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Article

Responses of a Submerged Macrophyte Potamogeton crispus and Epiphytic Biofilm to Humic-Substance Enrichment Coupled with Brownification in Freshwater Habitats

by
Xiang Wan
1,
Guoxiang Wang
1,
Fei Yang
2,* and
Yueming Zhu
2,*
1
School of Environment, Nanjing Normal University, Nanjing 210023, China
2
Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment of the People’s Republic of China, Nanjing 210042, China
*
Authors to whom correspondence should be addressed.
Water 2023, 15(16), 2860; https://doi.org/10.3390/w15162860
Submission received: 28 June 2023 / Revised: 31 July 2023 / Accepted: 7 August 2023 / Published: 8 August 2023
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Abstract

:
Brownification denotes increasing water color, partly caused by increasing dissolved organic matter of terrestrial origin in freshwater. Brownification has become a wide-spread environmental problem because water color alters the physicochemical environment and biological communities in aquatic ecosystems. However, our understanding of its ecological effects on aquatic macrophytes is limited. Here, an indoor mesocosm experiment with a common submerged macrophyte, Potamogeton crispus, along an increasing gradient of brownification was conducted over a period of 42 days. Results showed that P. crispus was able to overcome low degrees of brownification owing to the plasticity in morphological and physiological traits and P. crispus growth even benefitted from the concomitant nutrients along with brownification. However, the macrophyte growth was negatively affected by a 10-fold increase in water color beyond its current level. Additionally, collapse in antioxidant systems and potent photosynthesis inhibition implied that P. crispus could not adapt to the low-light stress generated under the high degree of brownification. Epiphytic bacteria are more sensitive to brownification than their hosts. Any degree of brownification initially caused a decrease in microbial diversity of epiphytic biofilm, whereafter the concomitant nutrients under brownification favored the growth of epiphytic microorganisms. The shading effect of a large number of epiphytic biofilms under brownification may further aggravate the low-light stress on macrophytes. Overall, the study provides new insights into the comprehensive effects and underlying mechanisms of brownification on macrophytes.

1. Introduction

Submerged macrophytes, as primary producers, play an indispensable role in maintaining the structure and function of aquatic ecosystems [1]. Submerged macrophytes provide food for small aquatic animals and serve as a habitat for periphyton [2]. Moreover, macrophyte species compete with other primary producers (e.g., phytoplankton) and further inhibit the massive proliferation of algae, thereby improving water clarity [3,4]. The growth of submerged macrophytes can be limited by many environmental factors, and there have been many studies on the mutual effects of submerged macrophytes and their ambient environments [5,6,7,8]. Among the many influencing factors, light availability is considered to be a key driving factor for the abundance and distribution patterns of submerged macrophytes in shallow lakes [9,10]. Previous studies have reported that the light limitation resulting from climate change and human impact has caused the decline of submerged macrophytes in global lakes [11]. Extreme climate events such as heavy precipitation events and human activities (e.g., dam construction) cause increases in the water level of shallow lakes, which in turn make the underwater light conditions more and more unfavorable for the growth of submerged macrophytes [12,13]. Additionally, eutrophication leads to the occurrence of severe algae blooms in global lakes, thus reducing the water transparency and leading to the rapid decline of submerged macrophytes [10,11].
Over recent decades, an increase in dissolved organic matter (DOM) of terrestrial origin reaching inland waters has been reported throughout the Northern Hemisphere, which has led to the increasing water color, known as “brownification” [14,15]. Brownification of aquatic ecosystems is expected to intensify further in the future due to global warming [16], increased precipitation [17] and land-utilization changes [18]. Brownification has complex consequences on underwater light conditions in aquatic ecosystems. On the one hand, brownification can attenuate UV-B radiation, which endangers macrophyte growth [19]. On the other hand, brownification hinders light penetration into the water column and reduces the depth of the euphotic zone in lakes [20]. Previous studies have reported that phytoplankton and macrophytes that are sensitive to light conditions were severely influenced by brownification [21,22,23]. Generally, brownification and lake primary productivity have a unimodal relationship based on the response of these aquatic biota to brownification [24]. The biological productivity of aquatic ecosystems will initially benefit from brownification through the attenuation of UV-B penetration, CO2 provision and the binding of harmful substances [24,25]. However, the subsequent brownification will reduce primary producers [19,26]. In all, the effects of brownification on aquatic ecosystems are very complex, and our understanding of those effects is fairly scarce. Presently, one large knowledge gap is the link between increasing terrestrial DOM and aquatic macrophytes [19]. In addition, macrophytes and their epiphytic biofilm constitute a highly interactive unit with the provision of the substrate, and competition for resources (e.g., light and nutrients), and thus these interactions have broad implications for ecosystem structure, function and integrity [27]. However, how the interaction between epiphytic biofilm and their host plants responds to brownification in freshwaters remains poorly characterized. Therefore, more research on the effects of brownification on aquatic ecosystems is needed.
In this study, Potamogeton crispus was selected as a model submerged plant, which has a widespread distribution in most shallow freshwater ecosystems [28]. P. crispus is the dominant species in winter and spring since most aquatic plants decay during this period, and thus the clear state of water bodies is mainly dependent on the growth of this aquatic plant [29] However, in recent decades, P. crispus has been reported to rapidly grow, spread and cover entire lakes, causing a negative impact on water environments in summer when large amounts of plant debris decomposed [28,30]. Given the two-sided effects of P. crispus towards aquatic environments, the response of the aquatic plant to the changing environment deserves more research. The main objective of this study was to examine the effects of humic-substance addition and concomitant increase in water color on the submerged macrophyte P. crispus. We hypothesized that brownification and P. crispus would also exhibit a unimodal relationship because of the impact of humic substances on light availability. The focus mainly included three aspects: (1) investigation of the effects of terrestrial humic substance import on lake-water quality and water color using mesocosms; (2) examination of the growth and physiological and photosynthetic responses of submerged macrophytes to different degrees of brownification by a gradient of humic substance addition; and (3) exploration of the response of epiphytic biofilm microbial communities to brownification. The findings of this work will provide new insights into the comprehensive ecological effects and underlying mechanisms of brownification on macrophytes.

2. Materials and Methods

2.1. Experimental Materials

P. crispus turions, lake water and sediments were all collected from an urban lake in Jiangsu province, China (119°51′15.34″ N, 31°40′4.51″ E) in November 2018. These turions were washed with tap water and then pre-cultured in tanks containing lake water. During the pre-culture, the medium was refreshed every two weeks. The next spring, healthy and uniform apical-shoot segments, with a length of approximately 15 cm, were chosen for the experiment.

2.2. Indoor Mesocosms

The experiment was conducted in a greenhouse. The indoor mesocosms were set up using twelve tanks (height: 70 cm, upper diameter: 56 cm, bottom diameter: 44 cm). Each tank was filled with 60 cm of lake water (dissolved total nitrogen (DTN): 2.53 mg/L, dissolved total phosphorus (DTP): 0.24 mg/L, dissolved organic carbon (DOC): 3.85 mg/L, pH: 8.2). Brownification treatments were achieved by adding a humic substance (purity ≥ 90%) purchased from Aladdin (CAS. No.: 1415-93-6, Shanghai, China), which is composed of approximately 43% organic carbon. In this experiment, to create a brownification gradient, the purchased humic substance was added at three levels: low concentration with 10 mg/L (LH) of the humic substance, medium concentration with 20 mg/L (MH) and high concentration with 40 mg/L (HH), all in triplicate. In addition, three mesocosms without addition of the humic substance were considered as controls (CK). In this study, DOC ranged from 3.61 to 14.52 mg/L in all treatments. In general, DOC in most lakes is less than 10 mg/L, whereas it can reach up to 20 mg/L in forested lakes of the Northern Hemisphere. Thus, the DOC concentration in this experiment after the addition of different concentrations of the humic substance was within the range of that reported in boreal lakes [22,31]. After the addition of the humic substance, all the mesocosms were mixed well and left to stand for one week. P. crispus shoots were planted in a pot (diameter: 15 cm, height: 10 cm) that was filled with a kilogram of sediment (TN: 20.52 g/kg, TP: 2.07 g/kg, total organic carbon: 0.58 g/kg). Three pots with nine plant shoots each were suspended in every tank with ropes, and the distance between the upper edge of the pot and the water surface was 30 cm. In this experiment, the filtered surface lake water and sediment were mixed well to ensure homogeneity before use. The experiment was performed between 10 April and 21 May in 2019, and lasted for a total of 42 days. During the experiment, the average water temperature and pH among different treatments were 22.4~22.6 °C and 8.20~8.49, respectively (Table 1). The daylength during the culture period was about 14 h. Photosynethically available radiation in various treatments was from 1.53 to 4.67 MJ/m2. Ultrapure water was added to make up for evaporation losses. Plant and water samples (at a depth of 20 cm) in different treatment groups were collected weekly for further analysis.

2.3. Determination of Plant Growth and Root Vigor

Once a week, we lifted the pot and harvested three P. crispus plants from each tank (an individual P. crispus per pot) to measure morphological and physiological traits, including plant height, fresh weight and root vigor. Plant height was the total length of the main shoot and root. Plant fresh weight was measured by a Sartorius Analytic A210P balance (precision: 0.0001 g) after removing the surface water with filter paper. The relative growth rate (RGR) of P. crispus was calculated with the following equation [25]:
RGR = (ln W2 − ln W1)/Δt
where W1 and W2 are the initial and total fresh weight, respectively, and Δt represents the total number of days. Additionally, root vigor was detected by the triphenyltetrazole chloride (TTC) method. Dehydrogenase activity can be used as an important root vigor indicator, and the dehydrogenase activity was defined as the reduction in TTC content at 485 nm wavelength [32].

2.4. Chlorophyll Fluorescence Parameters

We randomly selected mature leaves at the same morphological positions and used a portable chlorophyll fluorometer with WinControl−3 software (Diving-Pam, Walz, Effeltrich, Germany) to measure chlorophyll fluorescence [33]. Before measurement, the leaves from different treatments were kept in darkness for 15 min. The maximum quantum yield of PSII (Fv/Fm) was measured and recorded with WinControl software. In addition, the rapid light curves (RLCs) were run with Diving-Pam for evaluating the photosynthetic status of macrophytes. The paired data from RLCs running electron transport rate (ETR) as a function of photosynthetic active radiation (PAR) were fitted based on the empirical equation documented by Platt et al. [34]. The fitting parameters, including the initial slope of RCLs (α), the maximum relative electron transport rate (rETRmax) and the minimum saturating irradiance (Ek), were calculated using the equations reported by Ralph and Gademann [35]. The equations for chlorophyll fluorescence parameters calculating and rapid light curves (RLCs) fitting are as follows:
F v / F m = ( F m − F 0 ) / F m
Y = ( F m ′ − F t ) / F m ′
r E T R = Y × P A R × 0.5 × 0.84
r E T R = r E T R m ( 1 − e − α × P A R / r E T R m ) × e − β × P A R / r E T R m
E k = r E T R m / α

2.5. Determination of Water Quality Parameters and Color

Water temperature (WT) and pH were measured in situ using a YSI 6600 V2 multi-sensor sonde (Yellow Springs Instruments Inc., Yellow Springs, OH, USA). DTN and DTP were measured weekly using filtered water samples after potassium persulfate digestion at 121 °C for 30 min. Dissolved organic carbon (DOC) analysis was carried out with a total organic carbon (TOC) analyzer (multi N/C 3100 TOC, Germany). To quantify the water color, the absorbance of the filtrated water through GF/C (Whatman, Maidstone, UK) filters was measured at 420 nm using a spectrophotometer (Shimadzu, Kyoto, Japan).

2.6. Analysis of Biochemical Indicators

Fresh leaves were homogenized in potassium phosphate buffer solution (100 mM, pH 7.0) on ice to prepare a crude enzyme solution. The homogenates were centrifuged at 10,000× g for 15 min at 4 °C, and the supernatant obtained was used for superoxide dismutase (SOD) activity and malondialdehyde (MDA) content analysis. The experimental procedures were according to the instructions of commercial test kits designed by Nanjing Jiancheng Bioengineering Institute (Nanjing, China), including the Total Superoxide Dismutase Assay Kit (hydroxylamine method) and MDA test kit (thiobarbituric substance method).

2.7. Epiphytic Biofilm Microbial Community

The extraction of epiphytic biofilm was according to Zhang et al. [36]. Leaf samples of P. crispus were gathered together from three biological replicates. At the start of the experiment and on days 21 and 42, one gram of fresh leaf samples was accurately weighed and added into 30 mL of potassium phosphate buffer solution (100 mM, pH 7.0). The mixture was ultrasonicated for 1 min (40 KHz) and then centrifuged at 10,000× g for 5 min at 4 °C. Sediments were collected and stored at −80 °C for further DNA extraction. DNA extraction and high-throughput sequencing was performed by Majorbio BioPharm Technology Co., Ltd. (Shanghai, China). In this study, the universal barcoded primer set (338F, 5′-ACTCCTACGGGAGGCAGCAG-3′; 806R, 5′-GGACTACHVGGGTWTCTAAT-3′) was used for the amplification of 16S rRNA genes. After high-throughput sequencing on the Illumina MiSeq PE300 platform, a bioinformatics analysis was used to describe the microbial diversity and community structure [37].

2.8. Statistical Analyses

SPSS 22.0 software (IBM Corp., Chicago, IL, USA) was used to test the difference between different treatments, using a one-way analysis of variance (ANOVA) with Duncan’s test. Before ANOVA, homogeneity of variance among groups was tested by Levene’s test. When necessary, data were transformed for normalization to reduce heterogeneity of variances. The difference was statistically significant when p value was less than 0.05. The Ciros figure and heatmap were completed by R 4.1.3, and other graphs were performed using the OriginPro 2023 software (OriginLab, Northampton, MA, USA).

3. Results and Discussion

3.1. Water Quality and Color

In this study, the humic substance addition had measurable effects on the water quality. As shown in Table 1, compared to CK, no significant difference in DTP concentration was observed in humic treatments, whereas the DTN concentration significantly increased (p < 0.05) in all treatments with additional humic substances. Similar to our finding, Zheng et al. [38] reported the added humic substances ranging from 10 mg/L to 20 mg/L caused a pronounced increase in TN content instead of TP content in overlying water, which was attributed to a considerable percentage of organic nitrogen contained in the humic substance. Compared with the changes in nutrients, humic substance significantly altered the DOC concentration and water color in lake water (p < 0.05). The average water colors in LH, MH and HH were 3, 6 and 10 times, respectively, larger than that in CK. Moreover, compared to CK, the mean DOC concentration increased by approximately 90%, 170% and 300% in LH, MH and HH, respectively. In our study, the mean A420 in LH treatment was 0.043 ± 0.004, which was fairly close to the water color level reported in boreal forest lakes [22,31]. From the perspective of this mesocosm experiment, the increased input of exogenous DOM-like humic substance may lead to brownification coupled with eutrophication in freshwater ecosystems.

3.2. P. crispus Growth Response to Humic Substance Addition

As shown in Figure 1, brownification significantly affected the growth of P. crispus (p < 0.05). During the whole period, the plant height and biomass of P. crispus in all treatments (except HH) increased linearly, while plants of HH stopped growing after day 35 (Figure 1A,B). In comparison to CK, growth of P. crispus in LH and MH was slightly inhibited at the early stage of culturing (before the 7th day), while macrophytes recovered soon and grew faster than those of CK with the increasing culturing time (Figure 1, Table 2). On the contrary, the RGR results indicated that a high degree of brownification severely inhibited the macrophyte growth of HH throughout the experiment. However, smaller RGR values in late stages of all treatments indicated that nutrient depletion may also affect plant growth in the late stages of the experiment.
The root is the primary organ by which submerged macrophytes absorb nutrient and mineral elements, and root activity is a physiological indicator that can reflect the life activities of the plant root [32]. Overall, a similar trend to that of plant height was observed for the root weight and vigor of P. crispus with increasing culturing time (Figure 1C,D). Compared with CK, the mean peak root fresh weight on day 35 in LH and MH increased by 26.8% and 52.4%, respectively. However, the peak root weight of the HH treatment group was significantly lower than that of the control (63.4% that of the control, p < 0.05). Root vigor of the plants in the LH and MH treatment groups increased rapidly and remained at a high level with increasing culturing time when compared to CK. In contrast, the root vigor in the HH treatment was significantly inhibited throughout the experiment. Therefore, a high degree of brownification severely inhibited root development and activity, which might have interfered with the nutrient absorption and organic matter synthesis processes of submerged macrophytes.
Our results suggest that submerged macrophytes in freshwater will initially benefit from humic substance input coupled with brownification, partly ascribed to the increased concomitant nutrients (i.e., carbon and nitrogen) and attenuation of UV-B radiation, which harms macrophyte growth [19,38]. Similar to this study, Choudhury et al. [25] reported that charophytes benefited from brownification until a 2-fold increase in brownification above today’s levels (A420 ≈ 0.008 in their research). By contrast, P. crispus appeared to have a solid adaptation to the increased color because, in this study, it could grow normally until a 10-fold increase in water color of CK (A420 = 0.011). Therefore, slight brownification might further stimulate P. crispus growth in freshwater, aggravating the adverse effects of this plant on water environments in summer when large amounts of plant debris decompose. Overall, the adverse effects of brownification on submerged macrophytes had a threshold that differed greatly among various macrophytes. Brownification will reduce the primary productivity and potentially promote the selection of a few macrophytes that are adapted to increased color in freshwater, causing regime shifts in the macrophyte communities. Moreover, a previous study has reported that water brownification might help an invasive submerged macrophyte, overwhelming the native one [39].

3.3. P. crispus Chlorophyll Fluorescence Response to Humic Substance Addition

Fv/Fm is a valuable parameter that reflects the photosynthetic response of plants to environmental changes, and a higher Fv/Fm value indicates a higher light conversion efficiency of plants [32]. Figure 2A shows that the Fv/Fm of plants in all humic treatments decreased at the early stage when compared with CK, which indirectly suggested the decreased underwater light availability owing to the increased water color. In comparison to CK, Fv/Fm of LH and MH on day 7 decreased slightly by 3.9% and 8.8%, respectively. With the increasing culturing time, Fv/Fm in LH and MH gradually recovered to the level of CK, which implied that submerged macrophytes had overcome low light availability caused by a relatively low degree of brownification. Previous studies have reported submerged macrophytes can modify their morphological and physiological traits, such as extending stems towards the water surface and a larger leaf area, to make themselves adaptive to a decreased underwater light intensity [6,7,25]. Nevertheless, Fv/Fm of macrophytes in HH treatment significantly decreased (t-test, p < 0.001) compared with CK during the whole experiment, demonstrating a consistent inhibition in photosynthesis due to the high degree of brownification. The RLCs differed greatly among different treatment groups (Figure 2B), and the fitted parameters of RLCs (i.e., α, rETRmax and Ek) are provided in Table 3. The fitted parameters of RLCs all presented a similar pattern of increasing in LH and MH, but decreasing in HH compared with CK. Overall, our results further confirmed that the adverse effect of brownification on submerged macrophyte photosynthesis has a threshold based on chlorophyll fluorescence response. Additionally, the intra- and inter-species competition relative to plant canopy structure could also aggravate the light limitations under brownification.

3.4. Antioxidant System Response to Humic Substance Addition

Many exogenous pollutants and environmental changes can stimulate plants to produce excessive reactive oxygen species (ROS), which results in severe oxidative damage to plants [40]. Antioxidant mechanisms in plants help them to defend against ROS, and SOD is the first defence line of the antioxidant system [41]. Figure 3A shows that SOD activity in P. crispus increased in a concentration-dependent manner with the addition of a gradient of humic substance before day 28. However, SOD activity of plants in HH was significantly lower than that of CK, which implied a collapse of the antioxidant system of the plants at the end of the experiment. MDA, derived from the reaction of excessive ROS and polyunsaturated fatty substances, can be used as an indicator to judge the degree of oxidative damage [42]. As can be seen from Figure 3B, there was no significant difference in MDA content among varying treatments except for HH. In contrast, the MDA concentration of HH was several times larger than that in other treatments. A previous study also showed that the decreased light availability in deep waters induced an imbalance of antioxidant systems in functional organs of submerged macrophytes [6]. The promoted enzyme activity could help scavenging ROS generated under stress conditions, thereby avoiding or reducing oxidative damage [43]. By contrast, the significant decrease in antioxidant enzyme activity and the greatest accumulation of MDA of HH implied that P. crispus could not adapt to the low-light stress generated under the high degree of brownification.

3.5. Microbial Community Diversity and Composition in Epiphytic Biofilm

3.5.1. Analysis of Microbial Diversity

To the best of our knowledge, this is the first attempt to explore the response of the epiphytic biofilm of macrophytes under brownification. In order to investigate the microbial diversity of leaf epiphytic biofilm, α-diversity analysis was conducted (Table 4). The Chao and Shannon indices can reflect the richness and diversity of epiphytic biofilm, respectively [36]. Higher Chao and Shannon indices reflect higher microbial community richness and diversity. In this study, our results showed that the Chao and Shannon indices of CK were lower than those of the humic treatments at the beginning of the experiment, which indicated that humic substance addition initially reduced the microbial community richness and diversity. Similarly, Zheng et al. [38] recently conducted a mesocosm experiment to investigate the diversity of microbial communities in freshwater environments under humic-induced brownification, and results showed that the richness and diversity of the microbial communities in the overlying water decreased as the degree of brownification increased. Previous studies have reported that the structural characteristics of macrophytes directly influenced the epiphytic biofilm structure [27]. However, the structural features of macrophytes at the beginning of our experiment were almost the same. Thus, we speculated that the effect of brownification on microbial diversity was not due to this reason. To a certain extent, the increases in DOC concentration, resulting from brownification might serve as an additional resource for macrophyte growth [44]. However, the existence of excessive DOC would harm aquatic plants when circumstances become extreme because DOM leads water to turn brown and further weakens light penetration into the water column, causing a decrease in the abundance and diversity of these photosynthetic microorganisms and aquatic organisms at deeper depths [19]. In this study, the decreased microbial community diversity at the start of the experiment was observed for all degrees of brownification, indicating that epiphytic biofilm on the macrophytes was more sensitive to increased water color than their morphological traits. The shading effect of epiphytic biofilm has been previously reported to be a primary reason for the dramatically reducing submerged vegetation in light-limited environments [45]. In this study, the Chao and Shannon indices of brownification treatments were even higher than those of CK at the late stage, demonstrating that the concomitant nutrients under brownification favored the growth of epiphytic microorganisms. Therefore, the existence of a large number of epiphytic biofilms in a high degree of brownification may further reduce the light availability to submerged macrophytes, thereby resulting in a potent inhibition in photosynthesis.

3.5.2. Taxonomic Distribution of Bacteria at the Phylum and Genus Levels

Microbial community composition at the phylum level is exhibited in Figure 4A, and the results indicate that the microbial community composition in the epiphytic biofilm was largely affected by humic substance addition coupled with brownification. The top five phyla with the highest relative abundance in biofilm were Firmicutes (9.1–71.4%), Proteobacteria (6.3–31.0%), Chloroflexi (10.4–36.9%), Acidobacteriota (2.3–12.6%) and Actinobacteriota (3.3–14.7%). The presence of these primary phyla has been previously reported in submerged macrophyte biofilms [36,37]. The stability of a biofilm community is determined by the dominant microbial species on the biofilm. According to Figure 4A, at the beginning of the experiment, the dominant phyla in the initial lake water differed greatly from those in humic treatments. Humic substance addition sharply promoted the domination of Firmicutes, and its relative abundance ranged from 55.3% to 71.4% among varying humic treatments. The sharp increase in Firmicutes may have been primarily derived from the addition of humic substances. As the culturing time increased, the relative abundance of Firmicutes decreased (16.1–27.0%), but Proteobacteria clearly increased (26.3–31.0%), which may be due to the ability of Proteobacteria to degrade organic macromolecules in humic treatments [46]. Additionally, the sum of the relative abundance of the top five phyla decreased with increasing time, while the relative abundance of other phyla (e.g., Bacteroidetes and Ignavibacteriae) increased. However, the microbial community composition among different concentrations of humic substance addition was small.
The microbial composition was also analyzed at genus level, with blue representing low abundance and red representing high abundance (Figure 4B). Overall, the microbial composition at genus level was primarily influenced by the culturing time, followed by the humic substance treatment. At the early stage, humic-substance addition led to an increase in the relative abundance of some genera, such as Bacillus, Pullulanibacillus and Tumebacillus, while some bacteria, like Anaerolinea, decreased. At the late stage, Pullulanibacillus and Tumebacillus decreased, whereas many bacteria, including Substanceovorax, Methylotenera and Geothrix increased. Epiphytic biofilm plays an active role in the functional processes of freshwater ecosystems [27]. For example, brownification has been reported to promote nitrogen-cycling-microorganism growth in freshwater [38]. However, the response of other functional microorganisms under brownification remains poorly understood.

4. Conclusions

Multiple drivers may have already caused excessive input of terrestrial DOM, including humic substances, into aquatic systems. In this study, we comprehensively investigated the response of submerged macrophytes to humic substance enrichment coupled with brownification (Figure 5). The results showed that submerged macrophytes would initially benefit from low degrees of brownification in freshwaters because more concomitant nutrient availability and plant plasticity helps macrophytes to overcome light limitations. However, further brownification will cause P. crispus to decline when crossing certain threshold levels for light limitations, which may cause regime shifts in the macrophyte communities. Although the extrapolation of the experimental results to natural freshwaters has its limitations, the present study underlined the importance of restricting the import of terrestrial DOM into lakes. In future, passive remote-sensing monitoring should be developed to distinguish the factors responsible for increases in DOM within aquatic ecosystems at the catchment scale, thereby improving lake brownification management strategies. For example, riparian zones managing and increasing the hydraulic retention time can effectively reduce the terrestrial export to aquatic ecosystems. Moreover, epiphytic bacteria are more sensitive to brownification than their hosts, and more research is needed to focus on the changes in functional microorganisms under brownification, which will provide new insights into aquatic ecosystem responses to large-scale environmental changes.

Author Contributions

X.W., conceptualization, investigation and writing—original draft; Y.Z., conceptualization and review and editing; G.W., conceptualization, funding acquisition, review and editing and supervision; F.Y., funding acquisition and review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was jointly supported by the National Natural Science Foundation of China (grant number 41971043), national water pollution control and management technology major projects (grant number 2017ZX07202006) and the innovative team project of Nanjing Institute of Environmental Sciences, MEE (GYZX200101).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Water 15 02860 g001
Figure 1. Plant height (A), fresh weight (B), root fresh weight (C) and root vigor (D) of Potamogeton crispus under different brownification treatments over a period of 42 days.
Figure 1. Plant height (A), fresh weight (B), root fresh weight (C) and root vigor (D) of Potamogeton crispus under different brownification treatments over a period of 42 days.
Water 15 02860 g001
Water 15 02860 g002
Figure 2. The maximum quantum yield of PSII (Fv/Fm) (A) and rapid light curve (RLC) fitting (B) of Potamogeton crispus under different brownification treatments over a period of 42 days.
Figure 2. The maximum quantum yield of PSII (Fv/Fm) (A) and rapid light curve (RLC) fitting (B) of Potamogeton crispus under different brownification treatments over a period of 42 days.
Water 15 02860 g002
Water 15 02860 g003
Figure 3. Superoxide dismutase (SOD) activity (A) and malondialdehyde (MDA) content (B) of Potamogeton crispus under different brownification treatments over a period of 42 days.
Figure 3. Superoxide dismutase (SOD) activity (A) and malondialdehyde (MDA) content (B) of Potamogeton crispus under different brownification treatments over a period of 42 days.
Water 15 02860 g003
Water 15 02860 g004
Figure 4. High−throughput sequencing analysis of microbial community composition under different brownification treatments over a period of 42 days. The Circos figure (A) reflects the composition proportion and distribution proportion of dominant phyla among different groups. The heatmap (B) presents the composition proportion among different groups at genus level.
Figure 4. High−throughput sequencing analysis of microbial community composition under different brownification treatments over a period of 42 days. The Circos figure (A) reflects the composition proportion and distribution proportion of dominant phyla among different groups. The heatmap (B) presents the composition proportion among different groups at genus level.
Water 15 02860 g004
Water 15 02860 g005
Figure 5. A diagram reflecting the comprehensive effects of different degrees of brownification on a submerged macrophyte Potamogeton crispus in idealistic scenarios where no adjacent macrophytes exist.
Figure 5. A diagram reflecting the comprehensive effects of different degrees of brownification on a submerged macrophyte Potamogeton crispus in idealistic scenarios where no adjacent macrophytes exist.
Water 15 02860 g005
Table 1. Mean values (±S.D.) of water quality parameters and water color for the indoor mesocosm.
Table 1. Mean values (±S.D.) of water quality parameters and water color for the indoor mesocosm.
ParametersCKLHMHHH
WT (° C)22.6 ± 1.7 a22.6 ± 1.6 a22.5 ± 1.6 a22.4 ± 1.2 a
pH8.20 ± 0.07 a8.22 ± 0.08 a8.35 ± 0.13 a8.49 ± 0.19 a
DTN (mg/L)2.05 ± 0.16 b2.62 ± 0.26 ab2.99 ± 0.38 a3.42 ± 0.64 a
DTP (mg/L)0.19 ± 0.04 a0.18 ± 0.03 a0.17 ± 0.02 a0.22 ± 0.04 a
DOC (mg/L)3.61 ± 0.12 d6.82 ± 0.25 c9.65 ± 0.52 b14.52 ± 0.76 a
A420 (cm−1)0.011 ± 0.001 d0.043 ± 0.004 c0.079 ± 0.009 b0.121 ± 0.016 a
Note: A420: absorbance at 420 nm wavelength. Different letters represent statistically significant differences (p < 0.05) among treatments for this parameter.
Table 2. The relative growth rate (mg/d) of P. crispus under different treatments and over different time periods.
Table 2. The relative growth rate (mg/d) of P. crispus under different treatments and over different time periods.
TimeCKLHMHHH
0~14 d31.4 ± 6.7 a21.5 ± 7.6 ab25.4 ± 6.4 ab14.3 ± 1.0 b
15~28 d30.3 ± 1.3 a32.9 ± 8.5 a42.9 ± 12.8 a3.3 ± 9.5 b
29~42 d23.6 ± 4.2 a25.4 ± 7.8 a38.3 ± 6.2 a−19.4 ± 7.1 b
Total28.4 ± 1.4 b29.0 ± 1.2 b33.3 ± 1.6 a14.1 ± 0.2 c
Note: Different letters represent statistically significant differences (p < 0.05) among treatments in different time periods.
Table 3. Parameter values of rapid light curve (RLC) fitting.
Table 3. Parameter values of rapid light curve (RLC) fitting.
GroupsrETRm
μmol/(m2·s)		αEk
μmol/(m2·s)
CK15.22 ± 1.35 b0.22 ± 0.02 b70.24 ± 2.61 b
LH21.03 ± 1.74 a0.26 ± 0.03 b86.23 ± 2.01 a
MH24.82 ± 2.21 a0.33 ± 0.02 a90.18 ± 1.94 a
HH 6.58 ± 0.88 c0.08 ± 0.01 c48.29 ± 1.01 c
Note: Different letters represent statistically significant differences (p < 0.05) among treatments for this parameter.
Table 4. Microbial diversity of epiphytic biofilm under different brownification treatments.
Table 4. Microbial diversity of epiphytic biofilm under different brownification treatments.
SamplesChaoShannon
CK-0d1730.66.22
LH-0d1600.73.17
MH-0d1696.34.03
HH-0d1498.02.95
CK-21d1874.55.67
LH-21d1710.55.58
MH-21d1765.65.67
HH-21d1696.75.94
CK-42d1763.55.60
LH-42d1931.25.86
MH-42d1727.85.71
HH-42d1765.15.83
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Wan, X.; Wang, G.; Yang, F.; Zhu, Y. Responses of a Submerged Macrophyte Potamogeton crispus and Epiphytic Biofilm to Humic-Substance Enrichment Coupled with Brownification in Freshwater Habitats. Water 2023, 15, 2860. https://doi.org/10.3390/w15162860

AMA Style

Wan X, Wang G, Yang F, Zhu Y. Responses of a Submerged Macrophyte Potamogeton crispus and Epiphytic Biofilm to Humic-Substance Enrichment Coupled with Brownification in Freshwater Habitats. Water. 2023; 15(16):2860. https://doi.org/10.3390/w15162860

Chicago/Turabian Style

Wan, Xiang, Guoxiang Wang, Fei Yang, and Yueming Zhu. 2023. "Responses of a Submerged Macrophyte Potamogeton crispus and Epiphytic Biofilm to Humic-Substance Enrichment Coupled with Brownification in Freshwater Habitats" Water 15, no. 16: 2860. https://doi.org/10.3390/w15162860

APA Style

Wan, X., Wang, G., Yang, F., & Zhu, Y. (2023). Responses of a Submerged Macrophyte Potamogeton crispus and Epiphytic Biofilm to Humic-Substance Enrichment Coupled with Brownification in Freshwater Habitats. Water, 15(16), 2860. https://doi.org/10.3390/w15162860

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