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Determining accurate rates of benthic nitrogen (N) removal and retention pathways from diverse environments is critical to our understanding of process distribution and constructing reliable N budgets and models. The whole-core N-15 isotope pairing technique (IPT) is one of the most widely used methods to determine rates of benthic nitrate-reducing processes and has provided valuable information on processes and factors controlling N removal and retention in aquatic systems. While the whole core IPT has been employed in a range of environments, a number of methodological and environmental factors may lead to the generation of inaccurate data and are important to acknowledge for those applying the method. In this review, we summarize the current state of the whole core IPT and highlight some of the important steps and considerations when employing the technique. We discuss environmental parameters which can pose issues to the application of the IPT and may lead to experimental artifacts, several of which are of particular importance in environments heavily impacted by eutrophication. Finally, we highlight the advances in the use of the whole-core IPT in combination with other methods, discuss new potential areas of consideration and encourage careful and considered use of the whole-core IPT. With the recognition of potential issues and proper use, the whole-core IPT will undoubtedly continue to develop, improve our understanding of benthic N cycling and allow more reliable budgets and predictions to be made.

This report is related to the BONUS project “Nutrient Cocktails in COAstal zones of the Baltic Sea” alias COCOA. The aim of BONUS COCOA is to investigate physical, biogeochemical and biological processes in a combined and coordinated fashion to improve the understanding of the interaction of these processes on the removal of nutrients along the land-sea interface. The report is especially related to BONUS COCOA WP 6 in which the main objective is extrapolation of results from the BONUS COCOA learning sites to coastal sites around the Baltic Sea in general. Specific objectives of this deliverable (D6.4) were to connect observed process rates with modelling data and ecosystem characteristics. In the report we made statistical analyses of observations from BONUS COCOA study sites together with results from the Swedish Coastal zone Model (SCM). Eight structural variables (water depth, temperature, salinity, bottom water concentrations of oxygen, ammonium, nitrate and phosphate, as well as nitrogen content in sediment) were found common to both the experimentally determined and the model data sets. The observed process rate evaluated in this report was denitrification. In addition regressions were tested between observed denitrification rates and several structural variables (latitude, longitude, depth, light, temperature, salinity, grain class, porosity, loss of ignition, sediment organic carbon, total nitrogen content in the sediment, sediment carbon/nitrogen-ratio, sediment chlorphyll-a as well as bottom water concentrations of oxygen, ammonium, nitrate, and dissolved inorganic phosphorus and silicate) for pooled data from all learning sites. The statistical results showed that experimentally determined multivariate data set from the shallow, illuminated stations was mainly found to be similar to the multivariate data set produced by the SCM model. Generally, no strong correlations of simple relations between observed denitrification and available structural variables were found for data collected from all the learning sites. We found some non-significant correlation between denitrification rates and bottom water dissolved inorganic phosphorous and dissolved silica but the reason behind the correlations is not clear. We also developed and evaluated a theory to relate process rates to monitoring data and nutrient retention. The theoretical analysis included nutrient retention due to denitrification as well as burial of phosphorus and nitrogen. The theory of nutrient retention showed good correlations with model results. It was found that area-specific nitrogen and phosphorus retention capacity in a sub-basin depend much on mean water depth, water residence time, basin area and the mean nutrient concentrations in the active sediment layer and in the water column.

Evidence of aerobic methane oxidation coupled to denitrification has been provided for different freshwater environments, whereas the significance of this process for the marine realm has not been adequately investigated. The goal of this study was to investigate the methane-related reduction of nitrate/nitrite in a marine environment (salinity 8.5). A water sample was collected from the oxic-anoxic transition zone of the Gotland Deep (central Baltic Sea) and the microorganisms contained therein were cultivated in a bioreactor under hypoxic conditions (0.5 μM O2). To enrich the microorganisms involved in the coupled process the bioreactor was continuously sparged with methane as the sole energy and carbon source and simultaneously supplied with a nutrient solution rich in nitrate and nitrite. The bioreactor experiment showed a relationship between the turnover of methane and the concomitant concentration decrease of nitrite and nitrate at the early stage of the experiment. This relationship indicates the role of methanotrophs, which may support heterotrophic denitrifiers by the release of organic compounds as an energy source. Besides, a mixture of uncultured microorganisms, aerobic methanotrophic and heterotrophic denitrifying bacteria were identified in the enrichment culture. Microbial incorporation of nitrite and methane was proven on the cellular and gene levels via 15NO−22-/13CH4 incubation experiments and subsequent analyses with nano secondary ion mass spectrometry (NanoSIMS) and stable isotope probing (SIP). The NanoSIMS showed the incorporation of 15N in almost all the bacteria and in 9% of those there was a concomitant enrichment in 13C. The relatively low abundance of methane-consuming bacteria in the bioreactor was further reflected in specific fatty acids indicative for type I methanotrophic bacteria. Based on pmoA gene analyses, this bacterium is different from the one that was identified as the only key player of methane oxidation in previous studies in the Gotland Deep, indicating the existence of other subordinate methanotrophic bacteria at that site. The results provide the first indications for the predisposition of a methane-related reduction of nitrate/nitrite under hypoxic conditions in the marine realm, supporting the assumption of an interaction between methanotrophic and denitrifying bacteria which hitherto has only been described for fresh water environments.

Methane is a powerful greenhouse gas that contributes significantly to global warming. In aquatic systems, microbes in anoxic sediments are the main methane producers. However, due to effective oxidative filters in the sediments and the water column, most of the methane produced does not end up in the atmosphere. This study explores methane dynamics in the Baltic Sea from the open sea, to estuaries and specific microbial processes. Major inflow cycles control methane dynamics in the open Baltic by bringing oxygen to the deep basins, where methane typically accumulates in large amounts during stagnation. The introduction of oxygen during a major inflow in 2014–2015 caused the disappearance of methane from the deep basins due to a combination of oxidation and displacement. However, the effects of the inflow were short-lived and methane started accumulating again in less than a year after the inflow. The coastal areas were more dynamic, and the primary source of methane varied with distance offshore. Near the river mouth of the studied estuarine system, methane brought in by the river was the most important source, whereas further offshore sedimentary methanogenesis fuelled by a legacy of eutrophication was the primary source. Atmospheric fluxes of methane were highest near the river mouth and decreased seawards, while bathymetry was the main control of sedimentary fluxes. Seasonality had a strong effect on methane dynamics, with methane concentrations generally increasing towards winter. However, as in the open Baltic, displacement also played a role at times, removing large amounts of methane at a time. While aerobic methane oxidation in the water column was the primary sink offshore, in the coastal areas anaerobic oxidation of methane (AOM) was by far the most important sink. Offshore, sulphate mediated AOM is expected to be the most important type of AOM. However in this study, metal mediated AOM was an equally important sink in the sediments. Both rates of AOM, and microbial community abundances, were highest below the main sulfidic zone in the sediment, pointing towards non-sulphate AOM pathways. Overall, eutrophication has had a large impact on methane dynam ics in the Baltic Sea. The legacy of past eutrophication fuels methanogenesis in the coastal areas to this day, despite reductions in nutrient and organic matter input from land, leading to enhanced atmospheric flux of methane. In the future, climate change will likely exacerbate methane emissions from the Baltic Sea. Metaani on voimakas kasvihuonekaasu, jolla on merkittävä vaikutus ilmaston lämpenemiseen. Vesiekosysteemeissä sitä muodostuu pääosin hapettomissa sedimenteissä, joista muodostuva kaasu pyrkii kohoamaan vesipatsaan läpi ilmakehään. Tehokkaat hapetusprosessit sedimentissä ja vesipatsaassa kuitenkin estävät suurinta osaa muodostuvasta kaasusta saavuttamasta ilmakehää. Tämä tutkimus tarkastelee Itämeren metaaniprosessien dynamiikkaa avomerellä, rannikkosedimenteissä ja metaania hapettavien mikrobiprosessien kannalta. Suolavesipulssit säätelevät metaanin esiintymistä Itämeren syvänteissä. Vuoden 2014 lopun suuri suolavesipulssi poisti tehokkaasti Gotlannin syvänteeseen aiemmin kertyneen metaanin, osin hapettumisen ja osin vesimassojen syrjäyttämisen kautta. Tämä vaikutus jäi kuitenkin lyhytaikaiseksi ja metaania havaittiin kertyvän jälleen syvänteisiin alle vuoden sisällä suolapulssista. Itämeren rannikkoalueet olivat metaaniprosesseiltaan avomerta dynaamisempia. Metaanin pääasiallinen lähde muuttui etäisyyden rannasta kasvaessa. Jokisuun lähistöllä jokiveden mukaan tuoma metaani oli tärkein metaanin lähde rannikkovesissä, siinä missä kauempana rannasta sedimenteissä tapahtuva metaanituotanto oli tärkein tekijä. Aiempien vuosikymmenten aikana tapahtuneen rehevöitymisen katsottiin vaikuttavan sedimentissä muodostuvia metaanimääriä lisäävästi. Eniten ilmakehään päätyi metaania jokisuun lähettyviltä ja määrät vähenivät kauempana rannasta. Sedimentistä vesipatsaaseen päätyvän metaanin määrään vaikuttivat eniten rannikon pohjatyyppi. Vuodenaikaisvaihtelu metaanin pitoisuuksissa niin vesipatsaassa kuin ilmakehäpäästöissäkin oli suurta, mutta yleisesti ottaen molemmat kasvoivat talvea kohti. Toisaalta kuten avomerelläkin, myös vesimassojen liikkeillä oli voimakas vaikutus veden metaanipitoisuuksiin. Siinä missä vesipatsaassa tapahtuva hapettuminen oli avomerellä tärkein metaanin nielu, rannikkoalueilla sedimenteissä tapahtuva hapetus olivat selvästi tärkein prosessi. Meriveden sulfaattipitoisuuden takia sulfaattipohjaisen metaanihapetuksen odotetaan yleensä voimistuvan korkeammissa suolapitoisuuksissa, mikä piti paikkansa tässäkin tutkimuksessa. Tämän lisäksi kuitenkin havaittiin myös, että syvemmällä sedimenteissä tapahtuvat metallipohjaiset metaanin hapetusreaktiot olivat vähintään yhtä tärkeitä tutkitulla alueella. Kaiken kaikkiaan rehevöitymisellä on suuri vaikutus Itämeren metaaniprosesseihin. Vaikka ravinnekuormitus valuma-alueilta onkin nykyisin vähäisempää, menneiden vuosikymmenten rehevöityminen vaikuttaa edelleen metaanin tuotantoa ja ilmakehäpäästöjä lisäävästi. Tämän lisäksi ilmastonmuutoksen aiheuttaman yhteisvaikutuksen voidaan odottaa lisäävän Itämeren metaanipäästöjä entisestään tulevaisuudessa.

The dynamics of phosphorus (P) and nitrogen (N) were examined in two (shallow and deep) eutrophic lakes. The effects of resuspension and macrophytes as well as oxygen availability were determined, as was the role of natural nitrogen (N) removal (denitrification). The effect of resuspension on the availability of N and P was examined in experimental columns in a shallow eutrophic lake. The release of soluble reactive P (SRP) from resuspended matter varied both seasonally and interannually, depending on the conditions in the lake. SRP release was most significant during periods of high primary production, probably due to pH-induced ligand-exchange reactions. The effect of resuspension on dissolved N was less clear, although resuspension also increased the concentrations of ammonium, nitrite and nitrate during some of the experiments. The studies also showed that strong resuspension decreases the total N to total P ratio in the water independently of the phase of the growing season. The nutrient dynamics were further studied among two different macrophyte stands (submerged and floating-leaved macrophytes) and in the open water. The concentration of combined nitrate and nitrite N (NOx N) decreased to the detection limit (PIENEMPI 2 µg l 1) at all of the stations during summer. Among the submerged macrophytes, the NOx N was depleted almost 2 months earlier than at the other stations, which restricted denitrification. The rate of denitrification was measured with the isotope-pairing technique in both lakes. In the shallow lake, denitrification was measured at the location of the resuspension experiments as well as at the macrophyte stations and adjacent open-water area. Denitrification was mainly dependent on the availability of NOx N and temperature, and also contributed to the changes observed in the total N to total P ratio in water during summer by removing substantial amounts of NOx N from the water. The effects of oxygen (O2) availability were studied in a deeper, stratifying lake, where the deeps were treated with continuous or pulsed aeration, or with no aeration. The SRP release in the deeps was linked with the O2 in the sediment and the SRP accumulated in the hypolimnion during the periods of anoxia. During the aeration pauses and pulses, the concentration of NOx N decreased and increased respectively and the concentration of ammonium varied contrastingly. Continuous aeration prevented the ammonium from accumulating and provided NOx N for denitrification to occur. Accordingly, denitrification showed higher rates during aeration. However, aeration also increased the temperature of the hypolimnion and thereby also the O2 consumption. Väitöskirjassa tutkittiin typen ja fosforin kiertoa kahdessa rehevässä järvessä. Matalassa Hiidenveden Kirkkojärvessä tehdyissä tutkimuksissa selvitettiin leville käyttökelpoisen fosforin ja typen saatavuutta tilanteessa, jossa järven pohjasedimenttiä sekoittuu vesipatsaaseen virtausten vaikutuksesta. Tutkimuksissa havaittiin, että vuodenajasta riippumatta sekoittuminen nosti ravinteiden kokonaispitoisuuksia vedessä, mutta kasveille sellaisenaan käyttökelpoisten liukoisten ravinteiden vapautumiseen vaikuttivat vedessä vallitsevat olosuhteet. Liukoisen fosforin vapautuminen oli merkittävintä loppukesällä, kun voimakkaat leväkukinnat olivat nostaneet veden pH-tasoa. Samalla järvellä tutkittiin myös vesikasvien (ulpukka ja kiehkuraärviä) vaikutusta ravinteiden kiertoon. Ravinnepitoisuudet olivat hyvin samankaltaisia ulpukkakasvustossa ja avovedessä, mutta ärviäkasvustossa veden ravinnepitoisuudet olivat huomattavasti muita pienempiä. Tulokset selittyvät kasvien erilaisilla ravinteidenoton mekanismeilla; ärviät voivat ottaa ravinteita myös lehdillään suoraan vedestä, kun taas ulpukat ottavat ravinteita pääsääntöisesti juuriensa avulla järven sedimentistä. Hapen vaikutuksia typen ja fosforin saatavuuteen tutkittiin Lahden Vesijärvessä, jonka syvänteitä hapetetaan Mixox-ilmastimilla. Tutkimuksessa havaittiin, että vaikka ilmastimet toimivat maksimiteholla, ei syvänteiden veteen alkanut kertyä happea. Ilmastuksen vaikutuksesta kuitenkin liukoinen typpi pysyi hapettuneessa nitraatti-muodossa. Yhdessä tutkimussyvänteessä tutkittiin myös pulssimaisen hapetuksen vaikutuksia. Pulssimaisesti käytettynä ilmastimet nostivat alusveden lämpötilaa vähemmän kuin jatkuva ilmastus, mutta ilmastustaukojen aikana veden nitraattityppi pelkistyi ammoniumtypeksi. Ammoniumtyppi jää veteen ja on näin ollen myöhemmin kasvien käytettävissä, kun taas nitraattimuotoinen typpi voi poistua vedestä luontaisen typenpoistoprosessin (denitrifikaatio) seurauksena. Luontaista typenpoiston nopeutta tutkittiin kummassakin järvessä. Tutkimusten perusteella typenpoistoon käytettävän nitraattimuotoisen typen saatavuus määräsi kesän aikana typenpoistonopeuden. Keväällä ja myöhään syksyllä kylmyys nousi nitraatin saatavuutta tärkeämmäksi rajoittavaksi tekijäksi.

The Baltic Sea is one of the largest brackish water bodies in the world. Primary production in the Baltic Sea is limited by nitrogen (N) availability with the exception of river outlets and the northernmost phosphorus limited basin. The excess human induced N load from the drainage basin has caused severe eutrophication of the sea. The excess N loads can be mitigated by microbe mediated natural N removal processes that are found in the oxic-anoxic interfaces in sediments and water column redoxclines. Such interfaces allow the close coupling between the oxic nitrification process, and anoxic denitrification and anaerobic ammonium oxidation (anammox) processes that lead to the formation of molecular nitrogen gas. These processes are governed by various environmental parameters. The effects of these parameters on N processes were investigated in the northern Baltic Sea sediments. During summer months when the sediment organic content is at its highest, nitrification and denitrification reach their maximum rates. However, nitrification had no excess potential, which was probably because of high competition for molecular oxygen (O2) between heterotrophic and nitrification microbes. Subsequently, the limited nitrate (NO3-) availability inhibited denitrification. In fall, winter and spring, nitrification was limited by ammonium availability and denitrification limited by the availability of organic carbon and occasionally by NO3-. Anaerobic ammonium oxidation (anammox) was not an important N removal process in the northern Baltic Sea. Modeling studies suggest that when hypoxia expands in the Baltic Sea, N removal intensifies. However, the results of this study suggest the opposite because bottom water hypoxia (O2< 2 ml l-1) decreased the denitrification rates in sediments. Moreover, N was recycled by the dissimilatory nitrate reduction to ammonium (DNRA) process instead of being removed from the water ecosystem. High N removal potentials were found in the anoxic water column in the deep basins of the Baltic Proper. However, the N removal in the water column appeared to be limited by low substrate availability, because the water at the depths at which the substrate producing nitrification process occurred, rarely mix with the water at the depths at which N removal processes were found. Overall, the natural N removal capacity of the northern Baltic Sea decreased compared to values measured in mid 1990s and early 2000. The reason for this appears to be increasing hypoxia. Rehevöitynyt Itämeri- Typpiravinteiden poistokyky Suomenlahdella on heikentynyt Itämeri on yksi maailman rehevöityneimmistä meristä. Rehevöityminen ilmenee vuotuisina laajoina sinileväkukintoina ja pohjan happikatona. Yksi tärkeimmistä rehevöitymistä aiheuttavista tekijöistä on valuma-alueelta tuleva liiallinen typpiravinteiden määrä. Typpiravinteiden rehevöittävän vaikutuksen on ajateltu vähenevän luonnollisesti meren mikrobiprosesseissa. Julkaistavan väitöstutkimuksen tulokset kuitenkin osoittavat, että Suomenlahdella luonnollisen typenpoiston merkitys on vähäinen. Lisäksi tulokset vahvistavat typpikuormituksen vähentämisen tarpeellisuutta rehevöitymisen ehkäisyssä. Väitöstutkimuksessa selvitettiin typpeä poistavien mikrobiprosesseiden merkitystä ja niitä sääteleviä tekijöitä Suomenlahdella ja eteläisellä Itämerellä. Tutkimuksen tulokset osoittivat, että pohjasedimentin luonnollinen typenpoistokyky Suomenlahdella vaihtelee huomattavasti vuodenajoittain ja on pienimmillään keväällä, jolloin typpikuormitus valuma-alueelta on suurinta. Yhdeksänkymmentäluvun puolivälissä, jolloin Suomenlahden pohjan happitilanne oli nykyistä parempi, luonnollinen typenpoisto vähensi noin kolmanneksen maalta tulevasta typpikuormasta. Tällä hetkellä typpikuormasta poistuu luonnollisesti vain noin kymmenesosa. Tutkimuksessa osoitettiin rehevöitymisestä aiheutuvan pohjan happikadon pienentävän luonnollista typenpoistokykyä ja kiihdyttävän typen varastoitumista pohjasedimentteihin. Tämä johtaa rehevöitymisen noidankehään, jossa rehevöitymisestä aiheutunut hapettomuus lisää typen saatavuutta kasviplanktonille kiihdyttäen niiden kasvua. Hajotessaan lisääntynyt kasviplankton taas kuluttaa happea lisäten pohjien hapettomuutta. Vastaava mekanismi fosforiravinteen suhteen osoitettiin jo 40-luvun alkupuolella. Luonnollista typenpoistoa tutkittiin myös eteläisen Itämeren hapettomissa syvänteissä. Mittaukset osoittivat, että Itämeren syvänteiden hapettomassa vedessä on huomattava typenpoistopotentiaali, mutta potentiaali toteutuu vain kun syvänteiden hapelliset ja hapettomat vesimassat sekoittuvat keskenään. Jotta typenpoiston merkitys vedessä pystyttäisiin selvittämään tarkasti, vesimassojen sekoittumista säätelevät fysikaaliset prosessit pitäisi tuntea paremmin.