Assembling plant diversity mitigates greenhouse gas emissions and achieves high nitrogen removal when treating the low-C/N wastewater by constructed wetlands

doi:10.21203/rs.3.rs-1446925/v1

The low carbon-to-nitrogen (C/N) ratio in wastewater will inhibit pollutant removal, and more seriously, it will cause an increment of nitrous oxide (N₂O) emissions of constructed wetlands (CWs). Raising the C/N ratio of wastewater is an effective way to solve this problem, while it may cause secondary pollution and is costly. Assembling plant diversity promotes N removal, while the effects of plant diversity and increasing C/N ratio on global warming potential (GWP) combined by N₂O and methane (CH₄) is lack of comparison. In this study, 108 CW microcosms were established to explore the effects of increasing the C/N ratio from 1 to 5 and assembling plant diversity on N removal and GHG emissions. Results showed that when the C/N ratio was 1, (1) increasing species richness reduced N₂O and CH₄ emissions then reduced the GWP by 70%; (2) the presence of Arundo donax in microcosms reduced GWP by 72%; (3) an A. donax × Tradescantia fluminensis × Reineckia carnea mixture resulted in a high N removal and decreased the GWP per g N removal by 92% with a cost increment of 0.05 USD per m³ wastewater treated; and (4) as the C/N ratio increasing to 5, the GWP per g N removal of monocultures was reduced by 96%, but the cost increased by at least 0.29 USD per m³ wastewater treated. In summary, configuring plant diversity in CWs is an efficient, clean and cost-effective measure to treat wastewater with a low C/N ratio.

Nitrous oxide

Methane

Species richness

Species identity

Plant uptake

Denitrification

Cost-effective

In recent years, the demand for wastewater treatment around the world has increased sharply, with 80% of wastewater being discharged without any treatment (UNESCO 2021). The carbon-to-nitrogen (C/N) ratio of wastewater from different sources ranges from 1 to 132, and the C/N ratio will affect the performance of wastewater treatment facilities (Vymazal 2014; Sylla 2020). Theoretically, the N removal will be limited if the C/N ratio of wastewater was under 2.86 (Itokawa et al. 2001; Pelaz et al. 2018). In addition, wastewater treatment is accompanied by greenhouse gas (GHG) emissions (IPCC 2021). The total GHG emissions around the word by treating wastewater are 775 Mt CO₂-eq (IPCC 2014). Low C/N ratio in wastewater also promotes nitrous oxide (N₂O) emissions due to incomplete denitrification (Duan et al. 2021). Therefore, the measurement for treating wastewater with low C/N ratio urgently needs to be addressed.

Constructed wetlands (CWs) are the green measure to treat wastewater from dispersed sources (Vymazal et al. 2021). The N₂O and methane (CH₄) emissions per kg N removed from CWs are only 3.4% and 0.1%, respectively, of those from wastewater treatment plants (Liu et al. 2012). However, CWs are commonly used to treat domestic wastewater with insufficient C sources (Sun et al. 2016; MEEPRC 2020). CWs are also used to treat piggery wastewater and landfill leachate, and the C/N ratio will be as low as 1 (Feng et al. 2020; Çakirgöz et al. 2021). The discharge of those wastewater with low C/N ratio is still increasing (Mander et al. 2014). Therefore, it is really urgent to optimize the CWs to meet the challenge posed by wastewater with a low C/N ratio.

Adding C sources is a common measure to enhance the wastewater treatment performance for wastewater with a low C/N ratio (Chen et al. 2020; Çakirgöz et al. 2021). Increasing the C/N ratio will enhance N removal (Nguyen et al. 2018) and reduce N₂O emissions (Duan et al. 2021). The lowest levels of N₂O emissions were reported at the C/N ratio of 5 (Wu et al. 2009) or 10 (Zhao et al. 2014; Guo et al. 2020). However, an excessive C/N ratio (such as 20) will also increase N₂O emissions (Wu et al. 2009). In addition, excessive C may promote CH₄ emissions (Bhullar et al. 2013). Moreover, increasing the C/N ratio of wastewater means continuous investment and transportation, which increases the cost (Hussain et al. 2019). For example, adding methanol increases the infrastructure cost by 25 ~ 31% (CDM 2007). Therefore, it is urgent to find a more economical and effective method to reach high N removal and low global warming potential (GWP) when treating the low-C/N wastewater.

As an important part of the ecological structure of CWs, plants play a key role in pollutant removal and GHG emissions (Brisson et al. 2020; Vymazal 2020). Optimizing the plant community may enhance N removal and reduce GHG emissions in CWs (Maucieri et al. 2017; Brisson et al. 2020). First, increasing plant richness promotes the complementary utilization of resources by plant communities and enhances N removal (Cardinale 2011; Brisson et al. 2020). Moreover, increasing plant species richness promotes plant biomass production to mitigate N₂O and CH₄ emissions (Du et al. 2020; Luo et al. 2020). However, increasing plant richness promotes N₂O emissions due to the increase in plant C secretion (Han et al. 2016). Notably, plant identities are also prominent (Abalos et al. 2014; Jesus et al. 2018). For example, CWs consisting of Arundo donax have higher N absorption than CWs consisting of other species (Kouki et al. 2012; Sylla 2020), and CWs with A. donax or Canna indica have lower N₂O emissions than those without them (Luo et al. 2020). Nevertheless, studies on the effects of plant diversity on CW ecosystem functioning have mostly been carried out in inorganic (C-free) wastewater treatment systems (Luo et al. 2016; Han et al. 2021). One study showed that the effects of mixing 4 plant species on N removal as well as N₂O emissions were equivalent to that of increasing the C/N ratio to 1 when treating C-free wastewater (Han et al. 2016). Whether assembling plant diversity is a suitable alternative to increasing the C/N ratio to a higher level (5) to effectively solve the problem raised by treating wastewater at a low C/N ratio still needs to be explored.

In this study, 108 simulated vertical flow CW microcosms were set up to compare the effects of increasing C/N ratio from 1 to 5 and assembling plant diversity (species richness and species identity) on ecosystem functioning (N removal and GHG emissions) in CWs. The aims were to study (1) the changes in the N mass removal rates and GHG emissions of microcosms with an increment in C/N ratio from 1 to 5; (2) effects of plant diversity on the N mass removal rates and GHG emissions of microcosms at C/N ratios of 1 and 5; and (3) the costs and benefits of species combinations with high N removal and low GWP when treating wastewater at a C/N ratio of 1.

2.1 Experimental design

In late March 2019, the total of 108 microcosms (46 cm × 35 cm × 22 cm) were set up in Sandun town (30° 21' N, 120° 02' E), Hangzhou city. Washed sand (diameter = 0.5 ~ 3 mm) was used as the substrate of each microcosm (Fig. S1).

The species richness in plant communities was 1, 3, or 4 (Fig. 1; Fig. S2). Two frequently used species in CWs, A. donax L. and Oenanthe javanica (Blume) DC., and two ornamental species, Reineckea carnea (Andr.) Kunth., and Tradescantia fluminensis Vell., were chosen (Fig. S2). The four species are morphologically different, and have different resources utilization mode (Table S1). The nine plant combinations (4 monocultures of each species, 4 three-species mixtures and 1 four-species mixture) and six replicates for each combination were set up based on a randomized block design. The planting density was 12 plants per microcosm. For the mixtures, the individual number of each species was the same, and different plant species were planted adjacent to each other to ensure the evenness of each species.

The synthetic wastewater was according to Hoagland and Arnon (1950), and sucrose was added to the water as the C source (Table 1). This study set the C/N ratio (calculated as COD/N) at 1 and 5 (Table 1). The research group has shown that plant diversity can improve N removal when treating low-C/N ratio wastewater, but there is a lack of research on GWP (Han et al. 2016; Du et al. 2020). Therefore, a C/N ratio of 1 was set in this study to further explore the various plant combinations in one treatment. The research group has also shown that in CWs, the promotion of increasing plant richness on N removal was equivalent to that of increasing the C/N ratio from 0 to 1 (Han et al. 2016). Previous studies suggested the C/N ratio of 5 was conducive to N removal and N₂O reduction at the same time (Wu et al. 2009). Therefore, in this study, a C/N ratio of 5 was set in another treatment for comparing the effect of plant diversity and increasing C/N ratio on N removal and GWP. The pH of wastewater was approximately 6. Each microcosm was fed with wastewater (7 L) once every 10 days. The experiment lasted until the beginning of July.

Table 1

Combination and concentration of nutrients in the simulated wastewater.
Macroelement	Concentration (g L^− 1)		Microelement	Concentration (mg L^− 1)
Macroelement	C/N = 1	C/N = 5	Microelement	C/N = 1	C/N = 5
Ca(NO₃)₂·4H₂O	0.71	0.14	H₃BO₃	2.86	0.57
CaCl₂·2H₂O	0.30	0.06	MnCl₂·4H₂O	1.81	0.36
(NH₄)₂SO₄	1.19	0.24	ZnSO₄·7H₂O	0.22	0.04
KH₂PO₄	0.14	0.03	CuSO₄·5H₂O	0.08	0.02
MgSO₄·7H₂O	0.49	0.10	H₂MoO₄·4H₂O	0.09	0.02
C₁₂H₂₂O₁₁	0.30	0.30	FeSO₄·7H₂O	5.56	1.11
			Na₂EDTA	7.44	1.49

2.2 Sample collection and measurements

GHGs mainly include carbon dioxide (CO₂), N₂O, and CH₄ (IPCC 2021). In this study, GHGs were represented by N₂O and CH₄ without considering CO₂, as CO₂ from herbaceous plants is a short-cycle gas and be not a contributor to the greenhouse effect (IPCC 2021). Gas samples were taken by closed static chamber method (Cheng et al. 2007) at the end of the experiment for horizontal comparison between the two treatments. Transparent PVC chambers (48 cm × 35 cm × 110 cm) enclosed all the plants and were made airtight by inserting the bottoms into water. During the pre-experiment, one microcosm was randomly selected for sampling and determination for each block, and sampling was performed at 0, 3, 5, 10, 20, 30, 40, 50 and 60 minutes after the chambers were made airtight. Results showed that the concentrations of N₂O and CH₄ increased linearly in the first 20 minutes. Therefore, during the formal test, the sampling time is determined as 20 minutes after the chambers were made airtight. A vacuum gas collection tube was used to collect gas samples from each microcosm. At the same time, the temperature and humidity in the chamber were measured by a hygrothermograph. Gas concentrations were determined by gas chromatography (Agilent 7820, Agilent Technologies Inc., USA).

After gas sampling, a water sample (100 mL) was taken from each microcosm. The concentrations of ammonium (NH₄⁺-N), nitrite (NO₂⁻-N), and nitrate (NO₃⁻-N) were determined by automated colorimetry (CleverChem-Anna, DeChem-Tech.GmbH, Germany). The total N (TN) concentration was analyzed by a nondispersive infrared detector (Multi N/C 3100, Analytik Jena AG, Germany). A multiparameter water quality meter (Bante 900P, Bante Instruments, China) was used to determine oxidation-reduction potential (ORP) and dissolved oxygen (DO).

After water sample collection, all the plants were harvested. The plants were oven-dried (65°C) to a constant weight to measure the aboveground and belowground biomasses. The N concentrations in plant tissues were detected by an elemental analyzer (Flash HT2000, Thermo Finnigan, Germany). To determine the N pool in the substrate, a fresh substrate sample from each microcosm was weighed at 150 g, and the N concentration of each sample was measured after extract by KCl solution (2 mol L^− 1). Bulk density was determined with the ring-cutting method.

2.3 Calculations

The calculation of GHG emissions followed the method described by Cheng et al. (2007),

$${E}_{i}=(\rho \times {d}_{c}\times V\times {T}_{0})/({d}_{t}\times A\times T)$$

1

where ${E}_{i}$ is the emissions rate of N₂O or CH₄ (mg m^− 2 day^− 1),$\rho$ is the density of every gas (mg m^− 3) under normal conditions, ${d}_{c}$ is the change in gas volume fraction (m³ m^− 3), $V$ is the volume (m³) of the air in chamber, ${T}_{0}$ is the reference temperature (273 K), ${d}_{t}$ is the sampling duration (min),$A$ is the bottom area (m²) of the chamber, and T is the experimental temperature (K).

The total GWP (g CO₂-eq m^− 2 day^− 1) was calculated as the sum of CH₄ and N₂O emissions multiplied by 27 and 273, respectively (IPCC 2021).

The TN mass removal rate (NR, mg m^− 2 day^− 1) was calculated as,

$$\text{N}\text{R}=({\text{c}}_{\text{i}}\times {\text{V}}_{\text{i}}-{\text{c}}_{\text{e}}\times {\text{V}}_{\text{e}})/(\text{S}\times \text{D})$$

2

where ${c}_{i}$ is the influent TN concentration (mg L^− 1), ${V}_{i}$ is the total influent volume during the whole operation, ${c}_{e}$ is the effluent TN concentration of the end of the last hydraulic cycle (mg L^− 1), ${V}_{e}$ is the total effluent water volume (L),$S$ is the microcosm’s bottom area (m²), and$D$ is the total experimental time (days).

The N budget of the microcosm was calculated by the mass balance method (Geng et al. 2019),

$${M}_{i}={M}_{e}+{M}_{p}+{M}_{s}+{M}_{d}+{M}_{a}$$

3

where ${M}_{i}$ is the amount of N supplied (mg), ${M}_{e}$ is the amount of N remaining in the effluent (mg), ${M}_{p}$ is the amount of plants N uptake (mg), ${M}_{s}$ is the amount of N adsorbed by the substrate (mg), ${M}_{d}$ is the amount of N used for denitrification, and ${M}_{a}$ is the amount of N lost by ammonia volatilization. Among these variables, ${M}_{a}$ was less than 1% of the total N removal in a previous study with the same influent N concentration, so it was ignored in this study (Luo et al. 2020).

The plant uptake (${N}_{p}$, mg N m^− 2 day^− 1), denitrification (${N}_{d}$, mg N m^− 2 day ^− 1), and substrate adsorption (${N}_{s}$, mg N m^− 2 day^− 1) rates were calculated as,

$${N}_{p}={M}_{p}/(S\times D)$$

4

$${N}_{d}={M}_{d}/(S\times D)$$

5

$${N}_{s}={M}_{s}/(S\times D)$$

6

where $S$ is the microcosm’s bottom area (m²), and$D$ is the total experimental time (days).

The transgressive under-emission effect was calculated according to Luo et al. (2016),

$${D}_{mix-GHG}=(Mix\left(Ei\right)-Min\left(Ei\right))/Min\left(Ei\right)$$

7

where $Mix\left(Ei\right)$ is the GHG emissions or GWP in the mixtures and $Min\left(Ei\right)$ is the lowest GHG emissions or GWP in the relevant monocultures. Similar to the transgressive under-depletion effect (Palmborg et al. 2005), ${D}_{mix-GHG}$ < 0 suggests that the GHG emissions in mixtures were lower than those in the monoculture with the lowest emissions, and this state is called transgressive under-emission (Luo et al. 2016).

2.4 Statistical analysis

The data were analyzed to investigate the impacts of plant species richness, identity, and composition on CW ecosystem functioning. At first, the data were subjected to the Kolmogorov-Smirnov test for normality and Levene's test for the variance equality. The GHG emissions data were natural logarithm transformed to meet the analysis of variance statistical assumptions. To meet the statistical requirements, the N₂O emissions data at a C/N ratio of 5 were added by one when taking the logarithm. The effects of species richness and composition on the N mass removal rate and N removal pathways were analyzed by the Kruskal-Wallis test. Independent sample t-tests were used to define the significant differences in GHG emissions or N removal under two C/N ratios as well as the differences in combinations with or without a particular species. The difference between D_mix−GHG and 0 was detected by a one-sample t-test. The analyses were performed in SPSS 20 (SPSS Inc., Chicago, IL, USA) and R 4.1.2. at a statistical significance level of α = 0.05.

3.1 Effects of plant diversity on N₂O and CH₄ emissions under two C/N ratios

When the C/N ratio was 1, the N₂O emissions of the microcosms decreased with increasing species richness (P < 0.05; Fig. 2a), and the lowest N₂O emissions occurred in the microcosms with the T. fluminensis × R. carnea × A. donax mixture, while the highest N₂O emissions were observed in the monoculture of T. fluminensis (P < 0.05; Fig. 2b). To allocate A. donax and R. carnea in the community reduced N₂O emissions (Table 2). When the C/N ratio was increased to 5, the N₂O emissions of all microcosms were reduced (P < 0.05; Fig. 2b), but there were no differences among the combinations (P > 0.05; Fig. 2b).

Table 2 Emissions of N₂O and CH₄ (mg m^-2 day^-1), the GWP (g CO₂ eq m^-2 day^-1), N removal rate (mg N m^-2 day^-1), plant uptake, denitrification, and substrate adsorption rate (mg N m^-2 day^-1) in the microcosms with or without the four plant species.

Similarly, increasing species richness reduced CH₄ emissions at a C/N ratio of 1, but not 5 (Fig. 2c). Regardless of whether the C/N ratio was 1 or 5, the CH₄ emissions were lowest for the mixture of O. javanica × A. donax × R. carnea, while those of the R. carnea monoculture were the highest (P < 0.05; Fig. 2d). To allocate O. javanica and A. donax in the community reduced CH₄ emissions (Table 2). For the R. carnea monoculture, the CH₄ emissions was declined when the C/N ratio was increased to 5 (P < 0.01; Fig. 2d).

3.2 Effects of plant diversity on GWP under two C/N ratios

For comprehensively evaluate GHG emissions, the N₂O and CH₄ emissions were integrated into the GWP. When the C/N ratio was 1, the GWP negatively responded to species richness (P < 0.05; Fig. 3a). The GWP of the microcosms with the T. fluminensis × R. carnea × A. donax mixture was the lowest, and that of the T. fluminensis monoculture was the highest (P < 0.05; Fig. 3b). Configuring the A. donax and R. carnea in community reduced the GWP when the C/N ratio was 1 (Table 2). The N₂O emissions composed the major part of GWP at a C/N ratio of 1, while CH₄ emissions were the driver at a C/N ratio of 5 (Fig. 3b). The GWPs of all the combinations was decreased as the C/N ratio increasing to 5, and the lowest GWP appeared in the O. javanica × A. donax × R. carnea mixture (P < 0.05; Fig. 3b).

The trend of GWP per g N removal was similar to that of GWP. The GWP per g N removal negatively responded to species richness only when the C/N ratio was 1 (P < 0.05; Fig. 3c). The GWP per g N removal of the microcosms with the T. fluminensis × R. carnea × A. donax mixture was the lowest (P < 0.05; Fig. 3d). Increasing the C/N ratio to 5 decreased the GWPs of the combinations except for the mixture of T. fluminensis × R. carnea × A. donax. The GWP per g N removal of the T. fluminensis × R. carnea × A. donax mixture under a C/N ratio of 1 was as low as the average value of monocultures under a C/N ratio of 5 (P > 0.05; Fig. 3d).

3.3 Effects of plant diversity on N removal and pathways under two C/N ratios

When the C/N ratio was 1, the TN mass removal rate was slightly improved with increasing species richness only when the C/N ratio was 5 (P < 0.001; Fig. 4a). The TN mass removal rate of O. javanica was lower than that of the other plant combinations (P < 0.05, Fig. 5a). Increasing the C/N ratio to 5 reduced the TN mass removal rate in each combination (P < 0.05, Fig. 5a). When the C/N ratio was 5, the monoculture of A. donax and the mixtures containing A. donax had the highest TN mass removal rates (P < 0.05; Fig. 5a). Regardless of whether the C/N ratio was 1 or 5, the presence of A. donax increased the TN mass removal rate (Table 2).

Plant uptake, denitrification, and substrate adsorption are three major N removal pathways. Species richness was positively correlated with plant uptake under the two C/N ratios (P < 0.001; Fig. 4b). When the C/N ratio was 1, the plant uptake was highest in the A. donax monoculture and the mixtures containing A. donax (P < 0.05; Fig. 5b). Increasing the C/N ratio to 5 resulted in a decrease in plant uptake in the A. donax monoculture and all the mixtures containing A. donax (P < 0.05; Fig. 5b). When the C/N ratio was 5, the A. donax × T. fluminensis × O. javanica mixture had the highest plant uptake (P < 0.05; Fig. 5b). Regardless of whether the C/N ratio was 1 or 5, the presence of A. donax increased plant uptake but inhibited denitrification (Table 2). Under the two C/N ratios, increasing species richness decreased denitrification (P < 0.001; Fig. 4c). Increasing the C/N ratio from 1 to 5 decreased denitrification in each combination (P < 0.01; Fig. 5c). Regardless of whether the C/N ratio was 1 or 5, the highest levels of denitrification occurred in the monocultures of T. fluminensis and R. carnea (P < 0.05; Fig. 5c). Substrate adsorption slightly decreased with increasing species richness only when the C/N ratio was 5 (P < 0.05; Fig. 4d), and increasing the C/N ratio to 5 led to a reduction in substrate adsorption (P < 0.01; Fig. 5d).

4.1 Increasing plant richness alleviates GHG emissions under a low C/N ratio

Low C/N ratio in wastewater, theoretically defined as lower than 2.86, usually leads to high N₂O emissions (Itokawa et al. 2001; Duan et al. 2021). In this study, when the C/N ratio was 1, the average N₂O emissions of monoculture microcosms accounted for 5% of the inflow TN mass (Table S2). This contribution is slightly higher than the scope of previous reports (0.001 ~ 4%; Table S2). Fortunately, increasing plant richness in CWs can make the mean N₂O emissions of mixtures (species richness at 3 or 4) 70% lower than that of monoculture microcosms when the C/N ratio is 1 (Fig. 2a). This result is different from the fact that increasing plant richness in CWs from 1 to 4 increased N₂O emissions by 68 ~ 267% when treating C-free wastewater (Fig. S4), but is consistent with the trend reported by Du et al. (2020) that the mean N₂O emissions of mixtures were 46% lower than those of monocultures at a C/N ratio of 1. Community denitrification is one of the important factors affecting N₂O emissions (Maucieri et al. 2017). In this study, increasing plant richness reduced the denitrifying N indirectly by promoting plant uptake by 2-fold compared with that of monocultures and then reduced N₂O emissions (Fig. 3c, d; Fig. S5). Furthermore, it was found that the higher the N in the ecosystem was, the stronger the effect of improving plant richness on reducing N₂O in the CW ecosystem (Fig. S4). The above results show that when CWs are used for treating wastewater with a low C/N ratio, improving plant species richness has great potential for N₂O emissions reduction.

In this study, the N₂O emissions of CWs account for 90% of GWP (calculated as the combination of N₂O and CH₄ emissions) when the C/N ratio is 1 (Fig. 3b). This result is similar to the values of 84% reported by Du et al. (2020) and 88% reported by Luo et al. (2020). Another study pointed out that N₂O emissions can account for up to 75% of the C footprint in wastewater treatment plants (Daelman et al. 2013), indicating that controlling N₂O emissions is the key factor in reducing GWP. However, there is often a trade-off between N₂O and CH₄ emissions (Saha et al. 2017), so reducing the emissions of one gas may be at the cost of adding another one. For example, Yao et al. (2012) found that although the application of N fertilizer in rice fields reduced CH₄ emissions by 53%, it increased N₂O emissions by 6-fold and was not conducive to reducing GWP. However, in this study, increasing species richness reduced CH₄ emissions by 56% on the premise of reducing N₂O emissions at a C/N ratio of 1 in the CW microcosms (Fig. 2c). Therefore, a simultaneous reduction in the emissions of the two GHGs was realized, so the system has great potential for a final reduction in GWP.

4.2 Species identity surpasses species richness in terms of the effect on GHG mitigation under a low C/N ratio

The plant species also affects community GHG emissions in CWs (Maucieri et al. 2017). A. donax was the most conducive to GHG emissions reduction among the species in this study (Table 2, Table S3). Compared with the microcosms without this species, the presence of A. donax in microcosms reduced N₂O by 71% under a low C/N ratio (Table 2, Table S3). The allocation of A. donax in the community also reduced N₂O emissions by 42% in the treatment of C-free wastewater (Luo et al. 2020). Configuring species with high plant uptake in the community is conducive to N₂O emissions reduction (Oram et al. 2020). A. donax has a high plant N uptake capacity that is 40% (Kouki et al. 2012) or 77% (Meng et al. 2015) higher than that of Typha latifolia, and its productivity is higher than that of 19 other species (Liu et al. 2012). The plant N uptake of A. donax was 2- to 23-fold higher than that of the other species in this study (Fig. 4b; Fig. S6). The allocation of A. donax in the community increased plant N uptake by 200% (Table S3) and indirectly reduced the N₂O emissions reflected by structural equation modeling (Fig. S5). In addition, A. donax has developed roots and a high oxygen secretion capacity, which is conducive to reducing denitrification and reducing N₂O (Lai et al. 2011; Kuypers et al. 2018). In this study, A. donax had the largest underground biomass production, and the presence of A. donax improved the DO concentration while reducing denitrification (Fig. S1; Table S3). An increase in DO may promote CH₄ oxidation (Bhullar et al. 2013), and the presence of A. donax reduced CH₄ emissions by 63% (Table 2; Table S3). In conclusion, A. donax is an excellent species for practical use since it has a high plant N uptake capacity and mitigates the emissions of two GHGs in the plant community.

It is worth noting that the total biomass production of a single plant of A. donax in mixtures was always higher than that in monoculture (Fig. S7, S8). This result means that A. donax can play a greater role in GHG emissions reduction in mixed communities than in monocultures. In fact, A. donax is also recommended as a high-yield biofuel production species to further reduce the GWP worldwide (Corno et al. 2014). Moreover, the presence of A. donax enhanced the TN mass removal by 8 mg m^− 2 day^− 1 in community (Fig. 4a; Table 2). Therefore, the combination containing A. donax is suitable for the wastewater treatment with a low C/N ratio to improve N removal while reducing GWP.

GHG emissions vary greatly among plant species, and monocultures increase the risk of high emissions. When the C/N ratio was 1, the N₂O emissions of the T. fluminensis monoculture were higher than those of other species in previous studies (1.3 ~ 293.8 mg m^− 2 day^− 1, Fig. S3, Table S2). In this study, NO₂⁻-N was positively correlated with N₂O emissions (Fig. 6a). The accumulation of NO₂⁻-N in this configuration (4.6 mg L^− 1, Fig. 6b) indicated incomplete denitrification so that N was released in the form of N₂O (Itokawa et al. 2001; Pan et al. 2013). Regarding CH₄ emissions, the monoculture of R. carnea yielded a high level in previous studies (-3.3 ~ 110.6 mg m^− 2 day^− 1, Table S2). However, no high emissions were observed in mixed communities containing these two species (Fig. 2b, c; Fig. 3b). Du et al. (2020) reported that species with a small biomass proportion in a mixture had little effect on community functioning, supporting the mass ratio hypothesis (Grime 1998). Similarly, in this study, the biomass proportions of T. fluminensis and R. carnea in mixtures only accounted for 8% and 3% on average, respectively (Fig. S7). Mixing plant species eliminated the disadvantage of high GHG emissions of T. fluminensis and R. carnea and was conducive to the ornamental value of the plant community (Hu and Gill 2015; Table S1). Therefore, although monocultures of T. fluminensis and R. carnea are not recommended for treating low-C/N ratio wastewater, they can be used in mixtures to ensure clean treatment.

Interestingly, A. donax combined with two high-GHG-emissions species (T. fluminensis and R. carnea) resulted in the most effective plant combination when treating a low-C/N wastewater (Fig. 2b, d). The plant species composition had a greater effect than plant species richness on GHG emissions reduction (Table 3; Table S6). This result is similar to the findings of Abalos et al. (2014) in grassland; that is, plant species composition explained a greater proportion of N₂O emissions. In this study, when the C/N ratio was 1, compared with the average GWP of monocultures, the mixture of T. fluminensis × R. carnea × A. donax reduced the GWP of the microcosm by 92% (Fig. 3b). The N₂O emissions of T. fluminensis × R. carnea × A. donax were 76% lower than those of the A. donax monoculture, leading to transgressive under-depletion of GWP in this community (Fig. 6c, d). Similarly, the NH₃ volatilization of Rumex japonicus × Cichorium intybus × Lolium perenne was found to be 60% lower than that in the monoculture with the lowest value (Luo et al. 2016). In general, allocating appropriate species combinations, rather than only improving richness, has a higher potential for GWP reduction.

Table 3

Results of two-way ANOVA of the effects of and interactions between plant species richness (SR) or species composition (SC) and C/N ratio on N₂O and CH₄ emissions (mg m^− 2 day^− 1), GWP (g CO₂ eq m^− 2 day^− 1), and GWP per g N removal (GWP/NR) (g CO₂-eq g N^− 1).
Source of effect	df	SS	MS	SS%	P
N₂O emissions
Block	5	3.60	0.72	0.95	0.308
C/N	1	274.75	274.75	72.12	< 0.001
SR	1	4.45	4.45	1.17	0.007
SC	7	22.48	3.21	5.90	< 0.001
C/N×SR	1	5.81	5.81	1.53	0.002
C/N×SC	7	19.55	2.79	5.13	< 0.001
CH₄ emissions
Block	5	2.30	0.46	3.04	0.312
C/N	1	2.26	2.26	2.99	0.017
SR	1	0.33	0.33	0.44	0.355
SC	7	29.64	4.23	39.21	< 0.001
C/N×SR	1	1.70	1.70	2.25	0.037
C/N×SC	7	7.03	1.00	9.30	0.016
GWP
Block	5	4.45	0.89	1.12	0.237
C/N	1	284.39	284.39	71.54	< 0.001
SR	1	3.59	3.59	0.90	0.020
SC	7	15.84	2.26	3.98	0.002
C/N×SR	1	7.80	7.80	1.96	< 0.001
C/N×SC	7	26.96	3.85	6.78	< 0.001
GWP/NR
Block	5	4.43	0.89	1.79	0.247
C/N	1	133.40	133.40	53.81	< 0.001
SR	1	3.60	3.60	1.45	0.021
SC	7	15.95	2.28	6.43	0.002
C/N×SR	1	7.89	7.89	3.18	< 0.001
C/N×SC	7	27.26	3.90	11.00	< 0.001

4.3 Manipulating plant combinations is a win-win measure compared to increasing the C/N ratio

Increasing the C/N ratio was found to reduce the N₂O emissions of monoculture CWs by 28 ~ 85% (Table S2). In this study, when the C/N ratio was increased from 1 to 5, N₂O emissions of monoculture CWs decreased by 1-fold in average (Fig. 2b). The N₂O emissions of each plant microcosm decreased to the same low level when the C/N ratio was 5 (0.28 mg m^− 2 day^− 1, Fig. 2b), and further increasing species richness no longer affected N₂O emissions under a high C/N ratio (Fig. 2a). However, in this study, the mass of N released in the form of N₂O from microcosms with a high C/N ratio was lower than 0.4%, which is at the low discharge level of CWs (0.001 ~ 4%, Table S2). Moreover, on the basis of reducing N₂O emissions, raising the C/N ratio further reduced the average CH₄ emissions of monocultures by 60% and finally reduced GWP by 98%. This effect is almost equivalent to the optimal emissions reduction obtained with plant combinations. The GWP per unit N removal is a suitable index to comprehensively measure the treatment performance of CWs (Du et al. 2020). The T. fluminensis × R. carnea × A. donax mixture reduced the GWP per unit N removal in the treatment of wastewater with a C/N ratio of 1 by 96%, and the value was as low as that of the monocultures with a C/N ratio of 5 (Fig. 3d). This mixture also provided a win-win combination of efficient N removal and low emissions under a low C/N ratio (Fig. 7a). The N removal efficiency of this combination was higher than 90%, which was higher than the average value of previous studies in CWs (55%, Table S4). These results showed that for the treatment of wastewater with a low C/N ratio, the configuration of an appropriate species combination has great potential and can be comparable to increasing the C/N ratio.

4.4 Plant diversity is a low-cost route to treat wastewater with a low C/N ratio in CWs

In the practical application of CWs, the trade-off between strengthening water treatment efficiency, reducing environmental impact, and increasing cost must be considered (Resende et al. 2019). The cost of wastewater treatment will rise when increasing the influent C/N ratio, which is generally achieved by adding external C sources (Sun et al. 2010). In wastewater treatment, the commonly used C sources include methanol, sucrose, glucose, and acetic acid, and their costs vary greatly (Table S5). Methanol is the cheapest (0.29 USD per m³ wastewater treated; Table S5) among the commonly used C sources; however, it is toxic (Ramírez et al. 2006). Sucrose is nontoxic and relatively economical (0.45 USD per m³ wastewater treated; Table S5). Under the same infrastructure, energy, and labor costs, when the C/N ratio was increased from 1 to 5 by adding C, the cost (seedlings, planting labor, and sucrose cost) per m³ wastewater treated reached 0.55 USD, while the cost (seedlings and planting labor) for assembling the best plant composition was only 0.15 USD per m³ wastewater treated (Table S5). This means that increasing plant diversity can save 73% of the cost compared with adding sucrose or save 62% of the cost compared with adding methanol (Table 4, Table S5). In addition, perennial plants can operate continuously in CWs (Table S1), and the harvested plant biomass can further effectively produce bioenergy (Liu et al. 2012; Tanaka et al. 2017). Therefore, assembling plant combinations can be more economical in the long terms. However, adding C requires continuous investment (Hussain et al. 2019). These findings showed that optimizing plant diversity will solve the problems, which raised by the low C/N ratio in wastewater, more economically than increasing C/N ratio.

Table 4

Cost analysis of increasing the C/N ratio or species diversity.
Cost (USD m^− 3 wastewater)	Increasing diversity		Data source
Construction
Infrastructure	104.00	104.00	Gu et al. 2016
Seedlings	0.08	0.06	Our data
Planting labor	0.07	0.04	Our data
Operation and maintenance
Energy and labor	0.01	0.01	Gu et al. 2016
Sucrose	0.00	0.45	Our data
Notes: The unit is US$ m^− 3 wastewater treated; the exchange rate from 1st November 2021 of 6.435 was used to convert US$ to RMB.

When treating wastewater with a C/N ratio as low as 1, both increasing the C/N ratio and assembling plant communities can make CWs efficient and green. Reasonable species combinations can mitigate the GWP of wastewater treatment by mitigating N₂O, and the reduction in GWP per g N removal achieved by this method is almost equivalent to that achieved by increasing the C/N ratio to 5. The excellent practical species A. donax has a high plant uptake ability, which provides an opportunity to improve efficiency while mitigating the environmental impact of treating wastewater with a low C/N ratio. Considering the economic costs of CWs, assembling optimal plant combinations, such as T. fluminensis × R. carnea × A. donax, is more cost-effective than increasing the C/N ratio. The biomass of high-yielding A. donax can be further considered to produce bioenergy to enhance the benefits of CWs. Future studies should expand the C/N ratio gradient, consider the time dynamics of GHG emissions, and explore species combinations suitable for different C/N ratios to optimize the treatment effect of CWs.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This work was funded by the National Natural Science Foundation of China (Grant No. 41901242).

Authors’ contributions

Jie Chang, Ying Ge, and Yuanyuan Du designed the experiments. Hang Jiang, Lichunxiao Wang, and Chenxu Xiang performed the experiments. Hang Jiang analyzed the data. Hang Jiang, Jie Chang, and Ying Ge wrote the first manuscript. Wenjuan Han and Yuanyuan Du provided editorial advice in the process of writing the article, and all authors provided insights throughout the completion of the manuscript.

Acknowledgements

We thank Yu Liu, Lei He, Guofu Yang, Bin Luo, Qian Wang, Xiao Wang, and Shaodan Niu for their assistance in the field and laboratory works.

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JiangHCNratioSIOK.docx

Assembling plant diversity mitigates greenhouse gas emissions and achieves high nitrogen removal when treating the low-C/N wastewater by constructed wetlands

Status:

Version 1

Abstract

Figures

1 Introduction

2 Methods

2.1 Experimental design

2.2 Sample collection and measurements

2.3 Calculations

2.4 Statistical analysis

3 Results

3.1 Effects of plant diversity on N₂O and CH₄ emissions under two C/N ratios

3.2 Effects of plant diversity on GWP under two C/N ratios

3.3 Effects of plant diversity on N removal and pathways under two C/N ratios

4 Discussion

4.1 Increasing plant richness alleviates GHG emissions under a low C/N ratio

4.3 Manipulating plant combinations is a win-win measure compared to increasing the C/N ratio

5 Conclusions

Declarations

References

Supplementary Files

Status:

Version 1

Assembling plant diversity mitigates greenhouse gas emissions and achieves high nitrogen removal when treating the low-C/N wastewater by constructed wetlands

Status:

Version 1

Abstract

Figures

1 Introduction

2 Methods

2.1 Experimental design

2.2 Sample collection and measurements

2.3 Calculations

2.4 Statistical analysis

3 Results

3.1 Effects of plant diversity on N2O and CH4 emissions under two C/N ratios

3.2 Effects of plant diversity on GWP under two C/N ratios

3.3 Effects of plant diversity on N removal and pathways under two C/N ratios

4 Discussion

4.1 Increasing plant richness alleviates GHG emissions under a low C/N ratio

4.3 Manipulating plant combinations is a win-win measure compared to increasing the C/N ratio

5 Conclusions

Declarations

References

Supplementary Files

Status:

Version 1

3.1 Effects of plant diversity on N₂O and CH₄ emissions under two C/N ratios