# Distribution of Dissolved Nitrogen Compounds in the Water Column of a Meromictic Subarctic Lake

Taisiya Ya. Vorobyeva, Anna A Chupakova, Artem V Chupakov, Svetlana A Zabelina, Olga Y Moreva, Oleg S Pokrovsky

► To cite this version:

Taisiya Ya. Vorobyeva, Anna A Chupakova, Artem V Chupakov, Svetlana A Zabelina, Olga Y Moreva, et al.. Distribution of Dissolved Nitrogen Compounds in the Water Column of a Meromictic Subarctic Lake. Nitrogen, 2021, 2 (4), pp.428-443. 10.3390/nitrogen2040029. hal-04842502

HAL Id: hal-04842502

https://ut3-toulouseinp.hal.science/hal-04842502v1

Submitted on 17 Dec 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Article

# Distribution of Dissolved Nitrogen Compounds in the Water Column of a Meromictic Subarctic Lake

Taisiya Ya. Vorobyeva<fnref n="1" /> ID, Anna A. Chupakova<fnref n="1" />, Artem V. Chupakov<fnref n="1" />, Svetlana A. Zabelina<fnref n="1" /> ID, Olga Y. Moreva<fnref n="1" /> and Oleg S. Pokrovsky<fnref n="1" />,<fnref n="2" />,* ID

<figure><img src="image_3.png" /></figure>

check for
updates

**Citation:** Vorobyeva, T.Y.; Chupakova, A.A.; Chupakov, A.V.; Zabelina, S.A.; Moreva, O.Y.; Pokrovsky, O.S. Distribution of Dissolved Nitrogen Compounds in the Water Column of a Meromictic Subarctic Lake. *Nitrogen* **2021**, *2*, 428–443. https://doi.org/10.3390/ nitrogen2040029

Academic Editor: Stephen Macko

Received: 30 June 2021

Accepted: 9 October 2021

Published: 13 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_4.png" /></figure>

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Abstract:** In order to better understand the biogeochemical cycle of nitrogen in meromictic lakes, which can serve as a model for past aquatic environments, we measured dissolved concentrations of nitrate, nitrite, ammonium, and organic nitrogen in the deep (39 m maximal depth) subarctic Lake Svetloe (NW Russia). The lake is a rare type of freshwater meromictic water body with high concentrations of methane, ferrous iron, and manganese and low concentrations of sulfates and sulfides in the monimolimnion. In the oligotrophic mixolimnion, the concentration of mineral forms of nitrogen decreased in summer compared to winter, likely due to a phytoplankton bloom. The decomposition of the bulk of the organic matter occurs under microaerophilic/anaerobic conditions of the chemocline and is accompanied by the accumulation of nitrogen in the form of N-NH₄ in the monimolimnion. We revealed a strong relationship between methane and nitrogen cycles in the chemocline and monimolimnion horizons. The nitrate concentrations in Lake Svetloe varied from 9 to 13 µM throughout the water column. This fact is rare for meromictic lakes, where nitrate concentrations up to 13 µM are found in the monimolimnion zone down to the bottom layers. We hypothesize, in accord with available data for other stratified lakes that under conditions of high concentrations of manganese and ammonium at the boundary of redox conditions and below, anaerobic nitrification with the formation of nitrate occurs. Overall, most of the organic matter in Lake Svetloe undergoes biodegradation essentially under microaerophilic/anaerobic conditions of the chemocline and the monimolimnion. Consequently, the manifestation of the biogeochemical nitrogen cycle is expressed in these horizons in the most vivid and complex relationship with other cycles of elements.

**Keywords:** nitrogen cycle; ammonium; nitrate; nitrite; stratification; boreal; meromictic lake

## 1. Introduction

Nitrogen is the most important nutrient, ranking fourth among biophilic elements after carbon, oxygen, and hydrogen. The biogeochemical nitrogen cycle is one of the main cycles in the biosphere [1–6]. The nitrogen cycle is an interconnected chain of reactions for the transformation of various forms of nitrogen compounds [7], the main role of which belongs to microorganisms [8–15]. The concentration of nitrogen compounds determines the water body’s biological productivity to a large extent. A change in the composition and ratio of the various forms and concentrations of nitrogen compounds indicate the direction of the dominant biological and biogeochemical processes, including the processes of self-purification of water bodies [2]. In addition to assessing water quality, information on the content and distribution of various forms of nitrogen is important for nutrient balance and the water’s chemical composition [16].

<footnote n="1">¹ N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, 23 Severnoi Dviny Emb., 163000 Arkhangelsk, Russia; vtais@yandex.ru (T.Y.V.); anna.a.ershova@gmail.com (A.A.C.); artem.chupakov@gmail.com (A.V.C.); svetzabelina@gmail.com (S.A.Z.); mapycr1@yandex.ru (O.Y.M.)</footnote>

<footnote n="2">² Géosciences Environnement Toulouse (GET), UMR 5563, CNRS-OMP-Université Toulouse, 14 Avenue Edouard Belin, 31400 Toulouse, France</footnote>

<footnote>* Correspondence: oleg.pokrovsky@get.omp.eu; Tel.: +33-5-61-33-2625</footnote>
Because the nitrogen cycle in aquatic systems is strongly dependent on redox condi-
tions, chemically stratified water bodies are particularly important for the biogeochemistry
of N in continental settings. Biogeochemical processes in meromictic water bodies are very
interesting from the point of view of limnology and aquatic ecology because lakes of this
type exhibit peculiar features: There is no water mixing between different layers and the
anaerobic zone persists throughout the whole year. There are three zones in the structure
of meromictic lakes: Mixolimnion—a zone in which convective and wind mixing of water
occurs; monimolimnion, which is an anaerobic zone, a non-mixing layer that does not
have contact with the atmosphere; and a chemocline, which is a layer of abrupt change of
hydrochemical characteristics at the boundary of the mixolimnion and monimolimnion,
where a complex microbial community is usually formed [17,18].

Of greatest interest in water bodies of this type are the processes occurring in the
chemocline and monimolimnion of lakes. The oxygen minimum zone (OMZ), formed
in the chemocline, is a site of intensive nitrogen turnover due to the influx of organic
material from the overlying water layers and the formation of a large amount of organic
matter in the chemocline zone. The OMZ contains unique microbial communities that
use alternative electron acceptors for respiration. The OMZ conditions provide an almost
complete nitrogen (N) cycle. Remineralization can occur both due to the reduction of
nitrate to nitrite and can be associated with the non-assimilatory reduction of nitrate to
ammonium, whereas the formation of gaseous nitrogen can occur due to heterotrophic
denitrification and anammox. Many microorganisms inhabiting the oxygen minimum
zones are capable of performing various functions in the nitrogen and other cycles of
elements [19].

Nitrifying bacteria (both ammonium and nitrite oxidants) are present throughout the
entire oxygen minimum zone. Even in waters with the lowest oxygen content, chemoau-
totrophic activity and oxidation of nitrogen compounds are usually detected in situ. The
greatest abundance of nitrifying bacteria and their greatest activity is found in the gradient
region at the upper boundary of the oxygen minimum [19,20]. A close relationship is
found between nitrification and denitrification in the oxygen minimum zone where the
rapid cycling of intermediate products of the nitrogen cycle, including nitrate, nitrite, and
nitrogen oxide (I), occurs [20].

An increase in exports from mixolimnion and the formation of a large amount of organic matter in the chemocline (hence, respiration within this zone) leads to the depletion of dissolved oxygen, followed by other main alternative electron acceptors (nitrate, sulfate, etc.). Some of the sulfide/sulfur oxidizers can use $NO_3^-/NO_2^-$ as electron acceptors and release gaseous $N_2$ and/or $N_2O$, while the released energy is used to fix inorganic carbon via so-called chemolitoautotrophic denitrification. The latter process can also be associated with hydrogenotrophy, methanotrophy, and iron oxidation [19].

Microorganisms of the non-biogeochemical nitrogen cycle are also capable of influenc-
ing its transformation under the specific conditions of meromictic lakes. Microorganisms
of the methane cycle (including both methanogenesis and methane oxidation) play an
important role in the binding of global carbon and nitrogen cycles in microaerophilic and
anoxic environments [21–26]. In addition to the fact that methanotrophic bacteria use
methane as a source of carbon and energy, they also modify the nitrogen cycle, especially
at the boundary of the aerobic–anaerobic interaction (chemocline zone) or just above or
below it [27]. Methanotrophic bacteria can assimilate nitrogen as ammonia or nitrate and
can compete with nitrifying bacteria for ammonia and oxygen [26,28,29].

Studies of small Red/Ox-stratified boreal lakes [24] have demonstrated that methan-
otrophs of boreal lakes can associate methane oxidation with $NO_x^-$ reduction under
hypoxic conditions. Recent studies of a shallow, seasonally stratified subalpine lake [30]
have shown that $NH_4^+$, being the main nutrient in this aquatic ecosystem, has a positive
correlation with methane-oxidizing bacteria (MOB). The latter can metabolize $NH_4^+$ for
growth. Investigations of the oxygen-stratified Lake Fohnsee (southern Germany) have
shown that anaerobic methane oxidation, denitrification, and anammox can simultaneously
occur in the anoxic water column [31]. Analysis of gas taken from various anaerobic envi-
ronments, where a significant amount of organic matter accumulates and decomposes with
the release of methane, demonstrated that free nitrogen of biological origin can constitute
up to 30% of all the released gas [7]. A relationship between methane oxidation and nitrate
reduction during hypoxia has recently been shown for the Gammaproteobacterium *Methy-
lomonas denitrificans*, attributing to this species a previously overlooked role in coupling
carbon and nitrogen cycles [32].

The methane cycle in Lake Svetloe is fairly well known [33–38]. The microbial commu-
nities participating in the methane cycle are represented by several genera of methanogens
and methanotrophs typical of freshwater lakes [35]. Methane oxidation was found in both
oxygen and anoxic conditions with a maximum in chemocline. Methanotrophic bacteria
*Methylobacter* sp. and methylotrophic *Methylotenera* sp. and *Methylophilus* sp. showed simi-
lar profiles of relative abundance throughout the epilimnion, chemocline, and hypolimnion
of Lake Svetloe [35,36]. However, in contrast to fairly good knowledge of the carbon and
methane cycle in Lake Svetloe, the knowledge of the nitrogen cycle remains highly limited.

This work presents the first data on the spatiotemporal dynamics of the concentra-
tions of organic and inorganic nitrogen forms in the subarctic meromictic Lake Svetloe
(NW Russia). The lake is a rare type of freshwater meromictic water body with high
concentrations of dissolved methane and ferrous iron, manganese, and low concentrations
of sulfates and sulfides in the hypolimnion [34,36,39,40]. Iron-rich and sulfur-depleted
meromictic lakes with conditions suitable for photoferrotrophy are considered modern
analogues of the ancient Archean Ocean [21,41]. The high transparency of the waters, low
water color, and the distribution of the photic layer throughout the depth create favorable
conditions for the development of phytoplankton and the specific microbial community
of the chemocline [33,36]. Our specific objectives were to (i) assess vertical and seasonal
dynamics of dissolved nitrogen forms and (ii) relate the spatial pattern of these concentra-
tions to already known cycles of carbon, oxygen, methane, iron, and manganese in the lake,
taking into account the microbiological control on the N cycle.

2. Study Site and Methods

2.1. *Site Description*

Lake Svetloe is located in the northern part of the boreal zone of European Russia (N 65°04.98', E 41°06.26'), 65 km NNE of Arkhangelsk, and its watershed is not subjected to any direct anthropogenic influence. The maximum depth of the lake is 39 m (Figure 1).

<figure><img src="image_5.png" /><figcaption>**Figure 1.** 3D model and bathymetric scheme of Lake Svetloe (scale bar in meters).</figcaption></figure>
Two pronounced relief depressions conditionally divide the lake into two parts with maximum depths of 39 and 20 m (Figure 1). In accordance with the meromictic status, three layers in the water column of the lake can be distinguished: (1) Mixolimnion (from 0 to 20 m), subject to convective mixing throughout the year and exhibiting aerobic conditions; (2) chemocline, which is a transitional zone at a depth of 20–24 m, where microaerophilic conditions are formed; in this zone, oxygen is produced by cyanobacteria and is actively consumed by other microorganisms [36]; and (3) monimolimnion (from 25 m to the bottom), which is an anaerobic layer [36,39]. Lake Svetloe is characterized by a predominance of autochthonous dissolved organic matter with a low content of dissolved organic carbon (83.3 to 333.3 µM, [39,40]) and high water transparency (12 to 16 m Secchi depth).

The water in the chemocline of Lake Svetloe has a faint pink color of varying intensity, due to the development of phototrophic communities inhabiting the boundary of the aerobic and anaerobic zones. Studies of phototrophic bacteria in the communities of the chemocline zone have demonstrated that the dominant bacteria are oxygenic phototrophic cyanobacteria of the genus Synechococcus (maximum development at a depth of 23 m) [33,36], which have positive chemotaxis to nitrogen sources [42–44] and are capable of fixing molecular nitrogen [45].

## 2.2. *Sampling and Analyses*

Sampling was carried out from 2010 to 2016 and included 30 survey campaigns during all hydrological seasons. Sampling was carried out over the entire water column from the surface to the bottom, with a step of 1 to 6 m. Water samples were taken at the deepest point of the lake approximately in the middle of the water body (N 65°4.975′, E 41°6.497′) from a PVC boat from May to October, and from the ice in winter (November to April) using a pre-cleaned polycarbonate horizontal water sampler (Aquatic Research Co, ID, New York, NY, USA).

A water sample for measurements of nitrite, nitrate, and total nitrogen was collected with a water sampler, to the tap of which a PVC outlet tube was attached. First, the bottle was rinsed 2–3 times with water from the sampler. Then the outlet tube of the sampler was lowered to the bottom of the bottle and began to fill it with water, passing several volumes of water, that is, until the water that was in contact with the air in the bottle was entirely displaced. As such, most of the water sample was not in direct contact with the atmosphere. Then the polypropylene containers were closed with screw caps with an inner cone with slight compression of the bottle itself. This technique minimized the possibility of having residual air in the vial. It should be noted that when examining the water samples in the laboratory after transportation, we did not observe the precipitation of Fe (III) hydroxide, which would inevitably form during the oxidation of Fe (II) in a neutral to slightly alkaline medium. Chemical analysis of water samples was carried out on the day of sampling.

A water sample for the determination of N-NH₄ was taken in a separate glass container in the same way as for other forms of nitrogen described above. Fixation of N-NH₄ with reagents was carried out immediately after filling the bottle with water. In addition, at the beginning of the study of Lake Svetloe, we carried out a parallel determination of the content of N-NO₂ and N-NO₃ directly on the lake shore, and afterwards, the samples were delivered to the laboratory. The results of parallel measurements were reproducible within the uncertainty of analyses.

The nitrogen forms' (N-NO₂, N-NO₃, N-NH₄, TN) determinations were based on colorimetric assays [40,46]. The indophenol blue method was used to measure ammonium (N-NH₄), with a relative error of up to 12% and a detection limit of 1 µM. A spectrophotometric method employing sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride was used for the analysis of nitrite (N-NO₂), with a relative error of up to 18% and a detection limit of 0.03 µM [47,48]. A spectrophotometric method employing sodium salicylate was chosen for the determination of nitrate (N-NO₃), with a relative error of up to 18% and a detection limit of 7 µM. The total dissolved organic nitrogen (Norg) was evaluated from the difference between the total dissolved nitrogen (TN, persulfate oxidation, relative
error up to 12%, detection limit of 18 µM) and the total dissolved inorganic nitrogen (DIN, or the sum of nitrite, nitrate, and ammonium nitrogen). All concentrations for the nitrogen compounds are given in micro mole per liter (µM) of nitrogen. Generally, we obtained a single result for each sample from a discrete horizon. The sample size and reliability of the mean estimates were achieved by the number of vertical surveys along the water column in different seasons (30 profiles).

## 3. Results

### 3.1. *Main Hydrochemical Characteristics*

The main hydrological parameters of the lake are presented elsewhere [34,39,40]. The vertical profile of oxygen, temperature, conductivity, and water density are shown in Figure S1 of the Supplementary Material. The numerical values of physicochemical parameters (oxygen, temperature, and pH) of the water column of Lake Svetloe in different seasons of the year are shown in Table S1 of the Supplementary Material. Seasonal water temperature variation occurs to a depth of 22 m; further below, the water temperature decreases with a gradient of about 0.7 °C/m, and below 27 m, the water temperature is in the range of 3.5–3.6 °C all year round. Specific conductivity of the surface horizons ranges from 150 to 250 µS/cm, whereas in the bottom horizons, it ranges from 340 to 380 µS/cm. In the mixolimnion, the pH varies from 7 to 8.4 (maximum values during periods of mass phytoplankton bloom), in the chemocline, the pH varies from 6.8 to 8, and in the monimolimnion, a narrower range of variation is observed (from 6.8 to 7.6). A general decrease in pH is observed with depth. At depths of 24–25 m, there is a slight increase in pH values associated with the development of anoxygenic bacteria or cyanobacteria.

The dissolved oxygen concentration in the summer period in the thermocline (3–7 m) reaches its maximum values (over 375 µM). In the lower layers, it gradually decreases to the minimum values in the chemocline. In winter, high oxygen content is noted throughout the mixolimnion with an abrupt increase above the chemocline. In the chemocline, at depths of 21 to 24 m, a layer with microaerophilic conditions is formed, which persists throughout the year. The absence of oxygen from a depth of 24–25 m down to the bottom was noted. In the chemocline layers, the sign of the redox potential changes. At the depth of 22–24 m, according to estimates (our data and [36]), Eh has a negative value, -107-(-168) mV.

The lake is a meromictic waterbody of iron-manganese type. The sulfide concentration is lower than that of iron and manganese, which is quite rare among meromictic lakes [39]. The most characteristic intervals (25–75%) of the concentrations of iron and manganese (dissolved fraction <0.45 µm) in the zones of mixolimnion, chemocline, and monimolimnion are equal, respectively: Fe—0.01–0.028, 0.6–41.7, 111–144 µM; Mn—0.006–0.028, 12–43, 52–54 µM. A sharp increase in the concentration of Fe and Mn also occurs when the Red/Ox conditions change.

Lake Svetloe exhibits a low concentration of dissolved organic carbon (DOC) (the minimum and maximum values were 68 µM and 358 µM, respectively); the average value over the entire water column was 167 ± 64 µM. The maximum values of the DOC concentration in the mixolimnion were observed in the surface layers during the phytoplankton bloom. The minimum and maximum values of dissolved inorganic carbon (DIC) were 1725 µM and 5704 µM, respectively; the average value over the entire water column was 3407 ± 130 µM.

### 3.2. *Nitrogen Compounds*

The various forms of nitrogen concentration of the water column of Lake Svetloe in different seasons of the year are shown in Table S1 of the Supplementary Material. The distribution of the N-NH₄ concentration in the water column of Lake Svetloe is characterized by a sharp increase in the chemocline during all seasons (Figures 2a and 3). The maximal concentration reaches, on average, 144 to 177 µM in the bottom layer. In the mixolimnion, N-NH₄ concentrations rarely exceeded 1.2 µM. In summer, the concentrations of N-NH₄ in the surface layers were lower (up to 0.7 µM) than in winter.
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Distribution of (a) ammonium, (b) nitrite nitrogen, and (c) nitrate nitrogen in the water column of Lake Svetloe (in µM) averaged over four seasons from 2010 to 2016. Middle point—median; box value—percentiles (25–75%); whisker value—min–max.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Seasonal dynamics of ammonium and the amount of nitrite and nitrate nitrogen in the water column of Lake Svetloe (in µM). Winter (November–April), spring (May), summer (June–August), autumn (September–October).</figcaption></figure>

The distribution of N-NO₃⁻ during all seasons demonstrated a slight decrease in concentration in the chemocline zone with a subsequent increase in the deeper layers (Figures 2c and 3). The concentration of N-NO₃⁻ in the monimolimnion did not exceed the values achieved in the mixolimnion and varied in the range of 9–13 µM. The TN concentration in the mixolimnion varied within the range of 9 to 23 µM (Figure 4).
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Various nitrogen forms' distribution in the water column of Lake Svetloe (in µM). Middle point—median; whisker value—percentiles (25–75%).</figcaption></figure>

There was a sharp increase in TN in the chemocline (by one order of magnitude). The TN concentration reached 293 µM near the bottom layer. The share of Norg in the mixolimnion varied from 16 to 75% of TN, and in the monimolimnion, it ranged from 12 to 95% of TN (Figure 4).

The vertical distribution of mineral forms of nitrogen (Figure 3) demonstrated that the N-NO₃ form always predominated in the mixolimnion of Lake Svetloe. The predominance of N-NH₄ is observed from a depth of 22 m; the content of N-NO₂ in all seasons was very low and varied within the range of 0–1.4 µM, which is 0.1–4% of the sum of inorganic forms of nitrogen (Figure 2b).

**4. Discussion**

Comparative analysis of inorganic forms of nitrogen concentration in some meromictic lakes (Table 1) showed that there is a similar tendency of an increase in the content of ammonium from the mixolimnion to the monimolimnion with a sharp increase in the N-NH₄ concentration in the chemocline. In the vertical distribution of nitrate concentration, there are similar features between lakes Svetloe (this study) and Pavin [49]. In both lakes, a decrease in the concentration of nitrate nitrogen in the chemocline zone is observed. In contrast to the compared meromictic lakes, the concentration of nitrates in the monimolimnion in Lake Svetloe remains at the level of the mixolimnion. A similar dynamic is observed only in the meromictic Lake Zug, where nitrate is present in the anoxic hypolimnion [50].

The surface horizon of Lake Svetloe (0.5–3 m) is almost always distinguished by an increased number of heterotrophic bacteria, including ammonifying ones, and a slight excess of ammonium concentrations in comparison with the lower layers of the epilimnion, which may indicate a more active ammonification. By the content of nitrogen compounds, the mixolimnion layer can be characterized as oligotrophic, which is also confirmed by the values of the isotopic composition of suspended organic carbon: For the upper oxygen layer, the values δ¹³Cₗorg of -27.2 to -30.2‰ are common for the phytoplankton of oligotrophic freshwater lakes [36].
<table><caption>**Table 1.** The content of mineral forms of nitrogen in Lake Svetloe (25–75%) and other meromictic lakes in the world. Concentrations are given in µM.</caption><thead><tr><th rowspan="2">Lake</th><th colspan="3">N-NH<sub>4</sub></th><th colspan="3">N-NO<sub>2</sub></th><th colspan="3">N-NO<sub>3</sub></th><th rowspan="2">Reference</th></tr><tr><th>Mix</th><th>Chem</th><th>Monim</th><th>Mix</th><th>Chem</th><th>Monim</th><th>Mix</th><th>Chem</th><th>Monim</th></tr></thead><tbody><tr><td>Lake Svetloe</td><td>0.5–1.2</td><td>13–91</td><td>144–177</td><td>0–0.07</td><td>0.3–0.8</td><td>0.6–1.4</td><td>5–9</td><td>5–8</td><td>9–13</td><td>This Research</td></tr><tr><td>Lake Matano</td><td>up to 20</td><td>20–300</td><td>200–300</td><td>&lt;0.1</td><td></td><td></td><td>all layers &lt; 0.1 only 90–100 m</td><td>0.1–0.2</td><td></td><td>[21]</td></tr><tr><td>Lake Pavin</td><td>–</td><td>–</td><td>max 389</td><td>–</td><td>–</td><td>–</td><td>max 32</td><td>–</td><td>–</td><td>[49]</td></tr><tr><td>Lake La Cruz</td><td>1.1–12.8</td><td>122.2</td><td>up to 3000</td><td>0.04–0.11</td><td>0</td><td>0</td><td>5–20</td><td>0</td><td>0</td><td>[18,51]</td></tr><tr><td>Kabuno Bay</td><td>–</td><td>–</td><td>–</td><td>up to 1</td><td>–</td><td>–</td><td>N-(NO<sub>2</sub> + NO<sub>3</sub>)<br>Up to 0.5 in dry season up to 1.5</td><td>up to 0.5</td><td></td><td>[52]</td></tr><tr><td>Lake Kuznechikha</td><td>1.4–3.5</td><td>17.8–50</td><td>83–121</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>[53]</td></tr><tr><td>Char lake</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>&lt;0.7</td><td>–</td><td>&lt;0.7</td><td>[54]</td></tr><tr><td>Lake Chernoe</td><td>–</td><td>–</td><td>up to 265</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>[55]</td></tr><tr><td>Hall Lake</td><td>0</td><td>–</td><td>305</td><td>–</td><td>–</td><td>–</td><td>13.3</td><td>–</td><td>4.4</td><td>[18,56]</td></tr><tr><td>Lake Zug</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>20–25</td><td>17–18</td><td>1–5</td><td>[50]</td></tr></tbody></table>

The microbial communities of the mixolimnion of Lake Svetloe are typical of those for aerobic lake environments [57] and are mainly represented by microbial groups participating in the initial stages of organic matter decomposition [35,36]. Measurements of the rate of dark CO₂ assimilation indicate low activity of heterotrophic bacterioplankton [35,36].

Aerobic nitrification of ammonia to nitrite and nitrate is a major process in the global nitrogen cycle. Low concentrations of N-NH₄ and high concentrations of nitrates were observed in the mixolimnion, except for the peak periods of phytoplankton development, when the concentration of nitrates can decrease below 1.6 µM. This testifies active processes of nitrification, which proceed to the final compound of nitrate in the water of Lake Svetloe. Low N-NO₂ concentrations in the mixolimnion and a large amount of N-NO₃ in this layer may indicate that nitrification processes proceed to the final stage in the presence of a sufficient amount of oxygen in the mixolimnion.

The peak in the development of phytoplankton communities (maximum oxygenic phototrophic activity [35,36]) is most often observed at a depth of 6–10 m, which is also evidenced by an increase in pH and dissolved oxygen concentration at this depth. There is also a slight increase in the concentration of organic nitrogen and a decrease in the inorganic nitrogen concentration, notably of nitrates.

The concentration of dissolved oxygen already starts to decrease at 16–17 m, reaching the minimum at 20–21 m. As a result, the intensity of aerobic processes sharply decreases and organic matter that is not completely mineralized enters the chemocline. In the mixolimnion of the lake, the suspended organic matter content and the total number of microorganisms are significantly lower than in the chemocline and monimolimnion [35,36].

The largest amount of organic matter is formed in the chemocline zones. The major peak of microbial processes in the chemocline coincides with the local maximum for carbon isotope fractionation [35,36]. Oxygenic phototrophic cyanobacteria, highly abundant in the chemocline, provide specific conditions for the functioning of the ecosystem. For example, the first peak of oxygenic photosynthesis is observed in the mixolimnion of the lake, and the second peak of oxygenic and anoxygenic photosynthesis is observed in the chemocline [35,36]. In accordance with previous works [58], we believe that the presence of constant stratification in such water bodies creates a “trap” for nutrients in the anaerobic zone. The organic matter formed in the chemocline enters the anaerobic layers, monimolimnion, where the anaerobic ammonification takes place.

Given that the majority of organic matter in Lake Svetloe undergoes biodegradation essentially under microaerophilic/anaerobic conditions of the chemocline and monimolimnion, the manifestation of the biogeochemical nitrogen cycle is expressed in these horizons in the most vivid and complex relationship with other cycles of elements. Due to the production of a significant amount of organic matter in the chemocline [36] and additional accumulation of OM in the chemocline linked to a sharp increase in the ver-
tical density gradient [18], a sharp increase in ammonium ions (up to 200 times) con-
centration occurs within this relatively thin water layer. A sharp increase in the con-
centration of ammonium ions in the chemocline of the lake in comparison with the
mixolimnion indicates intensive ammonification processes. As a result, at the bound-
ary of aerophilic/microaerophilic/anaerobic conditions, aerobic and facultative anaerobic
ammonifiers are likely to develop intensively.

The chemocline zone can be considered a very strong biological filter of methane.
Indeed, in this zone, the proliferating methanotrophic bacteria [35,36,59] use nitrogen in
the form of ammonia/ammonium or nitrate in the process of methane oxidation. Methan-
otrophic bacteria can assimilate nitrogen as ammonia or nitrate and can compete with
nitrifying bacteria for ammonia and oxygen [28]. It is possible that the nitrifying bacteria
development in the chemocline of Lake Svetloe is limited due to the active process of
methane oxidation since simultaneous nitrification and methane oxidation were observed
only while maintaining relatively high levels of ammonium and oxygen [27]. The use of
nitrate and/or suppression of the nitrification process is consistent with a slight decrease
in nitrate concentrations in the chemocline (Figure 2).

The oxidation of ammonia is carried out by both bacteria and archaea Crenarchaeota or Thaumarchaeota [60,61], which can play a dominant role in the oxidation of ammonia. The distribution of ammonium-oxidizing bacteria (AOB) and ammonium-oxidizing archaea (AOA) along the water column and during the seasons is influenced by environmental factors, such as oxygen concentration [62,63], ammonia concentration [64,65], pH, and concentrations of nitrites, nitrates, and phosphates [66].

Under conditions of low oxygen concentration, ammonium-oxidizing archaea are highly stable [67–69]. In addition, they are probably not influenced by methanotrophic bacteria, as in AOB. The presence of archaea Thaumarchaeota in the oxygen minimum zone [35] at a depth of 17–22 m is consistent with sufficient ammonium concentration and the presence of oxygen. As is known, under anaerobic conditions, ammonification is slow and does not lead to significant ammonia/ammonium production. Therefore, a sharp increase in the concentration of ammonium in the monimolimnion is not observed.

Gamma, δ-, and ε-proteobacteria have an ammonifying capacity, and some of them
are capable of other alternative respiratory pathways, such as dissimilatory iron, sulfate,
and sulfur reduction [70]. The ability to ammonify nitrates and nitrites has been also found
in chemolithotrophs such as oxidizers of Fe (II), hydrogen and sulfides, as well as anammox
bacteria [70,71]. Overall, the effect of high concentrations of ammonium on a decrease in
the assimilability of nitrates in bacteria, cyanobacteria, and archaea, including halophilic
species, was found. When microorganisms capable of assimilating nitrate are exposed to
ammonium, the cells' ability to use nitrate is drastically reduced [70,72–75].

There is another pathway for the formation of ammonium, the so-called dissimi-
latory nitrate reduction to ammonium (DNRA) process, also known as nitrate/nitrite
ammonification [19,70]. Under microaerophilic or anaerobic conditions, DNRA may occur
with the participation of bacteria, cyanobacteria, and archaea, including some halophilic
species [70,72–75]. For this, nitrate is transported into cells by an active transport system
and is reduced to ammonium by the sequential action of assimilatory nitrates (Nas) and
nitrite reductases (NiR) [73,76,77]. In natural environments, dissimilated nitrate reduction
occurs under a high ratio of ammonium nitrogen to nitrate nitrogen [7]. In Lake Svetloe, the
ratio of ammonium to nitrate strongly increases from a depth of 24 m (Figure 5), which may
indicate the predominance of dissimilatory nitrate reduction over denitrification under
anaerobic conditions.
<figure><img src="image_9.png" /><figcaption>**Figure 5.** The ratio of the concentrations of ammonium and nitrate nitrogen in the water column of Lake Svetloe. Middle point—median; box value—percentiles (25–75%); whisker value—min–max.</figcaption></figure>

The monimolimnion of Lake Svetloe remains a fairly stable environment. The decomposition of the main fraction of organic matter, which occurs under the anaerobic conditions of the monimolimnion, is accompanied by the accumulation of nitrogen in the N-NH₄ form, as evidenced by high concentrations of N-NH₄ in all layers regardless of the season. In the monimolimnion of lakes, nitrate is usually present in minimal concentrations, but for Lake Svetloe, rather high concentrations are observed up to the bottom sediments (Figure 2).

Nitrification usually does not occur under anaerobic conditions, whereas the denitrification processes are quite pronounced. Most likely, oxygen is still present in the chemocline and monimolimnion of Lake Svetloe. Microbiological studies of Lake Svetloe [35–37] reported a high relative abundance of cyanobacteria in the chemocline zone. Cyanobacteria were also found in the monimolimnion down to the bottom sediments, but in a lower relative abundance. Oxygen produced by photosynthetic cyanobacteria can be used by nitrifying bacteria, ammonium-oxidizing bacteria, or ammonium-oxidizing archaea, bacteria of the Nitrospirae family, found in small amounts in the chemocline of Lake Svetloe [35], representatives of which carry out the complete oxidation of ammonium into nitrate (comammox).

Comammox was discovered and described relatively recently [78,79]. After oxygen, the nitrate ion is the next preferred electron acceptor for respiration [80]. Moreover, it is the nitrate form of nitrogen that is the first preferred alternative electron acceptor among all nitrogen compounds. Consequently, nitrate ions are primarily subjected to assimilation/dissimilation under anaerobic conditions. The presence of nitrates down to the bottom horizons may indicate, as mentioned above, the presence of oxygen, which promotes the formation of nitrates.

It is also possible that under conditions of high concentrations of manganese and ammonium, anaerobic conditions occur with the formation of nitrates according to the reaction [81,82]:

$$4 \text{MnO}_2 + \text{NH}_4^+ + 6 \text{H}^+ \Rightarrow 4 \text{Mn}^{2+} + \text{NO}_3^- + 5 \text{H}_2\text{O}$$

Usually, the oxidation of Mn to form Mn particles occurs where Mn (II) and oxygen are present. Manganese oxides fall below the redox boundary, where there are very few sulfides acting as a reducing agent, but a sufficient amount of ammonium [50,83]. Manganese reduction can occur under oxidized conditions [84,85]; therefore, oxidation and reduction can occur simultaneously in the same water layer. For meromictic Lake Zug, it has been suggested that ammonium oxidation in the presence of manganese can be a source of nitrates, thus increasing their concentration up to 18 µM in the monimolimnion [50]. It
is important to note that in the anaerobic conditions in the monimolimnion layer of Svetloe Lake, nitrate concentration is still maintained at the 10 µM level, which is even higher than surface concentration. Considering the high concentration of iron and manganese, and in agreement with available literature information, it is most possible that redox-sensitive metals support ammonia oxidation to nitrite and nitrate.

However, we could not unambiguously identify the driver of manganese oxidation in the anaerobic horizons of the Lake Svetloe water column. It should be noted that a measured concentration value of zero does not mean the complete absence of a component. Oxygen can be found in concentrations below the detection limit of the method, for example, in a form not measurable by this method (e.g., peroxide or free radical). There may be an established balance of redox processes as a result of which the formed oxygen is immediately consumed in the reaction. As such, the scenario described above is quite possible.

Microorganisms associated with the process of nitrate/nitrite-dependent anaerobic oxidation of methane (ANME-2d) [35,37], that is, archaea of ANME 2d clusters, including *'CaNdidatus'*, Methanoperedens nitroreducens' and bacteria of the NC10 type (*'CaNdidatus'* Methylomirabilis oxyfera'), are absent or found in very small numbers in Lake Svetloe. Apparently, nitrate/nitrite-dependent anaerobic oxidation of methane is minimal [21,25].

A slight increase in nitrite in the bottom layers can occur due to the oxidation of ammonia to nitrite. In the 27–35 m horizons of Lake Svetloe, ammonia-oxidizing archaea *Thaumarchaeota* of the genus *Nitrosopumilus* are abundant, constituting up to 6.6% of all 16S rRNA gene sequences [35,37]. Ammonia-oxidizing archaea are ubiquitous in marine, freshwater, and terrestrial ecosystems. They are now considered to be a significant contributor to the carbon and nitrogen cycle. Representatives of this genus are autotrophs that receive energy from the aerobic oxidation of ammonia to nitrite and the fixation of inorganic carbon [66,86,87].

The permanent redoxcline and simultaneous presence of ammonia and nitrite pro-vide stable conditions for anaerobic ammonia oxidation ("anammox") [88]. Anammox is the anaerobic oxidation of ammonium with nitrite, resulting in the formation of molecular nitro-gen N₂. Although anammox is an anaerobic process, oxygen does not appear to completely suppress it up to a concentration of ~13.5 µM [89]. Anammox-conducting bacteria live in many natural ecosystems usually under conditions of limited ammonia and make a signifi-cant contribution to the nitrogen cycle [71]. The anammox reaction uses NH₄⁺ иNO₂⁻ in a stoichiometric ratio of about 1:1 [19]. In lake ecosystems, this process has been suggested for sediments [90] or for the chemocline of several meromictic lakes: Lake Tanganyika [91], Lake Rassnitz (Germany; [92]), and Lake Lugano (Switzerland, Italy; [93]). However, for Lake Svetloe, the anammox process is the least likely, since the bacteria Planctomycetes that carry out this process [71,94] have not been found in the lake monimolimnion [35]. It is possible that high ammonium content and low nitrite concentrations in the chemocline and monimolimnion are limiting factors for their development [19,71]. It is also not excluded that, in Lake Svetloe, anammox is additionally inhibited by methanol [89], formed during methane oxidation [95].

**5. Conclusions**

Analysis of the data on the spatio-temporal dynamics of the nitrogen forms and concentrations in the low-sulphide, ferro-manganese meromictic subarctic lake demonstrated the predominance of anaerobic processes in the nitrogen cycle. When the Red/Ox conditions change, the dominant form of mineral nitrogen changes from oxidized (NO₃) to reduced (NH₄). The N-NO₃ form always predominated in the mixolimnion of Lake Svetloe, and seasonal fluctuations in the nitrogen concentration were associated with phytoplankton consumption. Below the depth of 22 m, N-NH₄ predominated, whereas the concentration of N-NO₂ never exceeded 4% of the sum of inorganic forms of nitrogen. In the horizons of the chemocline and monimolimnion, there was a close relationship between the biogeochemical cycles of methane and nitrogen. This is indirectly confirmed by the
local minimum of the nitrate concentration in the chemocline, where the methanotrophic bacteria could use N-NH₄ or N-NO₃ for methane oxidation.

A peculiar feature of the nitrogen cycle in Lake Svetloe is high concentrations of nitrate in the anaerobic waters of monimolimnion. This phenomenon is possibly a consequence of the iron-manganese type of meromixia of the waterbody. Under conditions of high concentrations of manganese and ammonium, anaerobic oxidation of ammonium with the formation of nitrates could occur, where MnO₂ acts as an oxidizing agent. This process is described in the literature for other meromictic bodies. The functioning of the nitrogen cycle in the meromictic Lake Svetloe reflects the climatic features of the subarctic (long glacial season), geochemical factors of the catchment (low DOC content and high transparency), the type of meromixia of the lake (high concentrations of Fe and Mn), and the features of the carbon cycle (methanogenesis/methane oxidation). Further, a more comprehensive analysis of these relationships should provide the necessary information on the functioning of the ecosystem, and the biogeochemical nitrogen cycle provides such an opportunity. Further work is required in this direction, in particular, the assessment of the content of gas components of the nitrogen cycle such as N₂ and N₂O. In this regard, NO might be especially important among other processes as it is closely related to denitrification.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3390/nitrogen2040029/s1, Table S1: The various forms of nitrogen concentration and other physicochemical characteristics of the water column of Lake Svetloe in different seasons of the year. All concentrations are given in µM (for the nitrogen compounds µM nitrogen in L), Figure S1: Vertical and seasonal variability of values of temperature (A), oxygen concentration (B), specific electrical conductivity (C) and density (D).

**Author Contributions:** Conceptualization, T.Y.V. and O.S.P.; methodology A.A.C.; software, A.V.C.; validation, O.S.P. and S.A.Z.; formal analysis, A.A.C.; investigation, A.A.C., A.V.C. and O.Y.M.; writing—original draft preparation, T.Y.V., A.A.C. and A.V.C.; writing—review and editing, O.S.P. and S.A.Z.; supervision, O.S.P.; project administration, T.Y.V.; funding acquisition, T.Y.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the State Task AAAA-A18-118012390200-5.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the Supplementary Materials.

**Acknowledgments:** We are grateful to Klimov S.I. for the hydrological studies of Lake Svetloe.

**Conflicts of Interest:** The authors declare no conflict of interest.

## References

1. Campbell, N.A. *Biology*, 2nd ed.; The Benjamin/Cummings Publishing Company: Redwood City, CA, USA, 1990.

2. Bashkin, V.N. *Biogeochemistry*; Scientific World: Moscow, Russia, 2004; p. 584. (In Russian)

3. Downing, J.; McCauley, E. The nitrogen: Phosphorus relationship in lakes. *Limnol. Oceanogr.* **1992**, *37*, 936–945. [CrossRef]

4. Burt, T.P. Nitrogen Cycle. In *Encyclopedia of Ecology*, 2nd ed.; Fath, B., Ed.; Elsevier: Amsterdam, The Netherlands, 2013; Volume 4, pp. 135–142. [CrossRef]

5. Gruber, N.; Galloway, J. An Earth-system perspective of the global nitrogen cycle. *Nature* **2008**, *451*, 293–296. [CrossRef] [PubMed]

6. Mackenzie, F.; Ver, L.M.; Sabine, C.; Lane, M.; Lerman, A. C, N, P, S global biogeochemical cycles and modeling of global change. In *Interactions of C, N, P and S Biogeochemical Cycles and Global Change*; NATO ASI Series (Series I: Global Environmental Change); Wollast, R., Mackenzie, F.T., Chou, L., Eds.; Springer: Berlin/Heidelberg, Germany, 1993; Volume 4, pp. 1–61. [CrossRef]

7. Kuznetsov, S.I.; Dubinina, G.A. *Methods for Studying Aquatic Microorganisms*; Science: Moscow, Russia, 1989; p. 285. (In Russian)

8. Cabello, P.; Roldán, M.D.; Castillo, F.; Moreno-Vivián, C. Nitrogen Cycle. In *Encyclopedia of Microbiology*; Schaechter, M., Ed.; Elsevier Science Ltd.: Amsterdam, The Netherlands, 2009; pp. 299–321. [CrossRef]

9. Kuypers, M.; Marchant, H.; Kartal, B. The microbial nitrogen-cycling network. *Nat. Rev. Microbiol.* **2018**, *16*, 263–276. [CrossRef] [PubMed]

10. Pajares, S.; Ramos, R. Processes and Microorganisms Involved in the Marine Nitrogen Cycle: Knowledge and Gaps. *Front. Mar. Sci.* **2019**, *6*, 739. [CrossRef]
11. Hayatsu, M.; Tago, K.; Saito, M. Various players in the nitrogen cycle: Diversity and functions of the microorganisms involved in nitrification and denitrification. *Soil Sci. Plant Nutr.* **2008**, *54*, 33–45. [CrossRef]

12. Stein, L.Y.; Klotz, M.G. The nitrogen cycle. *Curr. Biol.* **2016**, *26*, R94–R98. [CrossRef] [PubMed]

13. Zehr, J.P.; Ward, B.B. Nitrogen cycling in the ocean: New perspectives on processes and paradigms. *Appl. Environ. Microbiol.* **2002**, *68*, 1015–1024. [CrossRef]

14. Kuznetsov, S.I.; Saralov, A.I.; Nazina, T.N. *Microbiological Processes of Carbon and Nitrogen Circulation in Lakes*; Science: Moscow, Russia, 1985; p. 216. (In Russian)

15. Gorlenko, V.M.; Dubinina, G.A.; Kuznetsov, S.I. *Ecology of Aquatic Microorganisms*; Science: Moscow, Russia, 1977; p. 289. (In Russian)

16. Camargo, J.A.; Alonso, Á. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. *Environ. Int.* **2006**, *32*, 831–849. [CrossRef]

17. Stewart, K.M.; Walker, K.F.; Likens, G.E. Meromictic Lakes. In *Encyclopedia of Inland Waters*; Likens, G.E., Ed.; Academic Press (Elsevier): Amsterdam, the Netherlands, 2009; pp. 589–602. [CrossRef]

18. Gulati, R.D; Zadereev, E.S.; Degermendzhi, A.G. (Eds.) *Ecology of Meromictic Lakes*; Springer International Publishing: Cham, Switzerland, 2017. [CrossRef]

19. Lam, P.; Kuypers, M.M. Microbial nitrogen cycling processes in oxygen minimum zones. *Ann. Rev. Mar. Sci.* **2011**, *3*, 317–345. [CrossRef]

20. Ward, B.B.; Glover, H.E.; Lipschultz, F. Chemoautotrophic activity and nitrification in the oxygen minimum zone off Peru. *Deep. Sea Res. Part A. Oceanogr. Res. Pap.* **1989**, *36*, 1031–1051. [CrossRef]

21. Crowe, S.A.; Katsev, S.; Leslie, K.; Sturm, A.; Magen, C.; Nomosatryo, S.; Pack, M.A.; Kessler, J.D.; Reeburgh, W.S.; Roberts, J.A.; et al. The methane cycle in ferruginous Lake Matano. *Geobiology* **2011**, *9*, 61–78. [CrossRef] [PubMed]

22. Oshkin, I.Y.; Beck, D.A.; Lamb, A.E.; Tchesnokova, V.; Benuska, G.; McTaggart, T.L.; Kalyuzhnaya, M.G.; Dedysh, S.N.; Lidstrom, M.E.; Chistoserdova, L. Methane-fed microbial microcosms show differential community dynamics and pinpoint taxa involved in communal response. *ISME J.* **2015**, *9*, 1119–1129. [CrossRef]

23. Chistoserdova, L. Methylotrophs in natural habitats: Current insights through metagenomics. *Appl. Microbiol. Biotechnol.* **2015**, *99*, 5763–5779. [CrossRef]

24. Rissanen, A.J.; Saarenheimo, J.; Tirola, M.; Peura, S.; Aalto, S.L.; Karvinen, A.; Nykanen, H. Gammaproteobacterial methanotrophs dominate methanotrophy in aerobic and anaerobic layers of boreal lake waters. *Aquat. Microb. Ecol.* **2018**, *81*, 257–276. [CrossRef]

25. He, Z.; Zhang, Q.; Feng, Y.; Luo, H.; Pan, X.; Gadd, G.M. Microbiological and environmental significance of metal-dependent anaerobic oxidation of methane. *Sci. Total Environ.* **2018**, *610*, 759–768. [CrossRef]

26. Cabrol, L.; Thalasso, F.; Gandois, L.; Sepulveda-Jauregui, A.; Martinez-Cruz, K.; Teisserenc, R.; Tananaev, N.; Tveit, A.; Svenning, M.M.; Barret, M. Anaerobic oxidation of methane and associated microbiome in anoxic water of Northwestern Siberian lakes. *Sci. Total Environ.* **2020**, *736*, 139588. [CrossRef]

27. Knowles, R. Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle. *Ecol. Eng.* **2005**, *24*, 441–446. [CrossRef]

28. Megmw, S.R.; Knowles, R. Active methanotrophs suppress nitrification in a humisol. *Biol. Fert. Soils* **1987**, *4*, 205–212. [CrossRef]

29. Raghoebarsing, A.A.; Pol, A.; van de Pas-Schoonen, K.T.; Smolders, A.J.P.; Ettwig, K.F.; Rijpstra, W.I.C.; Schouten, S.; Damsté, J.S.S.; Op den Camp, H.J.M.; Jetten, M.S.M.; et al. A microbial consortium couples anaerobic methane oxidation to denitrification. *Nature* **2006**, *440*, 918–921. [CrossRef]

30. Guggenheim, C.; Freimann, R.; Mayr, M.J.; Beck, K.; Wehrli, B.; Bürgmann, H. Environmental and Microbial Interactions Shape Methane-Oxidizing Bacterial Communities in a Stratified Lake. *Front. Microbiol.* **2020**, *11*, 579427. [CrossRef]

31. Einsiedl, F.; Wunderlich, A.; Sebilo, M.; Coskun, Ö.K.; Orsi, W.D.; Mayer, B. Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes. *Biogeosciences* **2020**, *17*, 5149–5161. [CrossRef]

32. Kits, K.D.; Klotz, M.G.; Stein, L.Y. Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium *Methylomonas denitrificans*, sp. nov. type strain FJG1: Denitrifying metabolism in M. denitrificans FJG1. *Environ. Microbiol.* **2015**, *17*, 3219–3232. [CrossRef] [PubMed]

33. Zabelina, S.A.; Kompantseva, E.I.; Chupakov, A.V.; Ershova, A.A. Seasonal dynamics of photosynthetic pigments in a freshwater meromictic lake. In *Biodiagnostics of the State of Natural and Natural-Technogenic SYSTEMS, Proceedings of the XII All-Russian scientific-Practical Conference with International Participation, Kirov, Russia, 2–3 December 2014*; Publishing House of LLC "Vesi": Kirov, Russia, 2014; pp. 119–123. (In Russian)

34. Kokryatskaya, N.M.; Chupakov, A.V.; Titova, K.; Chupakova, A.A.; Zabelina, S.A.; Moreva, O.Yu.; Neverova, N.V.; Zhibareva, T.A. Hydrological and hydrochemical characteristics of the iron-manganese meromictic freshwater Lake Svetloe (Arkhangelsk Region). *J. Sib. Fed. Univ. Biol.* **2019**, *12*, 147–159. [CrossRef]

35. Kallistova, A.; Kadnikov, V.; Rusanov, I.; Kokryatskaya, N.; Beletsky, A.; Mardanov, A.; Savvichev, A.; Ravin, N.; Pimenov, N. Microbial communities involved in aerobic and anaerobic methane cycling in a meromictic ferruginous subarctic lake. *Aquat. Microb. Ecol.* **2019**, *82*, 1–18. [CrossRef]
36. Savvichev, A.S.; Rusanov, I.I.; Zakharova, E.E.; Veslopolova, E.F.; Lunina, O.N.; Sigalevich, P.A.; Pimenov, N.V.; Gorlenko, V.M.; Kokryatskaya, N.M.; Zabelina, S.A.; et al. Microbial processes of the carbon and sulfur cycles in an ice-covered, iron-rich meromictic lake Svetloe (Arkhangelsk region, Russia). *Environ. Microbiol.* **2017**, *19*, 659–672. [CrossRef] [PubMed]

37. Kadnikov, V.V.; Mardanov, A.V.; Beletsky, A.V.; Ravin, N.V.; Savvichev, A.S.; Merkel, A.Y.; Pimenov, N.V. Microbial communities involved in the methane cycle in the near-bottom water layer and sediments of the meromictic subarctic Lake Svetloe. *Antonie Leeuwenhoek* **2019**, *112*, 1801–1814. [CrossRef]

38. Ershova, A.A.; Vorobyeva, T.Ya.; Moreva, O.Yu.; Chupakov, A.V.; Zabelina, S.A.; Neverova, N.V. Hydrochemical and microbiological research of a nitrogen cycle in freshwater meromictic lake Svetloe (the Arkhangelsk region). *Reg Environ.* **2015**, *5*, 44–50. (In Russian)

39. Chupakov, A.V.; Pokrovsky, O.S.; Shirokova, L.S.; Vorobyova, T.Ya.; Zabelina, S.A.; Kokryatskaya, N.M.; Moreva, O.Yu.; Ershova, A.A.; Shorina, N.V.; Klimov, S.I. Hydrochemical characteristics of meromictic freshwater Lake Svetloe (Arkhangelsk region). *Vestn. North. Arct. Fed. University. Ser. Nat. Sci.* **2013**, *1*, 20–31. (In Russian)

40. Chupakov, A.V.; Chupakova, A.A.; Moreva, O.Yu.; Shirokova, L.S.; Zabelina, S.A.; Vorobieva, T.Y.; Klimov, S.I.; Brovko, O.S.; Pokrovsky, O.S. Allochthonous and autochthonous carbon in deep, organic rich and organic-poor lakes of the European Russian subarctic. *Boreal Environ. Res.* **2017**, *22*, 213–230.

41. Canfield, D.E.; Rosing, M.T.; Bjerrum, C. Early anaerobic metabolisms. *Philos. Trans. R. Soc. Lond. B Biol. Sci.* **2006**, *361*, 1819–1836. [CrossRef]

42. Scanlan, D.J.; West, N.J. Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus. *FEMS Microbiol. Ecol.* **2002**, *40*, 1–12. [CrossRef]

43. Partensky, F.; Blanchot, J.; Vaulot, D. Differential distribution and ecology of Prochlorococcus and Synechococcus in oceanic waters. In *Reviews in Marine Cyanobacteria*. No. NS 19. *Bulletin de l'Institut Oceanographique Monaco*; Charpy, L., Larkum, A.W.D., Eds.; Musee Oceanographique: Monaco, 1999; Volume 19, pp. 457–475.

44. Willey, J.M.; Waterbury, J.B. Chemotaxis toward Nitrogenous Compounds by Swimming Strains of Marine Synechococcus spp. *Appl. Environ. Microbiol.* **1989**, *55*, 1888–1894. [CrossRef]

45. Steunou, A.S.; Jensen, S.I.; Brecht, E.; Becraft, E.D.; Bateson, M.M.; Kilian, O.; Bhaya, D.; Ward, D.M.; Peters, J.W.; Grossman, A.R.; et al. Regulation of nif gene expression and the energetics of N2 fixation over the diel cycle in a hot spring microbial mat. *ISME J.* **2008**, *2*, 364–378. [CrossRef]

46. Koroleff, F. Total and organic nitrogen. In *Methods for Seawater Analysis*; Grasshoff, K., Ehrhardt, M., Kremling, K., Eds.; Verlag Chemie: Weinheim, Germany, 1983; pp. 162–168.

47. Lozovik, P.A.; Efremenko, N.A. (Eds.) *Analytical, Kinetic and Computational Methods in Hydrochemical Practice*; Nestor-History: S-Pb, Russia, 2017; p. 272. (In Russian)

48. Golterman, H.L. *Methods for Chemical Analysis of Fresh Waters*; Blackwell Scientific: Oxford, UK, 1971; p. 166.

49. Biderre-Petit, C.; Taib, N.; Gardon, H.; Hochart, C.; Debroas, D. New insights into the pelagic microorganisms involved in the methane cycle in the meromictic Lake Pavin through metagenomics. *FEMS Microbiol. Ecol.* **2019**, *95*, fyi183. [CrossRef]

50. Oswald, K.; Milucka, J.; Brand, A.; Hach, P.; Littmann, S.; Wehrli, B.; Kuypers, M.M.M.; Schubert, C.J. Aerobic gammaproteobacterial methanotrophs mitigate methane emissions from oxic and anoxic lake waters. *Linnol. Oceanogr.* **2016**, *61*, S101–S118. [CrossRef]

51. Rodrigo, M.; Miracle, M.; Vicente, E. The meromictic Lake La Cruz (Central Spain). Patterns of stratification. *Aquat. Sci.* **2001**, *63*, 406–416. [CrossRef]

52. Llirós, M.; Garciá-Armisen, T.; Darchambeau, F.; Morana, C.; Triadó-Margarit, X.; Inceoğlu, Ö.; Borrego, C.M.; Bouillon, S.; Servais, P.; Borges, A.V.; et al. Pelagic photoferrotrophy and iron cycling in a modern ferruginous basin. *Sci. Rep.* **2015**, *5*, 13803. [CrossRef] [PubMed]

53. Umanskaya, M.V.; Tarasova, N.G.; Gorbunov, M.Y. Phototrophic plankton of siderotrophic meromictic Lake Kuznechikha (Republic Mari El, Russia). *Inland Water Biol.* **2017**, *10*, 158–167. [CrossRef]

54. Hove, P.V.; Belzile, C.; Gibson, J.A.E.; Vincent, W.F. Coupled landscape-lake evolution in High Arctic Canada. *Can. J. Earth Sci.* **2006**, *43*, 533–546. [CrossRef]

55. Stepanova, A.B.; Sharafutdinova, G.F.; Voyakina, E.Y. Hydrochemical features of Valaam Island small lakes. *Sci. Notes RSHU* **2010**, *12*, 97–110. (In Russian)

56. Culver, D.A. Biogenic meromixis and stability in a soft-water lake. *Linnol. Oceanogr.* **1977**, *22*, 667–686. [CrossRef]

57. Newton, R.J.; Jones, S.E.; Eiler, A.; McMahon, K.D.; Bertilsson, S. A guide to the natural history of freshwater lake bacteria. *Microbiol. Mol. Biol. Rev.* **2011**, *75*, 14–49. [CrossRef]

58. Melack, J.M.; Jellison, R. Limnological conditions in Mono Lake: Contrasting monomixis and meromixis in the 1990s. *Hydrobiologia* **1998**, *384*, 21–39. [CrossRef]

59. Zabelina, S.A.; Pokrovsky, O.S.; Klimov, S.I.; Chupakov, A.V.; Kokryatskaya, N.M.; Vorobyova, T.Y. The first results of the methane concentration in the stratified lakes of the River Svetlaya (the catchment area of the White Sea). In *Geology of Seas and Oceans, Proceedings of the XX International Scientific Conference (School) on Marine Geology, Moscow, Russia, 18–23 November 2013*; Lisitzin, A.P., Politova, N.V., Shevchenko, V.P., Eds.; GEOS: Moscow, Russia, 2013; Volume III, pp. 156–160. (In Russian)

60. Brochier-Armanet, C.; Boussau, B.; Gribaldo, S.; Forterre, P. Mesophilic crenarchaeota: Proposal for a third archaeal phylum, the Thaumarchaeota. *Nat. Rev. Microbiol.* **2008**, *6*, 245–252. [CrossRef] [PubMed]
61. Hugoni, M.; Etien, S.; Bourges, A.; Lepère, C.; Domaizon, I.; Mallet, C.; Bronner, G.; Debroas, D.; Mary, I. Dynamics of ammonia-oxidizing Archaea and Bacteria in contrasted freshwater ecosystems. *Res. Microbiol.* **2013**, *164*, 360–370. [CrossRef] [PubMed]

62. Lliros, M.; Gich, F.; Plasencia, A.; Auguet, J.C.; Darchambeau, F.; Casamayor, E.O.; Descy, J.P.; Borrego, C. Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo). *Appl. Environ. Microbiol.* **2010**, *76*, 6853–6863. [CrossRef] [PubMed]

63. Pouliot, J.; Galand, P.E.; Lovejoy, C.; Vincent, W.F. Vertical structure of archaeal communities and the distribution of ammonia monooxygenase A gene variants in two meromictic High Arctic lakes. *Environ. Microbiol.* **2009**, *11*, 687–699. [CrossRef] [PubMed]

64. Hatzenpichler, R.; Lebedeva, A.; Spieck, E.; Stoecker, K.; Richter, A.; Daimis, H.; Wagner, M. A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. *Proc. Natl. Acad. Sci. USA* **2008**, *105*, 2134–2139. [CrossRef]

65. Park, H.D.; Wells, G.F.; Bae, H.; Criddle, C.S.; Francis, C.A. Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors. *Appl. Environ. Microb.* **2006**, *72*, 5643–5647. [CrossRef]

66. Erguder, T.H.; Boon, N.; Wittebolle, L.; Marzorati, M.; Verstraete, W. Environmental factors shaping the ecological niches of ammonia-oxidizing archaea. *FEMS Microbiol. Rev.* **2009**, *33*, 855–869. [CrossRef]

67. Lam, P.; Jensen, M.M.; Lavik, G.; McGinnis, D.F.; Muller, B.; Schubert, C.J.; Amann, R.; Thamdrup, B.; Kuypers, M.M.M. Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea. *Proc. Natl. Acad. Sci. USA* **2007**, *104*, 7104–7109. [CrossRef]

68. Beman, J.M.; Popp, B.N.; Francis, C.A. Molecular and biogeochemical evidence for ammonia oxidation by marine Crenarchaeota in the Gulf of California. *ISME J.* **2008**, *2*, 429–441. [CrossRef]

69. Molina, V.; Belmar, L.; Ulloa, O. High diversity of ammonia-oxidizing archaea in permanent and seasonal oxygen-deficient waters of the eastern South Pacific. *Environ. Microbiol.* **2010**, *12*, 2450–2465. [CrossRef] [PubMed]

70. Simon, J. Enzymology and bioenergetics of respiratory nitrite ammonification. *FEMS Microbiol. Rev.* **2002**, *26*, 285–309. [CrossRef]

71. Kartal, B.; Kuypers, M.M.M.; Lavik, G.; Schalk, J.; Op den Camp, H.J.M.; Jetten, M.S.; Strous, M. Anammox bacteria disguised as denitrifiers: Nitrate reduction to dinitrogen gas via nitrite and ammonium. *Environ. Microbiol.* **2007**, *9*, 635–642. [CrossRef]

72. Flores, E.; Ramos, J.L.; Herrero, A.; Guerrero, M.G. Nitrate assimilation by cyanobacteria. In *Photosynthetic Prokaryotes: Cell Differentiation and Function*; Papageorgiou, G.C., Packer, L., Eds.; Elsevier: New York, NY, USA, 1983; pp. 363–387.

73. Martínez-Espinosa, R.M.; Marhuenda-Egea, F.C.; Bonete, M.J. Assimilatory nitrate reductase from the haloarchaeon *Haloferax mediterranei*: Purification and characterisation. *FEMS Microbiol. Lett.* **2001**, *204*, 381–385. [CrossRef]

74. Martínez-Espinosa, R.M.; Lledó, B.; Marhuenda-Egea, F.C.; Bonete, M.J. The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon *Haloferax mediterranei*. *Extremophiles* **2007**, *11*, 759–767. [CrossRef] [PubMed]

75. Martínez-Espinosa, R.M. Microorganisms and their metabolic capabilities in the context of the biogeochemical nitrogen cycle at extreme environments. *Int. J. Mol. Sci.* **2020**, *21*, 4228. [CrossRef]

76. Moreno-Vivián, C.; Cabello, P.; Martínez-Luque, M.; Blasco, R.; Castillo, F. Prokaryotic nitrate reduction: Molecular properties and functional distinction among bacterial nitrate reductases. *J. Bacteriol.* **1999**, *181*, 6573–6584. [CrossRef] [PubMed]

77. Richardson, D. Introduction: Nitrate reduction and the nitrogen cycle. *Cell. Mol. Life Sci.* **2001**, *58*, 163–164. [CrossRef] [PubMed]

78. Daimis, H.; Lebedeva, E.V.; Pjevac, P.; Han, P.; Herbold, C.; Albertsen, M.; Jehmlich, N.; Palatinszky, M.; Vierheilig, J.; Bulaev, A.; et al. Complete nitrification by Nitrospira bacteria. *Nature* **2015**, *528*, 504–509. [CrossRef]

79. Kits, K.D.; Sedlacek, C.J.; Lebedeva, E.V.; Han, P.; Bulaev, A.; Pjevac, P.; Daebeler, A.; Romano, S.; Albertsen, M. Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle. *Nature* **2017**, *549*, 269–272. [CrossRef]

80. Froelich, P.N.; Klinkhammer, G.P.; Bender, M.L.; Luedtke, N.A.; Heath, G.R.; Cullen, D.; Dauphin, P.; Hammond, D.; Hartman, B.; Maynard, V. Early oxidation of organic matter in pelagic sediments of eastern equatorial Atlantic: Suboxic diagenesis. *Geochim. Cosmochim. Acta* **1979**, *43*, 1075–1090. [CrossRef]

81. Luther, G.W.; Sundby, B.; Lewis, B.L.; Brendel, P.J.; Silverberg, N. Interactions of manganese with the nitrogen cycle: Alternative pathways to dinitrogen. *Geochim. Cosmochim. Acta* **1997**, *61*, 4043–4052. [CrossRef]

82. Hulth, S.; Aller, R.C.; Gilbert, F. Coupled anoxic nitrification/manganese reduction in marine sediments. *Geochim. Cosmochim. Acta* **1999**, *63*, 49–66. [CrossRef]

83. Jones, C.; Crowe, S.A.; Sturm, A.; Leslie, K.L.; MacLean, L.C.W.; Katsev, S.; Henny, C.; Fowle, D.A.; Canfield, D.E. Biogeochemistry of manganese in ferruginous Lake Matano, Indonesia. *Biogeosciences* **2011**, *8*, 2977–2991. [CrossRef]

84. Bratina, B.J.; Stevenson, B.S.; Green, W.J.; Schmidt, T.M. Manganese reduction by microbes from oxic regions of the Lake Vanda (Antarctica) water column. *Appl. Environ. Microbiol.* **1998**, *64*, 3791–3797. [CrossRef] [PubMed]

85. Canfield, D.; Kristensen, E.; Thamdrup, B. *Aquatic Geomicrobiology*; Advances Marine Biology Series; Southward, A.J., Tyler, P.A., Young, C.M., Fuiman, L.A., Eds.; Elsevier: San Diego, CA, USA, 2005; p. 640.

86. Konneke, M.; Bernhard, A.E.; de la Torre, J.R.; Walker, C.B.; Waterbury, J.B.; Stahl, D.A. Isolation of an autotrophic ammonia-oxidizing marine archaeon. *Nature* **2005**, *437*, 543–546. [CrossRef] [PubMed]

87. Walker, C.B.; de la Torre, J.R.; Klotz, M.G.; Urakawa, H.; Pinel, N.; Arp, D.J.; Brochier-Armanet, C.; Chain, P.S.J.; Chan, P.P.; Gollabgir, A.; et al. Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaeota. *Proc. Natl. Acad. Sci. USA* **2010**, *107*, 8818–8823. [CrossRef]

88. Mulder, A.; van de Graaf, A.A.; Robertson, L.A.; Kuenen, J.G. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. *FEMS Microbiol. Ecol.* **1995**, *16*, 177–184. [CrossRef]
89. Jensen, M.M.; Thamdrup, B.; Dalsgaard, T. Effects of specific inhibitors on anammox and denitrification in marine sediments. *Appl. Environ. Microbiol.* **2007**, *73*, 3151–3158. [CrossRef]

90. Hu, B.-I.; Shen, L.-D.; Xu, X.-Y.; Zheng, P. Anaerobic ammonium oxidation (anammox) in different natural ecosystems. *Biochem. Soc. Trans.* **2011**, *39*, 1811–1816. [CrossRef] [PubMed]

91. Schubert, C.J.; Durisch-Kaiser, E.; Wehrli, B.; Thamdrup, B.; Lam, P.; Kuypers, M.M.M. Anaerobic ammonium oxidation in a tropical freshwater system (Lake Tangayika). *Environ. Microbiol.* **2006**, *8*, 1857–1863. [CrossRef] [PubMed]

92. Hamersley, M.R.; Woebken, D.; Boehrer, B.; Schultze, M.; Lavik, G.; Kuypers, M.M.M. Water column anammox and denitrification in a temperate permanently stratified lake (Lake Rassnitzer, Germany). *Syst. Appl. Microbiol.* **2009**, *32*, 571–582. [CrossRef] [PubMed]

93. Wenk, C.B.; Zopfi, J.; Blees, J.; Veronesi, M.; Niemann, H.; Lehmann, M.F. Community N and O isotope fractionation by sulphide-dependent denitrification and anammox in a stratified lacustrine water column. *Geochim. Cosmochim. Acta* **2014**, *125*, 551–563. [CrossRef]

94. Strous, M.; Pelletier, E.; Mangenot, S.; Rattei, T.; Lehner, A.; Taylor, M.; Horn, M.; Daim, H.; Bartol-Mavel, D.; Wincker, P.; et al. Deciphering the evolution and metabolism of an anammox bacterium from a community genome. *Nature* **2006**, *440*, 790–794. [CrossRef]

95. Anthony, C. *The Biochemistry of Methylotrophs*; Academic Press: London, UK, 1982.