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The assimilation of inorganic carbon into biomass via heterotrophic inorganic carbon fixation (HICF) is estimated to contribute significantly to global carbon fixation, yet the controls on this process in strictly heterotrophic microorganisms remain poorly understood. To identify potential drivers of HICF, we cultivated well-characterized heterotrophic Gammaproteobacteria (\n                      <jats:italic toggle=\"yes\">Pseudomonas putida<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Pseudoalteromonas carrageenovora<\/jats:italic>\n                      ) at two temperatures (optimal vs colder) and resource treatments (high vs low organic carbon), all of which were amended with NaH\n                      <jats:sup>13<\/jats:sup>\n                      CO\n                      <jats:sub>3<\/jats:sub>\n                      . We performed bulk and single-cell stable carbon isotope probing (SIP) analysis to detect and quantify HICF in each condition, and assessed other measures of metabolic state, including C\/N ratio, natural abundance nitrogen isotope ratios, and cell size. We observed HICF in all treatments, with the percent of biomass originating from\n                      <jats:sup>13<\/jats:sup>\n                      C-dissolved inorganic carbon (DIC) for\n                      <jats:italic toggle=\"yes\">P. putida<\/jats:italic>\n                      (0.006% to 0.029%) and\n                      <jats:italic toggle=\"yes\">P. carrageenovora<\/jats:italic>\n                      (0.18% to 1.1%) on the lower end of estimates for the contribution of anaplerotic fixation to heterotrophic biomass (1% to 8%). HICF was generally highest during exponential phase, at the optimal growth temperature, and under low carbon conditions, and these trends were also observed at the single-cell level. Overall, temperature and resource availability appear to influence HICF rates. These findings suggest that HICF may be more relevant under environmental conditions where DIC and nitrogen are not limiting, and the flux of digestible organic carbon is limited or intermittent. Environmental applications of\n                      <jats:sup>13<\/jats:sup>\n                      C-DIC SIP in such systems may record heterotrophic contributions to community carbon fixation.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Heterotrophic microorganisms are abundant across ecosystems and play a central role in environmental carbon cycling by storing, transforming, and decomposing organic matter. However, heterotrophs can also incorporate inorganic carbon into biomass, but this is rarely considered in models of the carbon cycle. Our experiments on heterotrophic bacteria from soil (\n                        <jats:italic toggle=\"yes\">Pseudomonas putida<\/jats:italic>\n                        ) and marine environments (\n                        <jats:italic toggle=\"yes\">Pseudoalteromonas carrageenovora<\/jats:italic>\n                        ) show that inorganic carbon uptake is impacted by both resource availability and temperature and may be undetectable without stable isotope probing. By integrating single-cell techniques, our study measures variation in inorganic carbon uptake among individual cells across growth phases and conditions. Our study highlights heterotrophic inorganic carbon fixation as a variable source of\n                        <jats:sup>13<\/jats:sup>\n                        C-dissolved inorganic carbon incorporation into microbial biomass, underscoring the importance of considering this process in the design and interpretation of environmental stable isotope probing studies.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00628-26","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T13:01:19Z","timestamp":1790600479000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Heterotrophic inorganic carbon fixation varies with temperature and resource availability in marine and soil bacterial isolates"],"prefix":"10.1128","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6033-7113","authenticated-orcid":true,"given":"Emma K.","family":"Brown","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03efmqc40","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Earth and Space Exploration, Arizona State University","place":["Tempe, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kadin","family":"Pulliam","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03efmqc40","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Earth and Space Exploration, Arizona State University","place":["Tempe, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3985-9395","authenticated-orcid":true,"given":"Katelyn","family":"Weeks","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03r0ha626","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Geology & Geophysics, University of Utah","place":["Salt Lake City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8254-556X","authenticated-orcid":true,"given":"Carolynn M.","family":"Harris","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03efmqc40","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Earth and Space Exploration, Arizona State University","place":["Tempe, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7909-2475","authenticated-orcid":true,"given":"William D.","family":"Leavitt","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03r0ha626","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Geology & Geophysics, University of Utah","place":["Salt Lake City, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1023-6239","authenticated-orcid":true,"given":"Jaclyn K.","family":"Saunders","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02bjhwk41","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Marine Sciences, University of Georgia","place":["Athens, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3979-8676","authenticated-orcid":true,"given":"Elizabeth","family":"Trembath-Reichert","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03efmqc40","id-type":"ROR","asserted-by":"publisher"}],"name":"School of Earth and Space Exploration, Arizona State University","place":["Tempe, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,28]]},"reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.05702-11"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1042\/bj0300048"},{"key":"e_1_3_4_4_2","first-page":"610","article-title":"Heterotrophic assimilation of CO2 by bacterial flora of water","volume":"33","author":"Romanenko VI","year":"1964","unstructured":"Romanenko VI. 1964. 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