The study’s central innovation lies in its sampling method. Rather than lowering bottles from a vessel or relying on remote sensors, trained divers descended to specific underwater structures, including a small harbor basin, limestone drop-offs, a wooden shipwreck resting at 22 to 26 meters, and a submerged pipe extending from the shoreline to a depth of 47 meters. The pipe, officially designated as a decommissioned wastewater overflow, was a particular target of suspicion. Divers could visually identify the exact point where an effluent plume emerged, position sampling bottles directly within it, and document the surrounding environment on underwater slates, a level of spatial precision that neither autonomous vehicles nor surface-based sampling can match.

In total, the team collected 30 samples across depths ranging from half a meter to 47 meters, supplemented by four nearby reference dive sites. Every sample was brought to a mobile field laboratory within 30 minutes, where pH, salinity, and redox potential were measured before the water chemistry could shift. Trace metal samples were filtered, acidified with nitric acid, and stored cool, while samples for surfactant analysis were preserved with a small amount of acetonitrile to halt microbial degradation. Back in Germany, the metals were measured by inductively coupled plasma optical emission spectroscopy after solid phase extraction, a pre-treatment step essential for stripping away the interfering salt matrix of Adriatic seawater and concentrating the analytes to detectable levels.

The baseline hydrochemistry told a story of stability. Surface waters reached 25 degrees Celsius, with a distinct thermocline between 20 and 25 meters separating a warm, well-mixed upper layer from a cooler, more stagnant deep zone. pH held steady at 8.06, absolute salinity averaged 39.3 grams per kilogram, and redox potential of roughly 441 millivolts confirmed that the entire water column was well oxygenated, conditions that keep iron and manganese oxides stable and prevent the release of adsorbed metals from sediments.

One sample shattered that calm. At station SP05, drawn directly from the sewer pipe outlet at 47 meters, the water was turbid, yellowish-brown, and carried a distinct sewage odor. Its salinity of just 10.9 grams per kilogram marked it as freshwater-dominated effluent, its pH of 6.44 represented dramatic local acidification driven by carbon dioxide from microbial breakdown of organic matter, and its redox potential of nearly minus 99 millivolts revealed an anoxic micro-environment. That oxygen-starved plume matters chemically: the aerobic degradation of detergents requires molecular oxygen, so the plume’s own organic load effectively shields its pollutants from breakdown, allowing them to persist and spread into the stratified layers of the bay.

The surfactant results were the study’s most striking. Linear alkylbenzene sulfonates, or LAS, the world’s most widely used anionic detergents, are exclusively anthropogenic and therefore unambiguous tracers of domestic wastewater. At SP05, the divers measured a concentration of roughly 1469 parts per billion, nearly 300 times the background level. Every other sample in the bay, including the designated reference site, contained a consistent low-level signal of about 5 parts per billion. That ubiquitous background suggests either that the bay has reached a chemical steady state in which continuous inputs during the tourist season balance slow degradation, or that diffuse sources such as greywater runoff, contaminated karst groundwater, or discharges from recreational vessels are feeding the system. Because no comparable Mediterranean baseline datasets for marine surfactants exist, the authors say their values provide a critically needed reference for future monitoring along the Adriatic coast.

The trace metal analysis, by contrast, revealed contamination confined to specific spots rather than systemic pollution. Most elements were detected below 5 parts per billion, at or beneath concentrations reported for other Adriatic locations, confirming largely pristine ambient water. Zinc stood out with occasional values exceeding 20 parts per billion, consistent with antifouling paints, galvanized materials, and corrosion-protection coatings. Statistical analysis of the metal data revealed two clearly separated element clusters. An aluminum-cadmium-copper-zinc group points to maritime activity: copper and zinc leach from antifouling coatings, aluminum and zinc come from sacrificial anodes on hulls and harbor infrastructure, and cadmium travels as a characteristic impurity in zinc alloys. A second iron-manganese-nickel cluster reflects the region’s geology, where weathering of red-oxide-bearing limestone and terra rossa soils naturally releases these elements into the sea.

Principal component analysis sharpened this distinction, explaining 72 percent of the variance in two factors that separate total metal load from the contrast between the anthropogenic and geogenic clusters. The biplot flagged telling anomalies: samples taken beside the wooden shipwreck showed elevated iron, manganese, and nickel, consistent with the slow corrosion of the wreck’s iron fasteners, while a harbor sample displayed elevated copper, zinc, and cadmium, a signature of touristic boat traffic. One outlier station, where a parallel team had disturbed sediment shortly before sampling, illustrated how easily diving-based measurements in soft sediments can be skewed by resuspended pore water, a caution the authors fold into their methodological recommendations.

Crucially, the team emphasizes that none of these signals currently threatens the bay’s ecology. Outside the immediate outfall, metal concentrations and the pervasive 5 parts per billion surfactant background remain well below toxicological thresholds, and the Northern Adriatic’s high dilution capacity appears to buffer these localized pressures effectively. But the molecular warning is clear. The authors recommend modernizing the wastewater infrastructure at the submerged sewer pipe and expanding future work into multi-seasonal campaigns, mass-spectrometric separation of detergent homologs, and dive-computer-based three-dimensional mapping of sampling positions. Their broader message is that scientific diving, paired with rigorous multi-parameter chemistry, can detect subtle anthropogenic change in coastal waters long before it escalates into visible environmental damage, offering coastal managers an early-warning tool precisely where tourism pressure is growing fastest.

Subject of Research: Anthropogenic trace metal and surfactant contamination assessment of a Croatian coastal bay in the Northern Adriatic Sea using scientific diving

Article Title: Assessment of anthropogenic trace metal and surfactant influence on a coastal area in the Northern Adriatic Sea using scientific diving

Article References: Franke, E., Franke, N., Viehweger, C., & Vogt, C. (2026). Assessment of anthropogenic trace metal and surfactant influence on a coastal area in the Northern Adriatic Sea using scientific diving. Discover Oceans, 3(1), Article 47. https://doi.org/10.1007/s44289-026-00158-w

Image Credits: AI Generated

DOI: 10.1007/s44289-026-00158-w

Keywords: marine pollution, scientific diving, Northern Adriatic Sea, trace metals, linear alkylbenzene sulfonates, wastewater tracers, coastal monitoring, antifouling, principal component analysis, seawater chemistry, Croatia, tourism impact

The scale of the underlying process is staggering. Photoautotrophs, the phytoplankton of the sunlit ocean, account for roughly half of the planet’s primary production, fixing carbon dioxide into organic molecules that sustain nearly all marine life. Alongside them, chemoautotrophic microbes fix dissolved inorganic carbon throughout the entire water column, from deep-sea hydrothermal vents to shallow coastal shallows, at an estimated global rate of one to three petagrams of carbon per year. Although that is roughly ten times less carbon than photosynthesis fixes, it still dwarfs the roughly 0.4 to 0.5 petagrams of organic carbon carried into the ocean by all of the world’s rivers combined. Yet the researchers were interested in a third, even more shadowy contributor: heterotrophic organisms, which are normally considered net emitters of carbon dioxide, but which carry carboxylase enzymes that stitch carbon dioxide directly into their own biomass.

These carboxylation reactions, known as anaplerotic processes, replenish the intermediates of the tricarboxylic acid cycle, the metabolic hub from which cells draw building blocks for amino acids and other essential molecules. Laboratory studies suggest that anaplerotic carbon dioxide incorporation can account for between one and eight percent of heterotrophic cell carbon, and in some light-exposed bacteria bearing light-driven proteorhodopsin pumps, dark inorganic carbon fixation can supply up to a third of cellular carbon biomass. Cumulative evidence from Arctic surface waters, the mesopelagic North Atlantic, and even the hadal depths of the Hellenic Trench has increasingly shown that heterotrophic microbes take up inorganic carbon far more substantially than their textbook reputation implies. What remained almost entirely unknown was how this flux behaves over time in shallow, seasonally variable temperate waters, where sunlight, temperature, and river-borne nutrients swing dramatically through the year.

To fill that gap, the team sampled a long-term ecological research station in the Gulf of Trieste, a shallow basin less than 25 meters deep that is flushed by freshwater from the Isonzo and Timavo rivers. Salinity there oscillates between 29 and 38.5 and temperatures between roughly 4 and 29 degrees Celsius, producing strong summer stratification and complete winter mixing. From October 2018 to April 2021, the researchers collected seawater at one meter and fifteen meters depth each month, measuring dark inorganic carbon fixation rates with radiolabeled sodium bicarbonate, heterotrophic production with tritiated leucine, and the composition of the microbial community through 16S rRNA gene amplicon sequencing. They also quantified copies of the archaeal amoA gene, which encodes a subunit of ammonia monooxygenase, the enzyme that ammonia-oxidizing archaea use to kick-start nitrification and, in doing so, to power their own chemoautotrophic carbon fixation.

The rate measurements revealed a striking and repeatable seasonality. Dark inorganic carbon uptake sank to winter minima of around 0.09 micrograms of carbon per liter per day in February and March, then climbed steadily through spring, plateauing at summer maxima in surface waters and peaking sharply in bottom waters between September and October. Occasional spikes reached more than 1.8 micrograms per liter per day, as in November 2019. Heterotrophic carbon production traced a near-identical arc, rising from winter lows near 2 micrograms per liter per day to summer highs averaging over 13 micrograms in surface samples. The two processes were strongly correlated, and a partial least squares regression model identified temperature and heterotrophic production as the strongest positive predictors of dark carbon uptake, with dissolved organic carbon and nitrogen also contributing positively and ammonia concentration adding a smaller positive signal.

The implication, the authors argue, is that the bulk of dark inorganic carbon fixation in these shallow waters is fueling heterotrophic metabolism rather than chemoautotrophic growth. The ratio of carbon fixation to heterotrophic production in the study ranged from about 0.1 to 0.5, squarely within the 0.1 to 0.8 range reported for heterotroph-dominated systems worldwide. Notably, particulate organic carbon, which peaks during spring phytoplankton blooms, was actually a negative predictor of dark carbon fixation. That decoupling makes sense biochemically: freshly produced, labile phytoplankton organic matter requires few carboxylation steps to be assimilated, whereas the more refractory compounds that accumulate in the dissolved pool by late summer demand far greater metabolic effort, forcing microbes to lean harder on anaplerotic carbon dioxide incorporation. Photoheterotrophy may compound the summer pattern, since several taxa that bloom alongside the summer fixation maximum, including the OM60/NOR5 clade and HIMB11, carry the genomic machinery for light-driven energy harvesting and anaplerotic carbon fixation simultaneously.

Winter told a very different story. Quantification of the archaeal amoA gene revealed recurrent abundance peaks every December and January, rising as high as 2.26 times ten to the seventh copies per liter, and closely mirroring blooms of the ammonia-oxidizing archaeon Candidatus Nitrosopumilus, which can make up as much as ten percent of the winter community. These archaea perform the first and rate-limiting step of nitrification, oxidizing ammonia to nitrite and using the liberated electrons to fix carbon dioxide autotrophically. Their gene abundance correlated tightly with nitrite concentrations, and each winter the study area accumulated a distinct nitrite maximum, the coastal analogue of the ocean’s well-known primary nitrite maximum. Short winter days likely favor the archaea, which are photoinhibited, while simultaneously suppressing the phytoplankton that would otherwise consume the nitrite.

By back-calculating carbon fixation from observed nitrite accumulation, assuming the carbon yield of pure-culture Nitrosopumilus strains, the team estimated that nitrification could account for between 7.9 and 22.5 percent of measured dark inorganic carbon fixation in surface and bottom waters, respectively, and on average about 13.3 percent of wintertime uptake overall, with a peak estimate reaching 34 percent in bottom samples during winter blooms. A parallel calculation using a global euphotic nitrification rate yielded strikingly similar contributions of roughly fifteen percent. The authors caution that these figures likely overestimate the true autotrophic share, because carbon yields measured in idealized cultures probably exceed those in nature. Still, both independent approaches converged on the same conclusion: chemoautotrophic ammonia oxidation is a non-negligible, seasonally concentrated contributor to carbon cycling in shallow temperate seas, potentially supplemented by winter-active SUP05 cluster bacteria capable of sulfur-based autotrophy.

The broader significance of the work lies in what it says about the marine carbon budget. If heterotrophic anaplerotic fixation dominates dark carbon uptake in productive, sunlit coastal waters, then global estimates of chemoautotrophic carbon fixation, and of the carbon basis for microbial food webs, may need careful re-examination in these environments. Heterotrophic bacteria, long cast simply as recyclers that respire organic carbon back to carbon dioxide, emerge as genuine, if inadvertent, participants in carbon fixation, with inorganic carbon behaving as a ready co-substrate for their core metabolism. Meanwhile, the recurring winter nitrite maximum documented in the Gulf of Trieste suggests that archaeal nitrification imposes a seasonal biogeochemical fingerprint even in waters barely twenty meters deep. As ocean warming continues to raise metabolic rates, the calculated temperature sensitivity of dark carbon fixation in this study, with a Q10 of about 2.5, hints that this hidden carbon flux could intensify in a warmer sea, reshaping how coastal ecosystems process both nitrogen and carbon.

Subject of Research: Dark dissolved inorganic carbon fixation by coastal microbial communities and the relative roles of heterotrophic anaplerosis and archaeal nitrification.

Article Title: High contribution of dark dissolved inorganic carbon uptake to microbial carbon cycling in a shallow Mediterranean basin

Article References: Manna, V., Balestra, C., Banchi, E., Fonti, V., Kralj, M., & Celussi, M. (2025). High contribution of dark dissolved inorganic carbon uptake to microbial carbon cycling in a shallow Mediterranean basin. Ocean Microbiology, 1(1), Article 2. https://doi.org/10.1186/s44375-025-00002-0

Image Credits: AI Generated

DOI: 10.1186/s44375-025-00002-0

Keywords: dark DIC fixation, ammonia oxidation, amoA, chemoautotrophy, heterotrophic CO2 fixation, anaplerosis, nitrification, time-series, Mediterranean Sea, 16S rRNA, microbial community dynamics, northern Adriatic Sea