Cupriavidus necator as flexible platform organism for trehalose production

Cupriavidus necator as flexible platform organism for trehalose production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cupriavidus necator as flexible platform organism for trehalose production Jakub Gizewski, Tobias Kaiser, Luca Dallmann, Menglong Li, Dirk Holtmann This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7630680/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cupriavidus necator has emerged as a highly adaptable microbial chassis for sustainable bioproduction. In this study, we explore its potential as a flexible platform organism for trehalose production under heterotrophic, autotrophic, and formatotrophic conditions. Heterotrophic cultivation yielded the highest trehalose concentration of 7.2 g L⁻¹, demonstrating the organism’s efficiency when utilizing organic carbon sources. Autotrophic production, which relies solely on CO₂, achieved a trehalose concentration of 2.3 g L⁻¹, reflecting the limitations of carbon fixation pathways. Formatotrophic cultivation, using formate as a carbon and energy source, significantly improved by co feeding hydrogen resulting a doubled trehalose concentration. Compared with previously reported benchmarks, this study achieved a 15-fold increase in trehalose production, emphasizing the metabolic versatility of C. necator and its promise for scalable, carbon-efficient biomanufacturing. Sustainable production gas fermentation Cupriavidus necator trehalose hydrogen formate fructose Figures Figure 1 Highlights • Comparison of heterotrophic, autotrophic and formatotrophic trehalose production • Heterotrophically a trehalose concentration of 7.2 g L was reached • CO-based production resulted in a trehalose concentration of 2.3 g L • Hydrogen co-feeding doubled the formatotrophic trehalose production • Compared with previous benchmarks, a 15-fold increase in trehalose was achieved Introduction The increasing global demand for sustainable and eco-friendly bioprocesses has driven significant interest in the development of microbial production systems that utilize renewable resources. One promising approach involves the use of Cupriavidus necator , a metabolically versatile bacterium [[ 1 ] [ 2 ] 3], for the sustainable production of trehalose. Trehalose, a disaccharide with extensive applications in the food, pharmaceutical, and cosmetic industries, is valued for its unique properties, including its ability to stabilize proteins and preserve biological molecules [ 4 ]. C. necator is a metabolically versatile organism capable of growing under heterotrophic, autotrophic, and formatotrophic conditions. During autotrophic and formatotrophic growth, the Calvin cycle is used to fix carbon dioxide—a process that is energetically demanding and typically results in lower product concentrations and yields. To support this energy-intensive pathway, C. necator utilizes formate dehydrogenases and hydrogenases, which provide additional energy and reducing power [ 5 ]. It has recently been demonstrated that C. necator can grow using industrial flue gases as a source of CO₂, with no detectable negative effects on bacterial growth or product formation compared with a pure gas mixture [ 6 ]. While new industrial applications for trehalose continue to emerge, it is already incorporated into various products [ 4 ]. For instance, trehalose serves as a stabilizing agent in therapeutic protein formulations [ 7 ], aids in preserving restriction enzymes [ 8 ], prolongs food shelf life [ 9 ] and acts as a growth-enhancing compound that protects plants from drought stress [ 10 ]. Trehalose is currently produced by enzymes that convert starch [ 11 ], maltose [ 12 ], or maltooligosyltrehalose [ 13 ] into trehalose. These enzymatic pathways replaced the fermentative microbial processes that used Saccharomyces cerevisiae because the disadvantage of a complex extraction and purification process prevails [ 1 ]. In nature, microbes use trehalose as a protective substance, helping them cope with stress by stabilizing proteins and cellular structures [ 14 ]. Thus, under salt stress conditions, C. necator naturally accumulates trehalose. Löwe et al. transformed a plasmid from E. coli to C. necator encoding a sugar efflux transporter (setA), enabling the bacterium to export trehalose from the cell after being induced [ 2 ]. This genetically modified strain, was able to produce up to 0.31 g L − 1 under heterotrophic conditions when fructose was used as substrate and 0.47 g L − 1 under autotrophic conditions in shaking flasks [ 15 ]. Due to its metabolic flexibility, this paves the way for new trehalose production processes, which are important in a range of industries. This paper explores the potential of C. necator for sustainable trehalose production in bench top fermenter systems, investigating three distinct feeding strategies: 1) heterotrophic growth on fructose, 2) autotrophic growth on CO₂ and H₂, and 3) formatotrophic growth on formate, with and without additional H 2 . By harnessing the capabilities of C. necator , this research contributes to the development of environmentally friendly bioprocesses that support a circular bioeconomy. Experimental setup, media, and analytical methods All feeding strategies were tested across three separate experiments using a genetically modified strain of Cupriavidus necator H16 carrying the plasmid pSEVA228-setA [ 15 ]. This strain was transferred directly from the plate or from cryostock into a 500 ml shaking flask containing LB medium (50 mL with 5 g L − 1 tryptone, 5 g L − 1 NaCl and 2.5 g L − 1 yeast extract) and kanamycin (300 µg mL − 1 ), which provided a selection marker for bacteria containing the plasmid. The standard cultivation conditions in the incubator were set at 180 rpm and 30°C. The preculture was incubated overnight and then centrifuged (10 min, 4816 × g at 4°C). The liquid phase was discarded and the inoculum was diluted with minimal medium (Table 1 ) to achieve an OD of 2 in the reactor. Table 1 Standard minimal medium composition adapted from Sydow et al. [ 16 ]. Solutions 1–3 and the salt solution were sterilized by autoclaving, whereas solutions 4 and 5, along with the antibiotic and inducer, were sterilized by filtration. Stock solution Components Stock solution / g L − 1 Dilution factor 1 Na 2 HPO 4 28.95 10.0 NaH 2 PO 4 30.60 10.0 2 K 2 SO 4 1.70 10.0 CaSO 4 2 H 2 O 0.97 10.0 3 (NH 4 ) 2 SO 4 188.00 200.0 4 MgSO 4 7 H 2 O 500.00 625.0 5* FeSO 4 7 H 2 O 15.00 2500.0 MnSO 4 7 H 2 O 2.40 2500.0 ZnSO 4 7 H 2 O 2.40 2500.0 CuSO 4 5 H 2 O 0.48 2500.0 Na 2 MoO 4 2 H 2 O 1.80 2500.0 Ni 2 SO 4 6 H 2 O 1.50 2500.0 CoSO 4 7 H 2 O 0.04 2500.0 Antibiotic Kanamycin 50.00 170.0 Inducer 3-methylbenzoic acid 1.00 3.3 Salt NaCl 300.00 15.0 * Trace element solution dissolved in 0.1 M hydrochloric acid For the minimal medium 5 stock solutions were prepared and mixed under sterile conditions to achieve the concentrations shown in Table 1 . To maintain the plasmids, kanamycin was added at a final concentration of 300 µg mL − 1 . C. necator was inoculated from LB medium preculture to achieve an initial OD₆₀₀ of 0.1 for the formatotrophic experiment and 0.2 for both the heterotrophic and autotrophic experiments. For the heterotrophic experiments, additional fructose was added achieving 27 g L − 1 at the beginning of the experiment. Induction with 3-methylbenzoic acid and salt (NaCl) addition was carried out simultaneously once sufficient biomass had accumulated. The experiments were performed in a parallel stirred bioreactor system (Sixfors fermentation system, Infors AG) with a working volume of 200–260 ml. The gas phase composition was achieved by mixing air, hydrogen and carbon dioxide depending on the experiment with mass flow controllers (red-y smart series, Vögtlin Instruments GmbH). The gas composition (Table 2 ) varied based on the desired experimental conditions. The pH was regulated with phosphoric acid (4 M) and potassium hydroxide (3 M) and maintained between 6.6 and 7. Table 2 Experimental parameters for heterotrophic, autotrophic and formatotrophic conditions. For each experiment, four reactors were run under similar conditions. In the formatotrophic experiment Parameter Heterotrophic Autotrophic Formatotrophic Substrate Fructose CO 2 HCOO + HCOO + + H 2 Process Fed-Batch Batch Fed-Batch Gas flow 100 mL/min 50 mL/min 50 mL/min Gas mixture Air 100% Air 85% H 2 10% CO 2 10% Air 100% H 2 0% Air 95% H 2 5%* pH (controlled) 6.6–7.0 6.6–7.0 6.6–7.0 * two reactors with formate feed and additional H 2 The residual formate concentration in the fermentation samples was quantified via high-performance liquid chromatography (HPLC). The samples were first centrifuged at 17000 × g for 10 minutes. The HPLC system (Model 1100/1200, Agilent Technologies Deutschland GmbH) comprising a precolumn and an analytical column (Rezex™ ROA-Organic Acid H+ (8%) (300 × 7.8 mm), Phenomenex Ltd.), was operated at 65°C, with the refractive index detector maintained at 55°C and 210 nm. A 5 mM sulfuric acid solution was used as the mobile phase and was delivered at a flow rate of 0.5 mL/min. A 20 µL aliquot of the sample was injected into the system. Gas analysis was performed via a microGC (Inficon Holding AG) with one column for H 2 , N 2 and O 2 (PLOTU: 30 µm × 320 µm × 3 m, precolumn; Molsieve: 30 µm × 320 µm × 3 m, main column) and one for CO 2 (PlotQ: 10 µm × 320 µm × 10 m, main column). Microbial growth was traced via OD 600 measurements in a cuvette spectrophotometer within the range of 0.1–0.5 (Scientific™ Genesys 30, Fisher Scientific GmbH). If the OD 600 exceeded this range, the sample was diluted accordingly. Copilot was used to enhance the clarity and quality of the language. Results and discussion This study investigated trehalose production by C. necator in a laboratory-scale fermenter system, comparing heterotrophic, autotrophic, and formatotrophic growth conditions. The results are summarized in Fig. 1 . Across all conditions, a common trend is observed: following induction, the growth rate declines while trehalose production and extracellular secretion begins. Due to faster growth under heterotrophic conditions, salt and inducer were added earlier—at 21 h (indicated by arrow A)—compared to 45 h for both autotrophic and formatotrophic processes (indicated by arrow B). The results indicate that, for all conditions the provided carbon is not the limiting factor for trehalose production. Metabolic pathways that depend on the Calvin cycle—such as those in autotrophic and formatotrophic organisms—typically exhibit a prolonged lag phase (see Fig. 1 ). This delay occurs because cells must undergo a complete shift of their metabolism when transferred from LB medium to a minimal medium lacking conventional carbon [ 5 ]. Despite similar final optical densities (OD 600 ) between autotrophic and heterotrophic cultures, trehalose production is significantly greater under heterotrophic conditions. Under these conditions, trehalose levels increase linearly over time up to 7.2 g L − 1 . In contrast, autotrophic cultures exhibit a delayed but sharp increase in trehalose production approximately 67 hours after induction, followed by a subsequent decline and plateau at 2.3 g L⁻¹. Although carbon and electron sources remain available, the observed growth curve suggests a limitation in microbial activity, potentially attributable to transport or metabolic constraints [ 17 ]. Formatotrophic cultures presented the lowest optical density and trehalose concentration, even though formate contains more energy because of its additional hydrogen bond and, as a liquid substrate, should theoretically support better mass transport to the cells. This unexpectedly low performance may be due to formate toxicity in C. necator , although the concentration used was mostly below the toxic level of 100 mM [ 18 ]. As anticipated, the addition of hydrogen enhanced formatotrophic growth, likely due to the increased electron availability, which enables a greater proportion of formate-derived carbon to be directed toward product synthesis rather than energy generation [ 19 ]. Both the maximum optical density (OD₆₀₀) and product concentration approximately doubled from 4.5 to 8.1, and from 0.6 g L⁻¹ to 1.3 g L⁻¹, respectively. These findings suggest that trehalose concentration is directly correlated with cell dry weight. The most important performance indicators under the different conditions are summarized in Table 3 . Table 3 Key performance indicators: product concentration, cell dry weights and yields for cultivation with C. necator Parameter Heterotrophic Autotrophic Formatotrophic Substrate Fructose CO 2 HCOO + HCOO + + H 2 Substrate consumed g L⁻¹ 35.7 - - - Trehalose g L⁻¹ 7.2 2.3 0.6 1.3 Final OD 11.4 10.2 4.5 8.1 Biomass g L⁻¹ * 4.1 3.8 1.6 2.9 Space time yield / g L⁻¹ h − 1 0.25 0.08 0.02 0.04 Trehalose yield Y P/S , g g⁻¹ 0.2 - - - Produced Trehalose per biomass Y P/B , g g⁻¹ 1.8 0.6 0.40 0.43 *Correlation factor CDW per OD 600 = 0.363 g L⁻¹ [ 2 ] Key findings and future potential This study demonstrates the potential of C. necator as a platform organism for trehalose production under heterotrophic, autotrophic, and formatotrophic conditions. The autotrophic and formatotrophic approaches are particularly promising for reducing land use in biotechnological feedstock production, as they rely on CO₂ or formate—which can be synthesized electrochemically from CO₂ [ 20 ]—as carbon sources. In both systems, trehalose production was successfully confirmed, as previously shown by Loewe et al. [ 15 ]. The maximum trehalose concentration was further increased to 1.3 g L − 1 by adding hydrogen to the culture media. For industrial application the setA gene should also be incorporated into the megaplasmid to overcome limitations associated with induction. In this case the production of trehalose begins when salt stress increases. Two-phase cultivation with a growth phase and a production phase does make sense because microbial growth is decreased significantly by additional salt stress. It would be helpful to have a carbon and energy balance to enable a more detailed comparison of the yields of heterotrophic, autotrophic and formatotrophic processes. This comparison should also include electrotrophic trehalose production [ 6 ] [ 21 ]. Declarations Author Contribution J. G. led the conceptualization and experimental design as well as experimental work. T. K., L. D. and M. L. contributed the experimental work, data analysis and interpretation. D. H. supervised the project and provided critical revisions to the manuscript. Availability of Data and Material All data supporting the findings of this study are available within the paper and its Supplementary Information. References Pohlmann, A., Fricke, W., Reinecke, F. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat. Biotechnol. 2006; doi: 10.1038/nbt1244. Grunwald S, Mottet A, Grousseau E, Plassmeier JK, Popović MK, Uribelarrea JL, Gorret N, Guillouet SE, Sinskey. A. 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Mukai K, Tabuchi A, Nakada T, Shibuya T, Chaen H, Fukuda S, Kurimoto M, Tsujisaka Y. Production of Trehalose from Starch by Thermostable Enzymes from Sulfolobus acidocaldarius. Starch/Stärke. 1997; doi: 10.1002/star.19970490107 Nishimoto T, Nakano M, Nakada T, Chaen H, Fukuda S, Sugimoto T, Kurimoto M, Tsujisaka Y. Purification and Properties of a Novel Enzyme, Trehalose Synthase, from Pimelobacter sp. R48. Bioscience, Biotechnology, and Biochemistry. 1996; doi: 10.1271/bbb.60.640 Maruta K, Mitsuzumi H, Nakada T, Kubota M, Chaen H, Fukuda S, Sugimoto T, Kurimoto M. Cloning and sequencing of a cluster of genes encoding novel enzymes of trehalose biosynthesis from thermophilic archaebacterium Sulfolobus acidocaldarius. Biochim. Biophys. Acta. 1996; doi: 10.1016/S0304-4165(96)00082-7. Chen A, Tapia H, Goddard JM, Gibney PA. Trehalose and its applications in the food industry. Compr. Rev. Food Sci. Food Saf. 2022; doi: 10.1111/1541-4337.13048. 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A co-feeding strategy of formate and H2 for methanogens – Enhancing growth parameters and methane production. J. CO2 Util. 2025; doi: 10.1016/j.jcou.2025.103049 Stöckl M, Harms S, Dinges I, Dimitrova S, Holtmann D. From CO2 to Bioplastic – Coupling the Electrochemical CO 2 Reduction with a Microbial Product Generation by Drop-in Electrolysis. ChemSusChem . 2020; doi: 10.1002/cssc.202001235. Krieg T, Sydow A, Faust S, Huth I, Holtmann D. CO2 to Terpenes: Autotrophic and Electroautotrophic α-Humulene Production with Cupriavidus necator. Angew. Chem. Int. Ed. 2018; doi: 10.1002/anie.201711302. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":27224,"visible":true,"origin":"","legend":"\u003cp\u003eDatasets from three experiments with hetero (green ●), auto (blue x) and formato­trophic (black ▪, gray ▴) conditions showing OD\u003csub\u003e600\u003c/sub\u003e (top) and trehalose (bottom) over time in a parallel fermenter system under aerobic conditions. The formatotrophic dataset is split on the basis of the presence (black square) or absence (grey pyramid) of additional H₂ supply. The time of induction and NaCl addition is marked by arrow A, for the heterotrophic process and arrow B for both the auto- and formatotrophic processes. Hetero- and autotrophic data were obtained from four replicates, whereas formatotrophic data were based on duplicate measurements under both conditions.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7630680/v1/8cab8e6072583ded54b3f8f2.png"},{"id":99790149,"identity":"84540ac6-cc01-4f86-90d0-0f1c750b254d","added_by":"auto","created_at":"2026-01-08 12:56:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":576236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7630680/v1/641118ba-9b6c-4db1-8afe-35f6ef9991a3.pdf"},{"id":93502862,"identity":"3f6e7315-3cc6-4512-a616-62bceca094e4","added_by":"auto","created_at":"2025-10-14 14:14:03","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":34772,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7630680/v1/4c9045ca4ada75a33de6ae32.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cupriavidus necator as flexible platform organism for trehalose production","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; Comparison of heterotrophic, autotrophic and formatotrophic trehalose production\u003c/p\u003e\u003cp\u003e\u0026bull; Heterotrophically a trehalose concentration of 7.2 g L was reached\u003c/p\u003e\u003cp\u003e\u0026bull; CO-based production resulted in a trehalose concentration of 2.3 g L\u003c/p\u003e\u003cp\u003e\u0026bull; Hydrogen co-feeding doubled the formatotrophic trehalose production\u003c/p\u003e\u003cp\u003e\u0026bull; Compared with previous benchmarks, a 15-fold increase in trehalose was achieved\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe increasing global demand for sustainable and eco-friendly bioprocesses has driven significant interest in the development of microbial production systems that utilize renewable resources. One promising approach involves the use of \u003cem\u003eCupriavidus necator\u003c/em\u003e, a metabolically versatile bacterium [[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] 3], for the sustainable production of trehalose. Trehalose, a disaccharide with extensive applications in the food, pharmaceutical, and cosmetic industries, is valued for its unique properties, including its ability to stabilize proteins and preserve biological molecules [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. necator\u003c/em\u003e is a metabolically versatile organism capable of growing under heterotrophic, autotrophic, and formatotrophic conditions. During autotrophic and formatotrophic growth, the Calvin cycle is used to fix carbon dioxide\u0026mdash;a process that is energetically demanding and typically results in lower product concentrations and yields. To support this energy-intensive pathway, \u003cem\u003eC. necator\u003c/em\u003e utilizes formate dehydrogenases and hydrogenases, which provide additional energy and reducing power [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has recently been demonstrated that C. necator can grow using industrial flue gases as a source of CO₂, with no detectable negative effects on bacterial growth or product formation compared with a pure gas mixture [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile new industrial applications for trehalose continue to emerge, it is already incorporated into various products [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For instance, trehalose serves as a stabilizing agent in therapeutic protein formulations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], aids in preserving restriction enzymes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], prolongs food shelf life [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and acts as a growth-enhancing compound that protects plants from drought stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Trehalose is currently produced by enzymes that convert starch [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], maltose [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], or maltooligosyltrehalose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] into trehalose. These enzymatic pathways replaced the fermentative microbial processes that used \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e because the disadvantage of a complex extraction and purification process prevails [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In nature, microbes use trehalose as a protective substance, helping them cope with stress by stabilizing proteins and cellular structures [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, under salt stress conditions, \u003cem\u003eC. necator\u003c/em\u003e naturally accumulates trehalose. L\u0026ouml;we \u003cem\u003eet al.\u003c/em\u003e transformed a plasmid from \u003cem\u003eE. coli\u003c/em\u003e to \u003cem\u003eC. necator\u003c/em\u003e encoding a sugar efflux transporter (setA), enabling the bacterium to export trehalose from the cell after being induced [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This genetically modified strain, was able to produce up to 0.31 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under heterotrophic conditions when fructose was used as substrate and 0.47 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under autotrophic conditions in shaking flasks [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Due to its metabolic flexibility, this paves the way for new trehalose production processes, which are important in a range of industries.\u003c/p\u003e\u003cp\u003eThis paper explores the potential of \u003cem\u003eC. necator\u003c/em\u003e for sustainable trehalose production in bench top fermenter systems, investigating three distinct feeding strategies: 1) heterotrophic growth on fructose, 2) autotrophic growth on CO₂ and H₂, and 3) formatotrophic growth on formate, with and without additional H\u003csub\u003e2\u003c/sub\u003e. By harnessing the capabilities of \u003cem\u003eC. necator\u003c/em\u003e, this research contributes to the development of environmentally friendly bioprocesses that support a circular bioeconomy.\u003c/p\u003e"},{"header":"Experimental setup, media, and analytical methods","content":"\u003cp\u003eAll feeding strategies were tested across three separate experiments using a genetically modified strain of \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 carrying the plasmid pSEVA228-setA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This strain was transferred directly from the plate or from cryostock into a 500 ml shaking flask containing LB medium (50 mL with 5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tryptone, 5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl and 2.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yeast extract) and kanamycin (300 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), which provided a selection marker for bacteria containing the plasmid. The standard cultivation conditions in the incubator were set at 180 rpm and 30\u0026deg;C. The preculture was incubated overnight and then centrifuged (10 min, 4816 \u0026times; g at 4\u0026deg;C). The liquid phase was discarded and the inoculum was diluted with minimal medium (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to achieve an OD of 2 in the reactor.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStandard minimal medium composition adapted from Sydow \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Solutions 1\u0026ndash;3 and the salt solution were sterilized by autoclaving, whereas solutions 4 and 5, along with the antibiotic and inducer, were sterilized by filtration.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStock solution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eComponents\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStock solution / g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDilution factor\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCaSO\u003csub\u003e4\u003c/sub\u003e 2 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e188.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e200.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e 7 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e500.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e625.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003e5*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e 7 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMnSO\u003csub\u003e4\u003c/sub\u003e 7 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZnSO\u003csub\u003e4\u003c/sub\u003e 7 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCuSO\u003csub\u003e4\u003c/sub\u003e 5 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e 2 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNi\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 6 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCoSO\u003csub\u003e4\u003c/sub\u003e 7 H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2500.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibiotic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKanamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e170.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInducer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3-methylbenzoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSalt\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNaCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e300.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e* Trace element solution dissolved in 0.1 M hydrochloric acid\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor the minimal medium 5 stock solutions were prepared and mixed under sterile conditions to achieve the concentrations shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. To maintain the plasmids, kanamycin was added at a final concentration of 300 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. \u003cem\u003eC. necator\u003c/em\u003e was inoculated from LB medium preculture to achieve an initial OD₆₀₀ of 0.1 for the formatotrophic experiment and 0.2 for both the heterotrophic and autotrophic experiments. For the heterotrophic experiments, additional fructose was added achieving 27 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at the beginning of the experiment. Induction with 3-methylbenzoic acid and salt (NaCl) addition was carried out simultaneously once sufficient biomass had accumulated.\u003c/p\u003e\u003cp\u003eThe experiments were performed in a parallel stirred bioreactor system (Sixfors fermentation system, Infors AG) with a working volume of 200\u0026ndash;260 ml. The gas phase composition was achieved by mixing air, hydrogen and carbon dioxide depending on the experiment with mass flow controllers (red-y smart series, V\u0026ouml;gtlin Instruments GmbH). The gas composition (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) varied based on the desired experimental conditions. The pH was regulated with phosphoric acid (4 M) and potassium hydroxide (3 M) and maintained between 6.6 and 7.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental parameters for heterotrophic, autotrophic and formatotrophic conditions. For each experiment, four reactors were run under similar conditions. In the formatotrophic experiment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHeterotrophic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAutotrophic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eFormatotrophic\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFructose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHCOO\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHCOO\u003csup\u003e+\u003c/sup\u003e + H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProcess\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFed-Batch\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBatch\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eFed-Batch\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGas flow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100 mL/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50 mL/min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e50 mL/min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGas mixture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAir 100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAir 85%\u003c/p\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e 10%\u003c/p\u003e\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e 10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAir 100%\u003c/p\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e 0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAir 95%\u003c/p\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e 5%*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH (controlled)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.6\u0026ndash;7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.6\u0026ndash;7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e6.6\u0026ndash;7.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e* two reactors with formate feed and additional H\u003csub\u003e2\u003c/sub\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe residual formate concentration in the fermentation samples was quantified via high-performance liquid chromatography (HPLC). The samples were first centrifuged at 17000 \u0026times; g for 10 minutes. The HPLC system (Model 1100/1200, Agilent Technologies Deutschland GmbH) comprising a precolumn and an analytical column (Rezex\u0026trade; ROA-Organic Acid H+ (8%) (300 \u0026times; 7.8 mm), Phenomenex Ltd.), was operated at 65\u0026deg;C, with the refractive index detector maintained at 55\u0026deg;C and 210 nm. A 5 mM sulfuric acid solution was used as the mobile phase and was delivered at a flow rate of 0.5 mL/min. A 20 \u0026micro;L aliquot of the sample was injected into the system.\u003c/p\u003e\u003cp\u003eGas analysis was performed via a microGC (Inficon Holding AG) with one column for H\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e (PLOTU: 30 \u0026micro;m \u0026times; 320 \u0026micro;m \u0026times; 3 m, precolumn; Molsieve: 30 \u0026micro;m \u0026times; 320 \u0026micro;m \u0026times; 3 m, main column) and one for CO\u003csub\u003e2\u003c/sub\u003e (PlotQ: 10 \u0026micro;m \u0026times; 320 \u0026micro;m \u0026times; 10 m, main column). Microbial growth was traced via OD\u003csub\u003e600\u003c/sub\u003e measurements in a cuvette spectrophotometer within the range of 0.1\u0026ndash;0.5 (Scientific\u0026trade; Genesys 30, Fisher Scientific GmbH). If the OD\u003csub\u003e600\u003c/sub\u003e exceeded this range, the sample was diluted accordingly.\u003c/p\u003e\u003cp\u003eCopilot was used to enhance the clarity and quality of the language.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThis study investigated trehalose production by \u003cem\u003eC. necator\u003c/em\u003e in a laboratory-scale fermenter system, comparing heterotrophic, autotrophic, and formatotrophic growth conditions. The results are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Across all conditions, a common trend is observed: following induction, the growth rate declines while trehalose production and extracellular secretion begins. Due to faster growth under heterotrophic conditions, salt and inducer were added earlier\u0026mdash;at 21 h (indicated by arrow A)\u0026mdash;compared to 45 h for both autotrophic and formatotrophic processes (indicated by arrow B). The results indicate that, for all conditions the provided carbon is not the limiting factor for trehalose production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMetabolic pathways that depend on the Calvin cycle\u0026mdash;such as those in autotrophic and formatotrophic organisms\u0026mdash;typically exhibit a prolonged lag phase (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This delay occurs because cells must undergo a complete shift of their metabolism when transferred from LB medium to a minimal medium lacking conventional carbon [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite similar final optical densities (OD\u003csub\u003e600\u003c/sub\u003e) between autotrophic and heterotrophic cultures, trehalose production is significantly greater under heterotrophic conditions. Under these conditions, trehalose levels increase linearly over time up to 7.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In contrast, autotrophic cultures exhibit a delayed but sharp increase in trehalose production approximately 67 hours after induction, followed by a subsequent decline and plateau at 2.3 g L⁻\u0026sup1;. Although carbon and electron sources remain available, the observed growth curve suggests a limitation in microbial activity, potentially attributable to transport or metabolic constraints [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFormatotrophic cultures presented the lowest optical density and trehalose concentration, even though formate contains more energy because of its additional hydrogen bond and, as a liquid substrate, should theoretically support better mass transport to the cells. This unexpectedly low performance may be due to formate toxicity in \u003cem\u003eC. necator\u003c/em\u003e, although the concentration used was mostly below the toxic level of 100 mM [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As anticipated, the addition of hydrogen enhanced formatotrophic growth, likely due to the increased electron availability, which enables a greater proportion of formate-derived carbon to be directed toward product synthesis rather than energy generation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Both the maximum optical density (OD₆₀₀) and product concentration approximately doubled from 4.5 to 8.1, and from 0.6 g L⁻\u0026sup1; to 1.3 g L⁻\u0026sup1;, respectively. These findings suggest that trehalose concentration is directly correlated with cell dry weight. The most important performance indicators under the different conditions are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eKey performance indicators: product concentration, cell dry weights and yields for cultivation with \u003cem\u003eC. necator\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHeterotrophic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAutotrophic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eFormatotrophic\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFructose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHCOO\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHCOO\u003csup\u003e+\u003c/sup\u003e + H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate consumed g L⁻\u0026sup1;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrehalose g L⁻\u0026sup1;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFinal OD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiomass g L⁻\u0026sup1; *\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpace time yield / g L⁻\u0026sup1; h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrehalose yield Y\u003csub\u003eP/S\u003c/sub\u003e, g g⁻\u0026sup1;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProduced Trehalose per biomass Y\u003csub\u003eP/B\u003c/sub\u003e, g g⁻\u0026sup1;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Correlation factor CDW per OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.363 g L⁻\u0026sup1; [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Key findings and future potential","content":"\u003cp\u003eThis study demonstrates the potential of \u003cem\u003eC. necator\u003c/em\u003e as a platform organism for trehalose production under heterotrophic, autotrophic, and formatotrophic conditions. The autotrophic and formatotrophic approaches are particularly promising for reducing land use in biotechnological feedstock production, as they rely on CO₂ or formate\u0026mdash;which can be synthesized electrochemically from CO₂ [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u0026mdash;as carbon sources.\u003c/p\u003e\u003cp\u003eIn both systems, trehalose production was successfully confirmed, as previously shown by Loewe \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The maximum trehalose concentration was further increased to 1.3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by adding hydrogen to the culture media.\u003c/p\u003e\u003cp\u003eFor industrial application the setA gene should also be incorporated into the megaplasmid to overcome limitations associated with induction. In this case the production of trehalose begins when salt stress increases. Two-phase cultivation with a growth phase and a production phase does make sense because microbial growth is decreased significantly by additional salt stress. It would be helpful to have a carbon and energy balance to enable a more detailed comparison of the yields of heterotrophic, autotrophic and formatotrophic processes. This comparison should also include electrotrophic trehalose production [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ. G. led the conceptualization and experimental design as well as experimental work. T. K., L. D. and M. L. contributed the experimental work, data analysis and interpretation. D. H. supervised the project and provided critical revisions to the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePohlmann, A., Fricke, W., Reinecke, F. \u003cem\u003eet al.\u003c/em\u003e Genome sequence of the bioplastic-producing \u0026ldquo;Knallgas\u0026rdquo; bacterium \u003cem\u003eRalstonia eutropha\u003c/em\u003e H16. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e 2006; doi: 10.1038/nbt1244.\u003c/li\u003e\n\u003cli\u003eGrunwald S, Mottet A, Grousseau E, Plassmeier JK, Popović MK, Uribelarrea JL, Gorret N, Guillouet SE, Sinskey. A. 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Genet.\u003c/em\u003e 2019; doi: 10.1007/s00294-019-00938-2.\u003c/li\u003e\n\u003cli\u003eCollas F, Dronsella BB, Kubis A, Schann K, Binder S, Arto N, Claassens NJ, Kensy F, Orsi E. Engineering the biological conversion of formate into crotonate in \u003cem\u003eCupriavidus necator\u003c/em\u003e. \u003cem\u003eMetab. Eng.\u003c/em\u003e 2023; doi: 10.1016/j.ymben.2023.06.015.\u003c/li\u003e\n\u003cli\u003eSabel-Becker B, Jost NP, Kaster AK, Holtmann D. A co-feeding strategy of formate and H2 for methanogens \u0026ndash; Enhancing growth parameters and methane production. \u003cem\u003eJ. CO2 Util.\u003c/em\u003e 2025; doi: 10.1016/j.jcou.2025.103049\u003c/li\u003e\n\u003cli\u003eSt\u0026ouml;ckl M, Harms S, Dinges I, Dimitrova S, Holtmann D. From CO2 to Bioplastic \u0026ndash; Coupling the Electrochemical CO\u003csub\u003e2\u003c/sub\u003e Reduction with a Microbial Product Generation by Drop-in Electrolysis. \u003cem\u003eChemSusChem\u003c/em\u003e. 2020; doi: 10.1002/cssc.202001235.\u003c/li\u003e\n\u003cli\u003eKrieg T, Sydow A, Faust S, Huth I, Holtmann D. CO2 to Terpenes: Autotrophic and Electroautotrophic \u0026alpha;-Humulene Production with Cupriavidus necator. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e2018; doi: 10.1002/anie.201711302.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sustainable production, gas fermentation, Cupriavidus necator, trehalose, hydrogen, formate, fructose","lastPublishedDoi":"10.21203/rs.3.rs-7630680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7630680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eCupriavidus necator\u003c/em\u003e has emerged as a highly adaptable microbial chassis for sustainable bioproduction. In this study, we explore its potential as a flexible platform organism for trehalose production under heterotrophic, autotrophic, and formatotrophic conditions. Heterotrophic cultivation yielded the highest trehalose concentration of 7.2 g L⁻\u0026sup1;, demonstrating the organism\u0026rsquo;s efficiency when utilizing organic carbon sources. Autotrophic production, which relies solely on CO₂, achieved a trehalose concentration of 2.3 g L⁻\u0026sup1;, reflecting the limitations of carbon fixation pathways. Formatotrophic cultivation, using formate as a carbon and energy source, significantly improved by co feeding hydrogen resulting a doubled trehalose concentration. Compared with previously reported benchmarks, this study achieved a 15-fold increase in trehalose production, emphasizing the metabolic versatility of \u003cem\u003eC. necator\u003c/em\u003e and its promise for scalable, carbon-efficient biomanufacturing.\u003c/p\u003e","manuscriptTitle":"Cupriavidus necator as flexible platform organism for trehalose production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 14:13:59","doi":"10.21203/rs.3.rs-7630680/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9e44fa73-582c-4f78-a5c7-df09c8acffdc","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-04T19:38:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-14 14:13:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7630680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7630680","identity":"rs-7630680","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}