrifying bacteria and increase the number of Hao and NxrAB enzymes in the wetland (42.05%, 29.99%), as well as the number of NirK, NorB/NorC, and NosZ genes (28.69%, 20.82%, and 29.22%) [92]. Zhang et al. [93] found that the cumulative carbon release from plants could reach 119.78-172.84 mg/g when using the vertical subsurface flow constructed wetland to treat low C/N wastewater, and the addition of a plant carbon source led to an increase in TN removal efficiency by 49.62%-61.3%.

Organic matter liberated from plant carbon sources mainly consists of tryptophans, xanthohumic acids, and humic acids [94], which have a significant organic carbon release effect. Humic acid and fulvic acids contain a variety of functional groups, such as carboxyl, phenolic, and alcohol groups [95, 96]. Different microbial genera can selectively degrade and use these functional groups. For example, Pseudomonas and Thauera have outstanding degradation capability of phenolic groups in humic acid-soluble organic compounds and serve as substrates for growth and metabolism, thus effectively promoting denitrification reactions [97, 98]. Dechloromonas has good degradation capability to carboxyl and alcohol groups in humic acid [99]. Tao et al. [100] found that corn cobs can provide a large area for microorganisms to attach and metabolize, and at a dosage of 70 mg·m^-2, the average removal of TN, NH₄⁺-N, and NO₃^--N was 99.22%, 99.25%, and 99.54%, respectively, when an up-flow constructed wetland was used to treat secondary effluent from a wastewater plant, and the corn cobs released the equivalent of 2648.17 mg of COD organic matter. In addition, the addition of glucose or sodium acetate can also regulate the microbial community, accelerate the denitrification rate, and improve the nitrogen removal rate [101]. In the case of low C/N, although the addition of a carbon source can effectively improve the removal rate of nitrogen, it may also produce secondary pollution, which can be collected uniformly for harmless or resource treatment and utilization to avoid secondary pollution [102].

Improve Redox Conditions

Redox potential is an important factor affecting microbial communities; different redox potential gradients are favorable for redox processes; aerobic microorganisms tend to have high redox potential; and anaerobic microorganisms tend to have low redox potential [103]. Therefore, the redox environment is particularly important for nitrogen removal in constructed wetlands [104]. Sulfur can act as an electron donor or electron acceptor in various dissimilatory microbial reactions, and the sulfur flowing into the constructed wetland usually exists in the form of sulfate in the oxidizing environment and sulfide in the reducing environment, as in equations (5) and (6) below [105]. Sulfur-driven autotrophic denitrification can use inorganic electron donors rather than organic electron donors [106].

The addition of iron and manganese oxides can facilitate the nitrogen removal process. Iron can be an electron donor for the denitrification process, maintaining reducing and anoxic conditions for nitrogen removal, and dissolved iron ions can also facilitate bacterial growth [107]. Iron is also the active center of cytochrome and most NOx reductase enzymes and conductive materials such as iron can increase the rate of electron transfer and effectively improve the removal efficiency of pollutants [108, 109]. Manganese oxides have good oxidation capacity for NH₄⁺-N; Mn (II) can provide electrons for the ammonification of NH₄⁺-N and the reduction of NO₃^--N resulting from the oxidation reaction. Due to the redox cycle of Mn (II) ions, the created Mn (III) or Mn (IV) oxides can furthermore oxidize NH₄⁺-N. Therefore, strengthening the redox cycle of manganese can promote nitrogen removal [110]. Recently, researchers have proposed a process called Mnammox, which refers to the process by which manganese dioxide acts as an electron acceptor to mediate the oxidation of NH₄⁺-N to N₂, NO₃^--N, or NO₂^- -N [111], reaction equations as in (7)-(9) [112]. This new approach is of great significance for nitrogen removal, but there are few studies on it [113].

In addition, the bioelectrochemical system combined with the constructed wetland helps adjust the aerobic and anaerobic areas of redox reaction and electron flow balance. The system allows electrons from the anode to the cathode without resistance movement, improving the electron transfer efficiency. In the defect area, supplement electron donors and electron acceptors adjust the surrounding redox reactions, thus affecting the activity of microorganisms [114].

Microbial Enhancement Technology

Microbial enhancement technology refers to the addition of microorganisms, nutrients, and substrates with specific functions to constructed wetlands and the inoculation of natural or genetically modified microorganisms to enhance the efficacy of water purification in constructed wetlands [115, 116]. Microbial enhanced nitrogen removal, which can be achieved by directly adding microbial bacterial agents with nitrogen removal function. For example, the addition of heterotrophic nitrification-aerobic denitrifying bacteria to tidal flow-constructed wetlands can improve nitrification and denitrification-related genes (such as; nirS, amABC, napA, and hao genes). The addition of heterotrophic nitrification-aerobic denitrifying bacteria, which enhances the removal of NO₃^--N by the denitrification pathway by increasing the napA and nirS genes, can also stimulate the gltD and nirA genes of the nitrogen assimilation process, which may lead to direct removal of NH₄⁺-N [117]. Chen et al. [118] added denitrifying polyphosphate bacteria liquid to the subsurface flow constructed wetland, and the study found that, in the hypoxic environment, the denitrifying phosphorus-accumulating bacteria can use the polyhydroxyalkanoates in the body as the electron donor through nitrate reductase and the nitrate as the electron acceptor to provide energy for its growth. At the same time, the nitrate is finally reduced to nitrogen for removal through denitrification. It not only saves the carbon source, but also improves the nitrogen removal efficiency of the subsurface flow-constructed wetland in treating the tailwater of the sewage plant.

The dosing method can also be through immobilized microbial dosing, which is a way to fix the nitrogen removal bacteria on a suitable carrier to ensure their activity and make the nitrogen removal bacteria grow rapidly and multiply in large quantities under suitable conditions. Wang et al. [119] used a polyvinyl alcohol immobilized nitrification unit to treat ammonia-rich wastewater, and the immobilized nitrification unit could promote nitrification and improve ammonia nitrogen removal. Biochar is an excellent bacterial immobilization vector, and biochar can promote the expression levels of denitrification functional genes (napA and nirK) and electron-transferring genes (napB and napC) related to denitrification [120]. Zhao et al. [121] also found that biochar bacterial agents can substantially increase the abundance of genes associated with nitrogen removal (drsA, drsB, nirK, and nirS), thus improving the nitrogen removal efficiency of tidal flow-constructed wetland treatment of saline wastewater.

To ensure the nitrogen removal efficacy of constructed wetlands under certain extreme environments (such as low temperature and high salt), nitrogen removal microorganisms isolated and screened from the environment with the function of low temperature or salt tolerance can be added [122]. Low-temperature microorganisms can still maintain high activity in low-temperature environments, so the water purification efficiency and stability of wetland systems can be ensured. Zhao et al. [123] added compound microbial inoculants to the subsurface flow-constructed wetland to treat campus wastewater under low-temperature conditions, and the study showed that the removal rate of ammonia nitrogen and total nitrogen was increased by microbial inoculation, and the soil microbial community structure and nitrogen-related bacterial species balance were changed, and a new bacterial community balance was created in the original place. Combined with the growth characteristics of low-temperature microorganisms in low-temperature environments, it can be widely used for wastewater treatment in constructed wetlands in cold areas.

The addition of exogenous microorganisms can improve plant performance by enhancing nutrient utilization and decreasing plant stress responses to contaminants, and it can also facilitate plant uptake of contaminants by producing solubilizers or facilitate rhizosphere degradation of organic contaminants by secreting biosurfactants [124, 125]. Lingua et al. [126] inoculated arbuscular mycorrhizal fungi into reeds, which improved nitrate removal efficiency and promoted the growth of plant roots and stems, resulting in larger plants and more developed root systems.

Aeration

Aeration can be divided into continuous aeration and intermittent aeration. Although continuous aeration can significantly raise dissolved oxygen levels, it consumes too much energy. When intermittent aeration is used, dissolved oxygen levels rise as aeration progresses, and aerobic microorganisms use oxygen for the removal of organic matter, which is favorable for nitrification, when the aeration stops, the dissolved oxygen is gradually consumed by microorganisms, and the concentration decreases, which is conducive to the denitrification process. Intermittent aeration makes the dissolved oxygen alternately rise and fall, thus forming an alternating aerobic and anaerobic environment [127]. However, there were significant differences between aerated and non-aerated wetlands. Some studies showed that the nitrification intensity and denitrification intensification of the aerated wetland were 5.3 times and 1.3 times higher than those of the control wetland, and the copy density of the nosZ gene in the aeration wetland was higher, about 3.8 times that of the control wetland [128]. In addition, the aeration volume, aeration intensity, and frequency also influence nitrification and denitrification by generating different dissolved oxygen concentrations [129]. The aeration location can influence the spatial distribution of dissolved oxygen by affecting the diffusive path of oxygen [130]. Liu et al. [131] studied the treatment of polluted urban river water in horizontal subsurface flow-constructed wetlands with no aeration, continuous aeration, and intermittent aeration and demonstrated that the removal effectiveness of COD and NH₄⁺-N was improved by aeration, and the intermittent aeration method had the best effect, with a removal rate of up to 91.9% for TN. Therefore, intermittent aeration is one of the effective methods to increase the nitrogen removal effect.

Ammonification

Ammonification is the process by which ammoniated bacteria convert organic nitrogen to ammonia nitrogen [134]. Ammonia nitrogen includes ammonium ions (NH₄⁺) and free ammonia (NH₃) and mainly comes from nitrogen-containing organic matter in domestic wastewater [135]. The ammonification rate is related to physicochemical parameters. The ammonification ability of organic nitrogen is different under different parameter conditions; for instance, for temperature and pH values, the optimal reaction conditions are 40- 60 °C and 6.5-8.5, respectively [136], but the microbial populations involved in the ammonification process are not very different [137]. Ammonification is the first step in the removal of organic nitrogen, which is immediately linked to the follow-up nitrogen removal process and plays a significant part in the whole nitrogen removal process [138].

Nitrification and Denitrification

The process of nitrification involves the oxidation of ammonia nitrogen to nitrite nitrogen by ammonia-oxidizing bacteria in aerobic conditions. Afterward, nitrite nitrogen undergoes oxidation to nitrate nitrogen by nitrite-oxidizing bacteria. Denitrification is the use of nitrification-produced nitrate nitrogen, and under anaerobic conditions, denitrifying bacteria convert nitrate nitrogen into nitrogen [139]. Bacteria can complete the nitrogen cycle through a variety of pathways [140]. However, when oxygen transfer is restricted, nitrification is inhibited, and when an aerobic environment is generated, it is conducive to the nitrification reaction [141]. Denitrification is normally regarded as the primary and permanent process for nitrate removal, and organic carbon is a major factor limiting nitrate removal; organic carbon is an electron donor in denitrification, and its concentration affects the growth and activity of denitrifying bacteria, so insufficient organic carbon affects the effect of denitrification [142, 143]. In addition, the dissolved oxygen concentration has a large effect on the denitrification process, and the high oxygen conversion capacity will make the denitrification reaction difficult to occur [144].

Substrate-Microbe Coupling

The substrate can provide a carrier for the microbes to attach, support the development of the biofilm, and boost the removal of contaminants by providing electrons to the microbes [155]. For example, biochar, with its large surface area and porous structure, can provide rich attachment sites for microorganisms, creating a favorable environment for denitrifying bacteria to survive, and can release carbon sources to enhance denitrification. In addition, the high oxygen content and various electron-donor chemical structures (e.g., hydroxyl, carboxyl, and phenolic hydroxyl groups) in biochar can provide electrons to promote the activity of denitrifying bacteria [156, 157]. Biochar is a new multifunctional adsorption material extracted from waste biomass that has been widely used in sewage treatment in recent years and can be used as a new alternative material for constructed wetlands, which can remove pollutants more effectively than conventional substrates, modified biochar is also commonly used in sewage treatment of constructed wetlands [158]. The addition of biochar can enhance the diversity of wetland microbial communities and improve the performance of wastewater purification in constructed wetland ecosystems [159]. In addition, natural carbon source substrates such as wheat straw, apricot pits, and walnut shells can also improve the removal rate of total nitrogen by supplying a good environment for microorganisms to form biofilms and providing more electron donors for denitrification [160]. Inorganic substrates such as ceramics, gravel, and quartz sand have a large specific surface area and pore volume, which is also favorable for the survival of microorganisms [161]. Wan et al. [162] used ceramic grains as the substrate to treat domestic wastewater. The findings indicated that ceramic grains can help increase the percentage of beneficial microorganisms (nitrogen, sulfur, and phosphorus) in the wastewater treatment process, stimulate key microorganisms, and improve the efficiency and abundance of nitrifying bacteria.

Fig. 4.

Coupling between microorganisms, substrates and plants.

The nature of the substrate has a great impact on the microbial community structure, mainly reflected in the particle size, pore structure, and specific surface area of the substrate [19]. The smaller the substrate particle size, the larger the specific surface area per unit volume of substrate, the more microorganisms can be accommodated, and the better the purification efficiency of wastewater, but if the substrate particle size is too small, the porosity and hydraulic conductivity are low, and it is easy to block and affect the wastewater purification effect [163]. The larger the substrate particle size, the more oxygen enters the substrate pores, which is favorable for the growth of aerobic microorganisms, thereby enhancing the nitrification strength of the substrate. On the contrary, when the substrate particle size is small, the oxygen content in the substrate pore is less, which promotes the growth of anaerobic microorganisms and then strengthens the denitrification strength [20]. It has been found that the substrate with a larger pore volume or specific surface area generally has higher adsorption and removal performance for pollutants in constructed wetlands, and the porous structure of the substrate can improve the supply of dissolved oxygen, and promote the formation of biofilm and the propagation of microbial communities.

Significant differences in dissolved oxygen distribution and microbial community structure in different wetland depths [65]. In deep-constructed wetlands (d > 0.6 m), an alternating anaerobic-anoxic environment can be formed to create a favorable living environment for anaerobic ammonia-oxidizing bacteria. For the deeper bed, the oxygen transfer potential is low, and the anaerobic process will occupy a dominant position at the bottom of the bed [42]. When the concentration of nitrogen and organic matter is high, the constructed wetland can promote the heterotrophic nitrification of the upper aerobic zone and the denitrification and anaerobic ammonia oxidation processes in the lower anaerobic zone [136]. Chen et al. [164] found that when vertical flow-constructed wetlands were used to treat domestic sewage at a depth of 0-20 cm, the number of microorganisms was the largest and decreased with the increase of substrate depth, with ammonia-oxidizing bacteria and nitrifying bacteria primarily found at a depth of 0-40 cm and denitrifying bacteria primarily found at a depth of 60-80 cm.

Plant-Microbe Coupling

Plants ensure the stability of rhizosphere microorganisms, and their roots provide a good habitat environment for microorganisms [165]. Plants can form well-defined bacterial communities around the rhizosphere by inducing and stimulating the growth of specific bacterial communities [166], by influencing the distribution of microorganisms, thus affecting the nitrogen removal capability of constructed wetlands [167].

The rhizosphere of plants is a multifaceted and dynamic environment, and root exudates are good at attracting specific microorganisms. Plants can actively select specific key microbial species under spatiotemporal conditions, and the spatiotemporal dynamic changes of root exudates are accompanied by dynamic changes in the composition and function of rhizosphere microbial communities. As the rhizotrophic environment evolves, the composition of the microbial community will also generate corresponding dynamics [168]. Plant changes can lead to changes in sediment dissolved oxygen levels, nutrient availability, and organic matter content, all of which can affect microbial communities [169]. The root system of plants mainly affects the surrounding microorganisms through the role of oxygen delivery and secretion, and the root secretion can provide reducing carbon, nitrogen, and other nutrients for microorganisms, different plant root systems secrete different secretions, so there are differences in the types and numbers of microorganisms around the root system of different plants [170, 171]. For example, Phragmites australis can secrete glucose, sucrose, acetic acid, and succinic acid, and Cyperus alternifolius can secrete glucose, sucrose, malic acid, and malonic acid [172]. These can be utilized as a source of carbon for the denitrification process. Xu et al. [173] found that when using macrophytes to treat pretreated pig wastewater, root exudates had a positive effect on microorganisms, and the removal rate of ammonium nitrogen with root exudates treatment (90.4% and 69.3% on average) was significantly higher than that without root exudates treatment (87.3% and 62.0% on average).

In vertical-flow constructed wetlands, microorganisms in plant seedling roots preferentially utilize simple amino acids, while those in mature plant roots utilize more complex carbohydrates [174]. Prokaryotic and eukaryotic microorganisms are able to use organic pollutants as sources of carbon and energy, and organic pollutants can act as electron donors oxidized in aerobic and anaerobic environments, where various electron acceptors other than oxygen can achieve anaerobic respiration processes [175]. Plant litter can also be utilized as a solid carbon source to provide electrons for denitrification, which plays a role in mitigating carbon sources [176]. The growth and metabolism of microorganisms are also linked to pH; the optimal pH range for nitrification is 7.5-8.6, while for denitrification it is 7-8 [177]. However, studies have shown that plant roots can secrete hydrogen ions or hydroxide into the rhizosphere environment, such as hydrogen ions brought by organic acids secreted by some plants, thus playing a role in regulating the pH of constructed wetlands [178, 179].

The effect of plants on microorganisms is also reflected in different seasons. In winter, the light time is shorter, the temperature is lower, the enzyme activity in the plant is reduced, and the metabolic capacity of the plant is decreased. It had a significant effect on rhizosphere microbial diversity and significantly reduced denitrifying enzyme activity and functional microbial diversity [180]. Wang et al. [181] found in a research of subsurface flow-constructed wetland planted with phragmites: In summer, the appropriate temperature is favorable for the growing of plants and microorganisms, the respiration rate of plant roots is high, the level of metabolic flora is high, and the constructed wetland ecosystem is rich in functions, which can maintain a stable and good removal rate of nitrogen. In winter, although plant and microbial activity is affected by low temperatures and the release of plant oxygen is reduced, the existence of plants still has a positive impact on microbial communities and abundance.