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