Bioturbation Effects of Chironomid Larvae on Nitrogen Release and Ammonia-Oxidizing Bacteria Abundance in Sediments

The purpose of this work was to reveal the Chironomid larvae bioturbation impact on N release and to find the mechanism of bioturbation to N conversion at the SWI (sediment–water interface). Sampling at four points during a 35-day incubation experiment was conducted. Two in situ techniques (microelectrode and Peeper) were used to capture more realistic and accurate microenvironment information around U-shaped corridors. The results demonstrate that the concentrations of ammonia nitrogen (NH4) and nitrate nitrogen (NO3) decreased by 21.26% and 19.50% in sediment and increased by 8.65% and 49.82% in the overlying water compared to the control treatment, respectively. An inverse relationship was observed between NH4 and NO3 concentrations in pore water in Chironomid larvae treatment, and they were significantly negatively/positively correlated with AOB (ammonia-oxidizing bacteria) abundance, respectively. This study confirmed that the Chironomid larvae bioturbation promoted the N (NH4 and NO3) release from sediment by in situ techniques, and a part of NH4 converted into NO3 during their flow into the overlying water through the nitrification affected by AOB. Furthermore, the main depth of bioturbation influence is approximately 12 cm below the SWI and the most significant bioturbation effect was observed from days 15 to 25.


Introduction
Nitrogen (N) is an essential nutrient for aquatic plants and algae in freshwater ecosystems, and its significant role in water eutrophication has also attracted great attention over the last several decades [1].Some of the N taken up by aquatic plants can result from the ammonia nitrogen (NH 4 + ) release of the sediment, which is also called an internal load of N Sediment can contribute a substantial amount of N to the water column and support algal blooms, particularly after a reduction of the external N input in lakes [2].There is a range of environmental factors influencing the release of N, such as temperature, pH, Eh, and faunal hydro dynamics [3][4][5].However, a significant regulating factor-bioturbation-is almost neglected in freshwater.
According to the research of Lewandowski and Hupfer (2005) [6], bioturbation in aquatic environment is generated by the activity of animals within the upper sediment layers.The activity can alter the structure and properties of the sediment, which further influences the release of N from sediments and the flux of N to the overlying water [7][8][9].The bioturbation of benthic animals changes dissolved oxygen (DO), organic matter, redox conditions and microbial activity at the SWI [10,11].Hence, these changes likely influence N content and distribution in sediments and make the migration and conversion of N species more complicated in sediments.As a typical benthonic animal, Chironomid larvae have received extensive attention and research.Chironomid larvae can enhance the outflow of NH 4 + from the sediment to the overlying water across the sediment-water interface [12][13][14].While others show the opposite effect, Chironomid larvae increased the nitrate nitrogen (NO 3 − ) release rates [15].Moreover, exchange of inorganic nitrogen was slightly influenced by added Chironomid larvae [16].Nitrification and denitrification are important processes of N species conversion.In these studies, the results indicate that bioturbation can have a great impact on denitrification, and very low nitrification can occur [15,17].However, another study shows bioturbation related to ammonia oxidation, which is generally considered to be the critical rate-limiting step of the nitrification process [18].Since different phenomena behind the larvae bioturbation on N release have been observed, the apparently contradictory results may illustrate the relative importance of capturing more realistic and accurate information about the sediment microenvironment.Study of the N release process in sediments should be conducted at the real environmental status holding constant to monitor the spatial variations of N and the influencing factors (especially DO and Eh).However, in order to obtain the relevant information, previous studies have often used the invasive ex-situ sampling technique.This method will destroy the structure of the sediment layer, inevitably exposing the sediments to the air and will easily change the original properties of the samples, which results in a considerable inaccuracy of the N content [7].Peeper technology has provided promising alternatives to overcome this shortage with a slight disturbance of the sediment [19].It is an in situ technique able enough to satisfy the requirement of studying the Chironomid larvae bioturbation effects and can obtain more accurate and authentic N information from the sediment.In order to determine the environmental conditions around U-shaped corridors, the microelectrode measurement system was introduced, in which a microelectrode with a tip diameter can detect the micro-interfacial environment in a nondestructive or quasi-nondestructive manner [20,21].The application of a microelectrode thus improves the accuracy of our study.
This study aimed to investigate the effect of Chironomid larvae bioturbation on N release and conversion at the SWI.Two in-situ technologies were used in order to obtain more accurate and true information about the sediment's micro environment.The microelectrode measurement system was used to detect Eh and O 2 in sediments, and the Peeper technology was used to collect pore water for NH 4 + and NO 3 − detection.Then, the concentration of NH 4 + , NO 3 − and the abundance of ammonia-oxidizing bacteria (AOB) in sediments were analyzed at 1.5 cm intervals.All of the data assists in advancing the understanding of N release and conversion under the bioturbation in eutrophic lake sediments.

Experimental Microcosm Set up
Sediment used in the experiment was collected from the eutrophic Lake Dazong (31 • 30 31.1"N, 120 • 10 31.0"E) in the lower basin of the Huai River during November 2016.Twelve sediment cores (11 cm in diameter, 30 cm in length) were taken by a gravity corer (11 cm × 50 cm, Rigo Co., Chiba, Japan).Meanwhile, overlying water was collected with plastic vessels for simulation experiments in the laboratory.At the same sampling occasion, the Chironomid larvae were collected.All of the sediment cores were sectioned at a 1.5 cm interval with the same depth then pooled together, sieved with a 0.6 mm pore-size mesh, and thoroughly homogenized.Finally, the sediments were put into eight Plexiglas tubes at their original depth.Afterwards, every fourth sediment core was put into a tank with the addition of 40 cm of filtered lake water.The fourth instar Chironomid larvae were divided from the sediment, 72 larvae were equally added into four sediment cores approaching the population density in the sampling site (1887 ind./m 2 ), while the remaining four cores were kept as controls without the addition of Chironomid larvae.Single Chironomid larvae that were found dead were carefully removed and were replaced by individuals of approximately the same size.Chironomid larvae were precultured for 15 days.The temperature of the water in the tank was 15 • C, controlled by the circulating water system.The water was pumped with air for 10 min per hour to maintain the oxic environment.

Preparation of Peeper and Sampling
The Peeper technique was used to collect pore water for NH 4 + and NO 3 − detection.The principle of this technique is to use a dialysis membrane to separate multi-chambered receiver solutions from the surrounding pore water.The first dialysis pore water device, known as a Peeper, was developed by Hesslein (1976) [22] and Mayer (1976) [23].The Peeper probes were prepared according to Xu et al.
(2012) [19], and they have 75 chambers (18 mm × 1.0 mm × 1.0 mm, length × width × height) on a base plate.Each pair of adjacent chambers was separated horizontally by a 1 mm thick wall, producing a vertical resolution of 2.0 mm for sampling.The chambers were filled with deionized water and covered by a 0.10 mm PVDF membrane (Durapore ® , 0.45 µm pore size, Millipore, Burlington, MA, USA) to separate the inner chamber from the surrounding pore water.All the Peeper probes were soaked in purified water and deoxygenated with N 2 for more than 16 h before they were put into the sediment cores.The pore water was collected by the Peeper probes which had been deployed in sediments for 48 h.After removal from the sediments, the Peeper probes were immediately cleaned sequentially by wet filter papers and deionzied water.The water samples were collected by the transferpettor from the chamber in the Peeper probes and then analyzed as soon as possible.
On the 5th day, after the retrieval of Peepers probes, the sediment cores were obtained from the control tank and the Chironomid larvae treatment tank, respectively.The sediments were sliced into 10 sections in 1.5 cm intervals, and then the contents NH 4 + , NO 3 − and the abundance of AOB in sediments were measured.Then, the 5th day's procedure were repeated three times on the 15th, 25th and 35th days, respectively.

Analytical Methods
Water samples were kept in a cooler at 4 • C before analysis.The DO concentration and Eh were determined by a microelectrode system (OX 100 and Redox 100, Unisense, Aarhus, Denmark).The concentrations of NH 4 + and NO 3 − were detected by a multimode reader (M2e, Molecular Devices, San Jose, CA, USA) and a flow injection analyzer (Skalar SAN++, SKALAR, Breda, The Netherlands), respectively.The NH 4 + and NO 3 − in sediments were extracted with saturated KCl solution before detection.All analytical operations were conducted using of strict quality control guide lines and analysis of replicates.All samples were measured three times and the mean of the results were taken to eradicate any discrepancies.The basic sediment characteristics are shown in Table 1.The DNA was extracted from a 0.5 g freeze-dried and sieved sediment sample using an extraction and purification agent (FastDNA ® Spin Kit for Soil, MP Biomedical, Santa Ana, CA, USA).The samples were washed twice with precooled 70% alcohol, then were suspended in a sterilized Tris-EDTA (TE) buffer solution.The final volume of the sample was 50 µL.DNA was analyzed by agarose electrophoresis and the samples were stored below −20 • C. The DNA was extracted three times from each sample before consolidation to ensure the uniformity of the microbes.The consolidated DNA was diluted 10-fold and then used as a template.The AOB gene copies were quantified using primers amoA-1F (5'-GGGGTTTCTACTGGTGGT-3') and amoA-2R (5'-CCCCTCKGSAAAGCCTTCTTC-3') in a 25-µL amplification system (Corbett, RG65H0, Sydney, Australia) [24].The PCR product was analyzed by gel electrophoresis with a 1.0% agarose gel.The sample was treated with fluorescent dye (0.5 µg/mL) before a photo was taken with a gel imager (Bio-Rad, Chemi Doc XRS Total, Hercules, CA, USA).

Changes of DO and Eh in Sediment Cores
The distribution of DO in sediment cores measured at a 100 µm vertical resolution are shown in Figure 1.With the increase of the sediment depth, the content of DO decreased both the control treatment (i.e, non-bioturbated cores) and the Chironomid larvae treatment (i.e., bioturbated cores) at the four sampling times.Chironomid larvae bioturbation has little effect on the content of DO in the overlying water but has a great influence on the content of DO in sediment, and significantly increased the penetration depths of oxygen (OPD) (one-way ANOVA, treatment effect, p < 0.05), especially from 15th day to 25th day.From beginning to end, the OPD in the control were about 2.0 mm.However, the OPD values were 4.0, 6.5, 8.2 and 5.0 mm in the presence of Chironomid larvae, which were 2.0, 3.3, 4.1 and 2.5 times the size of the control, respectively.The result is similar to the report by Chen et al. ( 2015) [25], who has investigated the effects of Chironomid larvae bioturbation on the lability of phosphorus (P) in sediments.Chironomid larvae can bioirrigated oxygen-rich waters into their galleries and increased the penetration depth of oxygen [6,26], and the reason they divert overlying water into corridors is for food and oxygen (O 2 ) [27].
Water 2018, 10, x FOR PEER REVIEW 4 of 12 agarose electrophoresis and the samples were stored below −20 °C.The DNA was extracted three times from each sample before consolidation to ensure the uniformity of the microbes.The consolidated DNA was diluted 10-fold and then used as a template.The AOB gene copies were quantified using primers amoA-1F (5'-GGGGTTTCTACTGGTGGT-3') and amoA-2R (5'-CCCCTCKGSAAAGCCTTCTTC-3') in a 25-μL amplification system (Corbett, RG65H0, Sydney, Australia) [24].The PCR product was analyzed by gel electrophoresis with a 1.0% agarose gel.The sample was treated with fluorescent dye (0.5 μg/mL) before a photo was taken with a gel imager (Bio-Rad, Chemi Doc XRS Total, Hercules, CA, USA).

Changes of DO and Eh in Sediment Cores
The distribution of DO in sediment cores measured at a 100 μm vertical resolution are shown in Figure 1.With the increase of the sediment depth, the content of DO decreased both the control treatment (i.e, non-bioturbated cores) and the Chironomid larvae treatment (i.e., bioturbated cores) at the four sampling times.Chironomid larvae bioturbation has little effect on the content of DO in the overlying water but has a great influence on the content of DO in sediment, and significantly increased the penetration depths of oxygen (OPD) (one-way ANOVA, treatment effect, p < 0.05), especially from 15th day to 25th day.From beginning to end, the OPD in the control were about 2.0 mm.However, the OPD values were 4.0, 6.5, 8.2 and 5.0 mm in the presence of Chironomid larvae, which were 2.0, 3.3, 4.1 and 2.5 times the size of the control, respectively.The result is similar to the report by Chen et al. ( 2015) [25], who has investigated the effects of Chironomid larvae bioturbation on the lability of phosphorus (P) in sediments.Chironomid larvae can bioirrigated oxygen-rich waters into their galleries and increased the penetration depth of oxygen [6,26], and the reason they divert overlying water into corridors is for food and oxygen (O2) [27].

Changes of NH4 + and NO3 − in Overlying Water
The NH4 + concentrations in overlying water are shown in Figure 3a.The NH4 + concentrations of the control treatment were increased slowly during the whole experimental process, and the values were about 1.50, 1.62 1.74 and 1.85 mg•L −1 , respectively.The introduction of Chironomid larvae has a strong effect on NH4 + concentrations in the overlying water at four stages.The values of NH4 + concentrations were decreased on the first two stages and were increased on the last two stages in the larvae treatment, and the values were about 1.37, 1.22, 1.86 and 2.01 mg•L −1 , respectively.The smallest concentration appeared on the 15th day, with a decrease by 24.69% compared with the control treatment, followed by an increasing trend with incubation time, and the NH4 + concentrations increased by 8.65% relative to the control treatment at the end of the experiment.The maximum variation between the Chironomid larvae treatment was observed from day 15 to 25 (Tukey's HSD test, p < 0.001).

Changes of NH4 + and NO3 − in Overlying Water
The NH4 + concentrations in overlying water are shown in Figure 3a.The NH4 + concentrations of the control treatment were increased slowly during the whole experimental process, and the values were about 1.50, 1.62 1.74 and 1.85 mg•L −1 , respectively.The introduction of Chironomid larvae has a strong effect on NH4 + concentrations in the overlying water at four stages.The values of NH4 + concentrations were decreased on the first two stages and were increased on the last two stages in the larvae treatment, and the values were about 1.37, 1.22, 1.86 and 2.01 mg•L −1 , respectively.The smallest concentration appeared on the 15th day, with a decrease by 24.69% compared with the control treatment, followed by an increasing trend with incubation time, and the NH4 + concentrations increased by 8.65% relative to the control treatment at the end of the experiment.The maximum variation between the Chironomid larvae treatment was observed from day 15 to 25 (Tukey's HSD test, p < 0.001).Water 2018, 10, x FOR PEER REVIEW 6 of 12

Changes of NH4 + and NO3 − in Sediments
The distribution of NH4 + and NO3 − in sediments are shown in Figure 4.During the whole experiment process, NH4 + and NO3 − concentrations of the larvae treatment was lower than that of the control treatment.The values of NH4 + decreased by 3.46%, 13.12%, 18.68% and 21.26% relative to the control treatment, respectively.The values of NO3 -decreased by 9.92%, 18.35%, 18.55% and 19.50% relative to the control treatment, respectively.The largest difference of NH4 + and NO3 − contents with or without bioturbation occurred on the 35th day (Tukey's HSD test, p < 0.000).It was shown that the difference of NH4 + and NO3 − between the larvae treatment and control treatment is appeared from 0 to 15 cm (the maximum detection depth), and the main influence depth is approximately 12 cm below the SWI.The Chironomid larvae bioturbation was helpful to the decrease of NH4 + and NO3 − in sediments.In other words, it promotes the release of sediment N. The decrease of N content in sediments is mainly due to building caves behavior of the Chironomid larvae increase the surface area of SWI, and the biological diversion increased NH4 + and NO3 − diffuse into the pore water.

Changes of NH4 + and NO3 − in Pore Water
The ammonium nitrogen (NH4 + ) profiles in pore water collected by Peeper are shown in Figure 5a.The concentrations of NH4 + in the larvae treatment were lower than those in the control treatment for the duration of the experiment.In other words, the bioturbation of Chironomid larvae reduced the concentration of NH4 + in pore water, and the depth of bioturbation influence was up to 15 cm.At the set temperature (15 °C), as time went on, concentration differences of NH4 + with or without bioturbation were firstly increased and then decreased.This shows that the bioturbation effect on NH4 + first increases and then weakens.The bioturbation effects lasted more than 35 days, and the strongest bioturbation effect was observed on the 15th day (Tukey's HSD test, p < 0.000).On that day, NH4 + in the larvae treatment and the control treatment had the most obvious difference at the same layer.On the 25th and 35th day, the differences of NH4 + in the two treatments were gradually reduced.At the set temperature (15 • C), as time went on, concentration differences of NH 4 + with or without bioturbation were firstly increased and then decreased.This shows that the bioturbation effect on NH4 + first increases and then weakens.The bioturbation effects lasted more than 35 days, and the strongest bioturbation effect was observed on the 15th day (Tukey's HSD test, p < 0.000).On that day, NH 4 + in the larvae treatment and the control treatment had the most obvious difference at the same layer.On the 25th and 35th day, the differences of NH 4 + in the two treatments were gradually reduced.
Water 2018, 10, x FOR PEER REVIEW 7 of 12 The nitrate nitrogen (NO3 − ) profiles in pore water collected by Peeper are shown in Figure 5b.On the whole, the larvae bioturbation increased the concentration of nitrate nitrogen (NO3 − ).The depth of bioturbation influence is affected the whole 15 cm of the microcosm, and especially above 12 cm.Like NH4 + , concentration differences of NO3 − with or without bioturbation were firstly increased and then decreased.This shows that the bioturbation effect on NO3 − first increases and then weakens.The bioturbation effects lasted more than 35 days, and the strongest bioturbation effect was observed on the 25th day (Tukey's HSD test, p < 0.002).The maximal NO3 − concentration value in the larvae treatment was up to 4.42 mg•L −1 on the 15th day.Then, on the 35th day, the differences of NO3 − in the two treatments were gradually reduced.

Changes of Ammonia-Oxidizing Bacteria in Sediments
Ammonia-oxidizing bacteria (AOB) are important nitrifying bacteria, the distribution of which in the sediments was studied in this research.The abundance of AOB in sediments of control and larvae treatment are shown on Figure 6.The average AOB abundance values of larvae bioturbation were larger than those of the control at four stages.Obviously, there are significant differences between larvae treatment and control treatment.The AOB abundance of control treatment was little changed in the whole experimental process.In the early stage of the experiment, the AOB abundance of Chironomid larvae treatment increased quickly and reached its highest value at 7.16 × 107 copies/(g dw) on the 15th day, and then it decreased on the 25th day and the 35th day.This shows that the bioturbation effect on AOB abundance increases first and then weakens.The greatest difference of AOB between the Chironomid larvae sediments and the control sediments was observed on the 15th day (Tukey's HSD test, p < 0.020).The nitrate nitrogen (NO 3 − ) profiles in pore water collected by Peeper are shown in Figure 5b.
On the whole, the larvae bioturbation increased the concentration of nitrate nitrogen (NO 3 − ).The depth of bioturbation influence is affected the whole 15 cm of the microcosm, and especially above 12 cm.Like NH 4 + , concentration differences of NO 3 − with or without bioturbation were firstly increased and then decreased.This shows that the bioturbation effect on NO 3 − first increases and then weakens.
The bioturbation effects lasted more than 35 days, and the strongest bioturbation effect was observed on the 25th day (Tukey's HSD test, p < 0.002).The maximal NO 3 − concentration value in the larvae treatment was up to 4.42 mg•L −1 on the 15th day.Then, on the 35th day, the differences of NO 3 − in the two treatments were gradually reduced.

Changes of Ammonia-Oxidizing Bacteria in Sediments
Ammonia-oxidizing bacteria (AOB) are important nitrifying bacteria, the distribution of which in the sediments was studied in this research.The abundance of AOB in sediments of control and larvae treatment are shown on Figure 6.The average AOB abundance values of larvae bioturbation were larger than those of the control at four stages.Obviously, there are significant differences between larvae treatment and control treatment.The AOB abundance of control treatment was little changed in the whole experimental process.In the early stage of the experiment, the AOB abundance of Chironomid larvae treatment increased quickly and reached its highest value at 7.16 × 107 copies/(g dw) on the 15th day, and then it decreased on the 25th day and the 35th day.This shows that the bioturbation effect on AOB abundance increases first and then weakens.The greatest difference of AOB between the Chironomid larvae sediments and the control sediments was observed on the 15th day (Tukey's HSD test, p < 0.020).

Assessment of the Effects of the Larvae Bioturbation on Sediment N
This study has revealed considerable changes of NH4 + and NO3 − at the SWI with bioturbation in Chironomid larvae treatment and without bioturbation in control treatment, respectively.In-situ techniques used in this study could detect well the effects of larvae bioturbation.The results indicate that bioturbation by Chironomid larvae has a major impact on N release, reflected by the large decreases of NH4 + and NO3 − in the sediment (Figure 4), respectively.The mean concentrations of NH4 + and NO3 − in the bioturbation treatment were reduced by 21.26% and 19.50% compared to the control treatment, respectively.The phenomenon was also reported by Lewandowski et al. (2007) [28], and they found that the NH4 + content was reduced by 25% with C. plumosus bioturbation.Except for affecting N release, the bioturbation by Chironomid larvae can also result in N conversion, which can be reflected by the large increase of NO3 − concentrations in the overlying water and pore water, but NH4 + concentrations decreased in pore water and only slightly increased in the overlying water.An inverse relationship was observed between NH4 + and NO3 − concentrations in pore water in Chironomid larvae treatment.There is no relationship in control treatment but showed a significant negative correlation in Chironomid larvae treatment (Table 2), especially on the 15th day.The influence of larvae bioturbation on N release was mainly from days 15 to 25.In this stage, some extreme values appeared, including the minimum concentration of NH4 + and the maximum concentration of NO3 − in water, the most obvious concentration difference of NH4 + /NO3 − in pore water and the highest abundance of AOB.These phenomena may imply that AOB plays a significant role in the release of nitrogen from sediment.Then, we analyzed the correlation of NH4 + vs. AOB and NO3 − vs. AOB profiles in pore water of the Chironomid larvae treatment (Table 3).The  There is no relationship in control treatment but showed a significant negative correlation in Chironomid larvae treatment (Table 2), especially on the 15th day.The influence of larvae bioturbation on N release was mainly from days 15 to 25.In this stage, some extreme values appeared, including the minimum concentration of NH 4 + and the maximum concentration of NO 3 − in overlying water, the most obvious concentration difference of NH 4 + /NO 3 − in pore water and the highest abundance of AOB.These phenomena may imply that AOB plays a significant role in the release of nitrogen from sediment.Then, we analyzed the correlation of NH 4 + vs.
AOB and NO 3 − vs. AOB profiles in pore water of the Chironomid larvae treatment (Table 3).The results showed that there was a significant negative correlation between NH 4 + and AOB, and a significant positive correlation between NO 3 − and AOB.Especially on the 15th and 25th day, the correlation was the most significant, and the values were −0.838 ** and 0.785 **, −0.843 ** and 0.711 *, respectively.Therefore, we infer that nitrification process mediated by AOB plays an important role in N release and conversion in sediments, in which NH + was converted into NO 3 − .Furthermore, the depths of bioturbation effect on NH 4 + and NO 3 − concentrations in pore water and sediments affected the whole 15 cm of the microcosm: Especially above 12 cm, which was shorter than the 18 cm according to Caffrey [29] and deeper than the 7 cm according to Chen [25] and 100 mm according to Lewandowski [28].Thus, this proved that the application of these in situ technologies is reliable, and the main bioturbation influence depth for N release was about 12 cm below the SWI.

Bioturbation Mechanism on Sediment N
Chironomid larvae play an important role in the lake sediment environment via biological diversion, cave building, absorption, digestion, defecation and secretion [6,26].The action of diversion and the behavior of building caves caused dissolved substances in pore water transport to the overlying water easily and efficiently [30].Haruo Fukuhara [12] found that the larvae of Chironomus plumosus caused an enhancement of inorganic nitrogen release (mainly NH 4 + ).This finding is inconsistent with the result of our study.We found that Chironomid larvae did increase the N release (specifically, increasing NO 3 − release).This difference may be due to the reasons outlined below.
Chironomid larvae not only increase the surface area of SWI, but their burrows also become sites of high bacterial numbers and high metabolic activity compared to the surrounding sediment.Chironomid larvae bio-irrigated oxygen-rich overlying water into their U-shaped corridors, resulting in an increase in DO concentrations (Figure 1) in sediments.The imported oxygen diffused through the burrow walls into the surrounding sediments, leading to redox zones concentrically distributed around the tubes (Figure 2), which provided AOB with proliferation, activity sites, and enhanced the activity of AOB (Figure 6) [31,32].Combined with the NH 4 + (released from the sediment) and O 2 (bio-irrigated from the overlying water), AOB promoted the nitration reaction in the cave and the channel wall, which facilitated the conversion of NH 4 + in pore water into NO 3 − (Figure 7) [33].A part of NH 4 + released from the sediment flowed into the overlying water via molecular diffusion and another part was converted into NO 3 − through the nitrification affected by AOB.Hence, NH 4 + (Figures 4a and 5a) was decreased in the pore water and sediment, and the NO 3 − (Figures 3b and 5b) in the overlying water and pore water was increased.The studies indicate that the O 2 and AOB play important roles in controlling the release and conversion of N in sediments, i.e., NH 4 + is converted to NO 3 − in oxidized sediments, which is consistent with previous studies [34,35].
Water 2018, 10, x FOR PEER REVIEW 10 of 12

Conclusions
Through the use of two in situ technologies, we clearly found that the bioturbation of Chironomid larvae facilitated the release and conversion of N in sediments.The concentrations of NH4 + and NO3 − decreased by 21.26% and 19.50% in sediment, and increased by 8.65% and 49.82% in overlying water compared to the control treatment, respectively.The bioturbation of Chironomid larvae increased the release of NH4 + and NO3 − from sediments to the overlying water.The bioturbation also enhanced the nitration reaction, and a part of NH4 + released from the sediment to the overlying was converted into NO3 − by AOB with adequate DO.Furthermore, the main influence depths of Chironomid larvae bioturbation were approximately 12 cm below the SWI, and the most significant bioturbation effect was observed from days 15 to 25.The research results gained by in situ technologies can provide a more comprehensive and accurate understanding of the effect of bioturbation on N release at the SWI and also contribute to water eutrophication management.

Figure 1 .
Figure 1.DO profiles in sediment cores of the control and the Chironomid larvae treatment.The horizontal dashed line at zero indicates the sediment-water interface (SWI); over the line indicates overlying water; under the line indicates sediments.(e) DO changes in treatment minus changes in control on the 5th (a), 15th (b), 25th (c) and 35th (d) day, respectively.

Figure 1 . 12 Figure 2 .
Figure 1.DO profiles in sediment cores of the control and the Chironomid larvae treatment.The horizontal dashed line at zero indicates the sediment-water interface (SWI); over the line indicates overlying water; under the line indicates sediments.(e) DO changes in treatment minus changes in control on the 5th (a), 15th (b), 25th (c) and 35th (d) day, respectively.

Figure 2 .
Figure 2. Eh profiles in sediment cores of the control and the Chironomid larvae treatment.The horizontal dashed line at zero indicates the sediment-water interface (SWI); over the line indicates overlying water; under the line indicates sediments.(e) Eh changes in treatment minus changes in control on the 5th (a), 15th (b), 25th (c) and 35th (d) day, respectively.

3. 2 . 12 Figure 2 .
Figure 2. Eh profiles in sediment cores of the control and the Chironomid larvae treatment.The horizontal dashed line at zero indicates the sediment-water interface (SWI); over the line indicates overlying water; under the line indicates sediments.(e) Eh changes in treatment minus changes in control on the 5th (a), 15th (b), 25th (c) and 35th (d) day, respectively.

3. 4 .
Changes of NH 4 + and NO 3 − in Pore Water The ammonium nitrogen (NH 4 + ) profiles in pore water collected by Peeper are shown in Figure 5a.The concentrations of NH 4 + in the larvae treatment were lower than those in the control treatment for the duration of the experiment.In other words, the bioturbation of Chironomid larvae reduced the concentration of NH 4 + in pore water, and the depth of bioturbation influence was up to 15 cm.

Figure 5 .
Figure 5. Ammonium nitrogen (NH 4 + ) and nitrate nitrogen (NO 3 − ) profiles in pore water collected by Peeper of the control and the Chironomid larvae treatment.(a) NH 4 + and (b) NO 3 − .(a5) NH 4 + and (b5) NO 3 − in pore water changes in treatment minus changes in control on the 5th, 15th, 25th and 35th day, respectively.

12 Figure 6 .
Figure 6.Ammonia-oxidizing bacteria (AOB) profiles in sediments of the control and the Chironomid larvae treatment.Dw: dry sediment.(e) AOB abundance changes in treatment minus changes in control on the 5th (a), 15th (b), 25th (c) and 35th (d) day, respectively.

Figure 6 .
Figure 6.Ammonia-oxidizing bacteria (AOB) profiles in sediments of the control and the Chironomid larvae treatment.Dw: dry sediment.(e) AOB abundance changes in treatment minus changes in control on the 5th (a), 15th (b), 25th (c) and 35th (d) day, respectively.

1 .
Assessment of the Effects of the Larvae Bioturbation on Sediment N This study has revealed considerable changes of NH 4 + and NO 3 − at the SWI with bioturbation in Chironomid larvae treatment and without bioturbation in control treatment, respectively.In-situ techniques used in this study could detect well the effects of larvae bioturbation.The results indicate that bioturbation by Chironomid larvae has a major impact on N release, reflected by the large decreases of NH 4 + and NO 3 − in the sediment (Figure 4), respectively.The mean concentrations of NH 4 + and NO 3 − in the bioturbation treatment were reduced by 21.26% and 19.50% compared to the control treatment, respectively.The phenomenon was also reported by Lewandowski et al. (2007) [28], and they found that the NH 4 + content was reduced by 25% with C. plumosus bioturbation.Except for affecting N release, the bioturbation by Chironomid larvae can also result in N conversion, which can be reflected by the large increase of NO 3 − concentrations in the overlying water and pore water, but NH 4 + concentrations decreased in pore water and only slightly increased in the overlying water.An inverse relationship was observed between NH 4 + and NO 3 − concentrations in pore water in Chironomid larvae treatment.

Figure 7 .
Figure 7. Schematic illustration of the processes of Chironomid larvae bioturbation impact on N release and conversion at the SWI.

Figure 7 .
Figure 7. Schematic illustration of the processes of Chironomid larvae bioturbation impact on N release and conversion at the SWI.

3 −
Through the use of two in situ technologies, we clearly found that the bioturbation of Chironomid larvae facilitated the release and conversion of N in sediments.The concentrations of NH 4 + and NO decreased by 21.26% and 19.50% in sediment, and increased by 8.65% and 49.82% in overlying water compared to the control treatment, respectively.The bioturbation of Chironomid larvae increased the release of NH 4 + and NO 3 − from sediments to the overlying water.The bioturbation also enhanced the nitration reaction, and a part of NH 4 + released from the sediment to the overlying was converted into NO 3 − by AOB with adequate DO.Furthermore, the main influence depths of Chironomid larvae bioturbation were approximately 12 cm below the SWI, and the most significant bioturbation effect was observed from days 15 to 25.The research results gained by in situ technologies can provide a more comprehensive and accurate understanding of the effect of bioturbation on N release at the SWI and also contribute to water eutrophication management.

Table 1 .
Characteristics of sediment used in the simulation experiment.

Table 2 .
Correlation analyses between NH 4 + and NO 3 − profiles in pore water of the control and the Chironomid larvae treatment (p = 0.05).