微生物学报

Parameter	Xanthobacter autotrophicus	Cupriavidus necator	Rhodopseudomonas palustris	References
Energy source	CO₂, methanol, ethanol,N-butanol, organic acids, etc.	Chemolithoautotrophic growth using H₂ as electron donor, also capable of utilizing various heterotrophic carbon sources in combination with nitrate metabolism	Photoautotrophy (CO₂ as carbon source) or photoheterotrophy (obtaining energy and carbon from organic compounds)	[15-18,26-28,38-39]
Carbon metabolism	Carbon fixation capability with flexible metabolism under different electron donors and carbon sources	Chemolithoautotrophic growth using CO₂ as carbon source; metabolizes nitrate and nitrite to optimize growth rate	Strong carbon fixation capability; metabolizes organic acids and lignin degradation products	[15-18,26-28,40-41]
Nitrogen metabolism	Nitrogen fixation capability (potential for agricultural microbial modification)	Denitrification metabolic capacity (reduces nitrate to dinitrogen under anaerobic conditions)	Nitrogenase-related proteins; possesses photosynthetic nitrogen fixation capability applicable for nitrogen production optimization	[24,28,46]
Metabolic regulation	Environmental stresses such as magnetic fields, electric fields, and semiconductor materials; actively regulates membrane permeability to survive extreme conditions	High maximum specific growth rate under nitrate limitation; allocates protein resources on demand to adapt to environmental changes	High metabolic flexibility under illumination (can regulate photosynthetic electron transfer rate); thylakoid membrane structure limits CO₂ diffusion	[17,19-20,30-32,41]
Key metabolites	Carotenoids (pigments)	PHA/PHB or SCP; balance reducing power accumulation under anaerobic conditions	PHA production under optimized conditions;N-butanol synthesis and high-efficiency H₂ production	[25,36,38,45]
Environmental applications & potential	Degradation of environmental pollutants such as toluene and DCE (closely related to environmental remediation)	High-density fermentation of SCP; applicable for treating 3-HP and heavy metal contamination	Degradation of azo dyes (e.g., methyl red); enhances Fe²⁺ adsorption capacity through modified lignite	[21-22,25,36-37,43-44]

Parameter	Xanthobacter autotrophicus	Cupriavidus necator	Rhodopseudomonas palustris	References
Energy source	CO₂, methanol, ethanol,N-butanol, organic acids, etc.	Chemolithoautotrophic growth using H₂ as electron donor, also capable of utilizing various heterotrophic carbon sources in combination with nitrate metabolism	Photoautotrophy (CO₂ as carbon source) or photoheterotrophy (obtaining energy and carbon from organic compounds)	[15-18,26-28,38-39]
Carbon metabolism	Carbon fixation capability with flexible metabolism under different electron donors and carbon sources	Chemolithoautotrophic growth using CO₂ as carbon source; metabolizes nitrate and nitrite to optimize growth rate	Strong carbon fixation capability; metabolizes organic acids and lignin degradation products	[15-18,26-28,40-41]
Nitrogen metabolism	Nitrogen fixation capability (potential for agricultural microbial modification)	Denitrification metabolic capacity (reduces nitrate to dinitrogen under anaerobic conditions)	Nitrogenase-related proteins; possesses photosynthetic nitrogen fixation capability applicable for nitrogen production optimization	[24,28,46]
Metabolic regulation	Environmental stresses such as magnetic fields, electric fields, and semiconductor materials; actively regulates membrane permeability to survive extreme conditions	High maximum specific growth rate under nitrate limitation; allocates protein resources on demand to adapt to environmental changes	High metabolic flexibility under illumination (can regulate photosynthetic electron transfer rate); thylakoid membrane structure limits CO₂ diffusion	[17,19-20,30-32,41]
Key metabolites	Carotenoids (pigments)	PHA/PHB or SCP; balance reducing power accumulation under anaerobic conditions	PHA production under optimized conditions;N-butanol synthesis and high-efficiency H₂ production	[25,36,38,45]
Environmental applications & potential	Degradation of environmental pollutants such as toluene and DCE (closely related to environmental remediation)	High-density fermentation of SCP; applicable for treating 3-HP and heavy metal contamination	Degradation of azo dyes (e.g., methyl red); enhances Fe²⁺ adsorption capacity through modified lignite	[21-22,25,36-37,43-44]

Strains	Growth mode	Key enzymes	Engineering strategies	Synthetic products	References
X. autotrophicus	Chemolithoautotrophic	Hydrogenase, dehalogenase	Construction of gradient-copy number replicons and strong/weak promoter libraries; XanthoMoClo modular cloning; coupling of carbon fixation and nitrogen fixation; functional optimization of dehalogenase; CdTe quantum dot-microbe coupling; expansion of substrate spectrum	Degradation of chloroalkane/haloacid pollutants; biofertilizers (nitrogen-fixing inoculants); plant growth regulators; high-value pigments	[15,18,47]
C. necator	Facultative autotrophic	[NiFe]^- hydrogenase, RuBisCO	Knockout of CBB operons and competitive branches; co-expression of heterologous RuBisCO chaperones; expression regulation of hydrogenases (MBH/SH); carbon flux redirection; global reprogramming via ARTP mutagenesis; precise engineering of multi-omics identified targets	Bioplastics; high-value chemicals; biofuels; SCP	[26-27,32,48-49]
Hydrogenovibrio marinus	Obligate chemolithoautotrophic	Multiple RuBisCO isoforms	Synergistic expression of multiple RuBisCO isoforms; construction of salt-tolerant chassis; regulation of metabolic flux distribution; engineering optimization of promoter elements; enhancement of carbon fixation modules	Bioplastics; high-value solutes; biofuels	[50]
Acidithiobacillus ferrooxidans	Acidophilic chemolithoautotrophic	Hydrogenase, sulfur oxidase	Activity enhancement of hydrogenase and sulfur oxidase; engineering of extreme acid-base and heavy metal resistance; optimization of energy metabolic pathways; development of genetic tools; improvement of oxidative stress tolerance	Biohydrometallurgy; heavy metal removal from acid mine drainage; magnetic nanomaterials; barium sulfate crystals	[51]
Paracoccus denitrificans	Mixotrophic	Formate dehydrogenase, RuBisCO	Enhancement of heterotrophic nitrification-aerobic denitrification (HN-AD); engineering of formate dehydrogenase; optimization of pollutant degradation pathways; engineering of restriction-modification systems; synergistic regulation of carbon and nitrogen metabolism	Environmental governance; bioremediation; degradation of sulfonamide antibiotics and polycyclic aromatic hydrocarbons; carotenoids; PHB	[46]
Rhodococcus opacus	Dual chemolithoautotrophic and heterotrophic	NAD⁺-reducing hydrogenase, RuBisCO	Reconstruction of photosynthetic metabolic pathways; precise coupling of hydrogenase and carbon metabolism; knockout of competitive metabolic branches; functional enhancement of whole-cell biocatalysis; balanced regulation of carbon fixation and energy storage metabolism	Biofuels; PHA; fine chemicals	[52]

Strains	Growth mode	Key enzymes	Engineering strategies	Synthetic products	References
X. autotrophicus	Chemolithoautotrophic	Hydrogenase, dehalogenase	Construction of gradient-copy number replicons and strong/weak promoter libraries; XanthoMoClo modular cloning; coupling of carbon fixation and nitrogen fixation; functional optimization of dehalogenase; CdTe quantum dot-microbe coupling; expansion of substrate spectrum	Degradation of chloroalkane/haloacid pollutants; biofertilizers (nitrogen-fixing inoculants); plant growth regulators; high-value pigments	[15,18,47]
C. necator	Facultative autotrophic	[NiFe]^- hydrogenase, RuBisCO	Knockout of CBB operons and competitive branches; co-expression of heterologous RuBisCO chaperones; expression regulation of hydrogenases (MBH/SH); carbon flux redirection; global reprogramming via ARTP mutagenesis; precise engineering of multi-omics identified targets	Bioplastics; high-value chemicals; biofuels; SCP	[26-27,32,48-49]
Hydrogenovibrio marinus	Obligate chemolithoautotrophic	Multiple RuBisCO isoforms	Synergistic expression of multiple RuBisCO isoforms; construction of salt-tolerant chassis; regulation of metabolic flux distribution; engineering optimization of promoter elements; enhancement of carbon fixation modules	Bioplastics; high-value solutes; biofuels	[50]
Acidithiobacillus ferrooxidans	Acidophilic chemolithoautotrophic	Hydrogenase, sulfur oxidase	Activity enhancement of hydrogenase and sulfur oxidase; engineering of extreme acid-base and heavy metal resistance; optimization of energy metabolic pathways; development of genetic tools; improvement of oxidative stress tolerance	Biohydrometallurgy; heavy metal removal from acid mine drainage; magnetic nanomaterials; barium sulfate crystals	[51]
Paracoccus denitrificans	Mixotrophic	Formate dehydrogenase, RuBisCO	Enhancement of heterotrophic nitrification-aerobic denitrification (HN-AD); engineering of formate dehydrogenase; optimization of pollutant degradation pathways; engineering of restriction-modification systems; synergistic regulation of carbon and nitrogen metabolism	Environmental governance; bioremediation; degradation of sulfonamide antibiotics and polycyclic aromatic hydrocarbons; carotenoids; PHB	[46]
Rhodococcus opacus	Dual chemolithoautotrophic and heterotrophic	NAD⁺-reducing hydrogenase, RuBisCO	Reconstruction of photosynthetic metabolic pathways; precise coupling of hydrogenase and carbon metabolism; knockout of competitive metabolic branches; functional enhancement of whole-cell biocatalysis; balanced regulation of carbon fixation and energy storage metabolism	Biofuels; PHA; fine chemicals	[52]

Product	Strains	Fermentation conditions	Cultivation period	Titer	Process limits & mitigations	References
PHA	Cupriavidus eutrophusB-10646	Autotrophic cultivation with H₂:O₂:CO₂ (7:2:1) in the recycled-gas closed-circuit culture system	80 h	36 g/L	Limit: low cell density in chemostat, high mass transfer requirements for H₂/O₂ Mitigation: two-stage batch with nitrogen limitation achieved high cell density and polymer content; gas mixture recirculation improved substrate utilization	[63]
PHB	C. necator DSM 545	Cultivation with formic acid and acetic acid in fed-batch fermentations	63 h	58.5 g/L	Limit: O₂ transfer limits growth Mitigation: acetic acid is directly converted to acetyl-CoA; linear growth phase extends biomass accumulation	[64]
PHB	C. necator DSM 545	Cultivation with waste	48 h	66 g/L	Limit: exponential feeding alone caused over or under feeding due to parameter inaccuracies Mitigation: novel three-stage strategy maintained substrate within optimal range	[65]
L-threonine	Brevibacterium flavum	Cultivation with glucose, peptone, beef extract, and yeast extract in batch fermenters	44 h	12.14% (yield, g/100 mL)	Limit: pH drops during fermentation due to metabolic byproducts; high pH control increases ammonia cost Mitigation: optimal conditions determined via orthogonal design; maintaining pH at 6.0-7.0 is recommended to save ammonia costs with acceptable yield	[23]
SCP	X. variabilis NFM-97 and Shinella sp. NM-101	Cultivation with H₂:O₂:CO₂:N₂(10:2:10:78) in sealed bottles	72 h	0.6 g/L	Limit: energy-expensive N₂ fixation reduces biomass yield compared to NH₄⁺-based HOB Mitigation: coculture with heterotroph enhanced growth and protein quality via metabolic cooperation	[24]
N-butanol	R. palustris TIE-1	Cultivation in batch cultures under photoheterotrophic or photoautotrophic conditions	10 d	4.98 mg/L	Limit: deleting acetyl-CoA consuming pathways diverted carbon to acetone (byproduct), lowering N-butanol yield Mitigation: deleting the electron-consuming N-fixation pathway created a more reduced intracellular environment, enhancing N-butanol production and carbon conversion efficiency	[45]
H₂	R. palustris CGA009	Cultivation with glycerol and glutamate in batch bioreactors under anaerobic phototrophic conditions	Up to 212 h	~16 mL/h	Limit: overexpression of alternative nitrogenases caused a metabolic burden Mitigation: applying non-growing conditions reduced competition from cell division	[46]
SCP	HOB community	Autotrophic cultivation with H₂:O₂:CO₂(65:20:15)	90 d	24 g/L	Limit: Low productivity (0.08 g/L/h) due to predatory Bdellovibrio (33% abundance) Mitigation: high dilution rate selected for fast-growing Sulfuricurvum spp. (97% abundance), achieving stable, high-rate SCP production	[66]
SCP	NF-HOB community (92% Xanthobacter)	Autotrophic cultivation with H₂:O₂:CO₂:N₂(59:19:7:15-50:25:8:17)in airlift reactor	19.2 d	2.3 g/L	Limit: O₂>2% inhibits nitrogenase in start-up; low N₂ fixation efficiency wastes nitrogen gas Mitigation: two-stage gas supply reduced start-up lag from 20 to 4 days; CO₂ conversion efficiency>25%	[2]
SCP	C. necator H16	Autotrophic cultivation with H₂:O₂:CO₂:N₂(70:10:12:8) in a custom 300 L pressurized deep-jet bioreactor	35 h	72 g/L	Limit: heterotrophic inoculum caused 10 h lag phase; foaming at high cell densities Mitigation: enabling modified strains to outperform the wild-type with higher rates at later time points	[67]
SCP	C. necator R3	Autotrophic cultivation with H₂:O₂:CO₂(7:1:1-7:2:1) in fed-batch fermentations	5 d	13.8 g/L	Limit: low CO₂ fixation efficiency limits SCP productivity Mitigation: multi-omics revealed key upregulations; synonymous mutation in lysine-tRNA ligase enhances translation; process optimization maximized performance	[49]

Product	Strains	Fermentation conditions	Cultivation period	Titer	Process limits & mitigations	References
PHA	Cupriavidus eutrophusB-10646	Autotrophic cultivation with H₂:O₂:CO₂ (7:2:1) in the recycled-gas closed-circuit culture system	80 h	36 g/L	Limit: low cell density in chemostat, high mass transfer requirements for H₂/O₂ Mitigation: two-stage batch with nitrogen limitation achieved high cell density and polymer content; gas mixture recirculation improved substrate utilization	[63]
PHB	C. necator DSM 545	Cultivation with formic acid and acetic acid in fed-batch fermentations	63 h	58.5 g/L	Limit: O₂ transfer limits growth Mitigation: acetic acid is directly converted to acetyl-CoA; linear growth phase extends biomass accumulation	[64]
PHB	C. necator DSM 545	Cultivation with waste	48 h	66 g/L	Limit: exponential feeding alone caused over or under feeding due to parameter inaccuracies Mitigation: novel three-stage strategy maintained substrate within optimal range	[65]
L-threonine	Brevibacterium flavum	Cultivation with glucose, peptone, beef extract, and yeast extract in batch fermenters	44 h	12.14% (yield, g/100 mL)	Limit: pH drops during fermentation due to metabolic byproducts; high pH control increases ammonia cost Mitigation: optimal conditions determined via orthogonal design; maintaining pH at 6.0-7.0 is recommended to save ammonia costs with acceptable yield	[23]
SCP	X. variabilis NFM-97 and Shinella sp. NM-101	Cultivation with H₂:O₂:CO₂:N₂(10:2:10:78) in sealed bottles	72 h	0.6 g/L	Limit: energy-expensive N₂ fixation reduces biomass yield compared to NH₄⁺-based HOB Mitigation: coculture with heterotroph enhanced growth and protein quality via metabolic cooperation	[24]
N-butanol	R. palustris TIE-1	Cultivation in batch cultures under photoheterotrophic or photoautotrophic conditions	10 d	4.98 mg/L	Limit: deleting acetyl-CoA consuming pathways diverted carbon to acetone (byproduct), lowering N-butanol yield Mitigation: deleting the electron-consuming N-fixation pathway created a more reduced intracellular environment, enhancing N-butanol production and carbon conversion efficiency	[45]
H₂	R. palustris CGA009	Cultivation with glycerol and glutamate in batch bioreactors under anaerobic phototrophic conditions	Up to 212 h	~16 mL/h	Limit: overexpression of alternative nitrogenases caused a metabolic burden Mitigation: applying non-growing conditions reduced competition from cell division	[46]
SCP	HOB community	Autotrophic cultivation with H₂:O₂:CO₂(65:20:15)	90 d	24 g/L	Limit: Low productivity (0.08 g/L/h) due to predatory Bdellovibrio (33% abundance) Mitigation: high dilution rate selected for fast-growing Sulfuricurvum spp. (97% abundance), achieving stable, high-rate SCP production	[66]
SCP	NF-HOB community (92% Xanthobacter)	Autotrophic cultivation with H₂:O₂:CO₂:N₂(59:19:7:15-50:25:8:17)in airlift reactor	19.2 d	2.3 g/L	Limit: O₂>2% inhibits nitrogenase in start-up; low N₂ fixation efficiency wastes nitrogen gas Mitigation: two-stage gas supply reduced start-up lag from 20 to 4 days; CO₂ conversion efficiency>25%	[2]
SCP	C. necator H16	Autotrophic cultivation with H₂:O₂:CO₂:N₂(70:10:12:8) in a custom 300 L pressurized deep-jet bioreactor	35 h	72 g/L	Limit: heterotrophic inoculum caused 10 h lag phase; foaming at high cell densities Mitigation: enabling modified strains to outperform the wild-type with higher rates at later time points	[67]
SCP	C. necator R3	Autotrophic cultivation with H₂:O₂:CO₂(7:1:1-7:2:1) in fed-batch fermentations	5 d	13.8 g/L	Limit: low CO₂ fixation efficiency limits SCP productivity Mitigation: multi-omics revealed key upregulations; synonymous mutation in lysine-tRNA ligase enhances translation; process optimization maximized performance	[49]

氢氧化细菌设计构建研究进展

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张飘 ¹^,² , 卢子康 ¹^,³ , 李飞 ¹ , 马春玲 ¹^,^* , 朱之光 ¹^,²^,^*

微生物学报 | 综述 2026,66(9): 4420-4442

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微生物学报 |综述 2026 , 66 (9) : 4420 -4442

氢氧化细菌设计构建研究进展

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张飘¹^,², 卢子康¹^,³, 李飞¹, 马春玲¹^,^*, 朱之光¹^,²^,^*

作者信息

^1.中国科学院天津工业生物技术研究所，天津

^2.中国科学院大学，北京

^3.南开大学生命科学学院，天津

通讯作者:

马春玲, 朱之光

作者简介:

作者贡献声明

张飘：主要负责撰写1-3章内容，文献检索；卢子康：主要负责撰写3-6章内容，文献检索；李飞：基金获取；马春玲：完善修稿，文献检索；朱之光：提供概念，完善修稿及提供资源。

Research progress in the design and engineering of hydrogen-oxidizing bacteria

Piao ZHANG¹^,², Zikang LU¹^,³, Fei LI¹, Chunling MA¹^,^*, Zhiguang ZHU¹^,²^,^*

Affiliations

^1.Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

^2.University of Chinese Academy of Sciences, Beijing, China

^3.College of Life Sciences, Nankai University, Tianjin, China

Corresponding Author:

^*E-mail: MA Chunling, machl@tib.cas.cn

ZHU Zhiguang, zhu_zg@tib.cas.cn

出版时间: 2026-09-04 doi: 10.13343/j.cnki.wsxb.20260357

文章导航

摘要

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在全球气候变化和资源短缺的背景下，利用清洁能源驱动生物固碳技术逐渐兴起并得到广泛关注。H₂作为一种可再生的清洁能源，来源广泛且制备方式简便，可作为微生物固碳的能量和电子来源。本文综述了以氢氧化细菌(hydrogen-oxidizing bacteria, HOB)为代表的氢能驱动固碳底盘细胞的代谢基础，并对其中几种代表性菌株的研究进展进行介绍。探讨了HOB的工程改造方法，包括使能技术开发、代谢通路重构与强化策略、微生物-材料耦合等策略。此外，针对HOB现阶段存在的问题进行了探讨并提出未来发展方向。尽管当前面临一定的技术瓶颈，但随着代谢工程和系统生物学的推进，HOB将在碳循环利用和绿色生物制造中发挥重要作用。

关键词

氢氧化细菌 / 微生物固碳 / 化能自养菌 / 底盘细胞设计

Abstract

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Against the backdrop of global climate change and resource scarcity, biological carbon fixation technologies driven by clean energy have emerged and attracted widespread attention. Hydrogen (H₂), as a renewable and clean energy carrier with abundant sources and easy production, can serve as both the energy and the electron donor for microbial carbon fixation. This review summarizes the metabolic foundations of hydrogen-driven carbon-fixing chassis cells represented by hydrogen-oxidizing bacteria (HOB), and highlights recent advances in several representative strains. Furthermore, engineering strategies for HOB are discussed, including the development of enabling technologies, the reconstruction and reinforcement of metabolic pathways, and microbe-material coupling approaches. Additionally, this paper analyzes the current challenges facing HOB and proposes the future perspectives. Despite existing technical bottlenecks, with the advancement of metabolic engineering and systems biology, HOB are expected to play an important role in carbon recycling and sustainable biomanufacturing.

Key words

hydrogen-oxidizing bacteria / microbial carbon fixation / chemolithoautotrophs / chassis cell design

引用本文

张飘, 卢子康, 李飞, 马春玲, 朱之光. 氢氧化细菌设计构建研究进展. 微生物学报, 2026 , 66 (9) : 4420 -4442 . DOI: 10.13343/j.cnki.wsxb.20260357

Piao ZHANG, Zikang LU, Fei LI, Chunling MA, Zhiguang ZHU. Research progress in the design and engineering of hydrogen-oxidizing bacteria[J]. Acta Microbiologica Sinica, 2026 , 66 (9) : 4420 -4442 . DOI: 10.13343/j.cnki.wsxb.20260357

正文

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当前资源和环境问题日益严峻，工业生产导致的CO₂排放量持续增加，进一步加剧全球变暖^[1]。与此同时，随着人口增长，粮食安全问题日益突出，蛋白质需求量随之上升，未来这一需求还将进一步增加^[2]。然而，传统生产方式不仅能耗高、碳排放问题严重，还受到土地、水资源和环境污染等多重限制，难以满足可持续发展的需求^[3-4]。因此，发展可利用可再生能源驱动CO₂转化的新型生物制造系统，已成为绿色生物制造和合成生物学领域的重要研究方向。

H₂作为一种可再生清洁能源，可通过电解水实现绿色制备，同时可与风能、太阳能等可再生能源耦合。相较于其他形式的可再生能源，该能源在应用形式上与传统化石能源高度相似，更易获得公众认可^[5-6]；此外，与传统有机底物相比，以H₂为底物时，其氧化产物为水，不会引入碳排放^[6]。在生物进化过程中，早期地球处于缺氧环境，地质活动可持续产生H₂，这为以H₂为能源、CO₂为碳源的代谢方式提供了选择优势^[7-10]。在自然选择作用下，氢氧化细菌(hydrogen-oxidizing bacteria, HOB)保留了这一代谢特征。因此，HOB被认为是连接可再生能源利用与碳循环转化的重要微生物底盘。

HOB表现出灵活的代谢活性和较强的环境适应能力，可在自养、异养和混合营养模式间灵活切换^[11]，这为其作为固碳底盘细菌奠定了基础。近年来，多组学技术发展迅速，研究者开始尝试改造HOB，力求提升其碳固定与能量转化效率，进而合成高价值产物。然而，现有菌株在遗传操作工具、能量利用效率等方面仍存在局限，制约了其大规模生产与应用推广^[12]。因此，围绕H₂氧化、电子传递、CO₂固定以及特定碳流分配等关键模块，对微生物底盘进行系统设计与重构，是该领域当前面临的核心问题。本文紧扣HOB底盘细胞设计构建的核心主线，梳理HOB代谢基础与代表性菌株的研究进展，依次围绕使能技术开发、代谢通路重构与强化策略、微生物-材料耦合、微生物与电化学系统耦合等改造策略展开综述，展望HOB通过设计构建新型工程菌株，提升其在单细胞蛋白(single-cell protein, SCP)、生物材料合成及碳循环等领域的应用潜力。

1 HOB代谢基础

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HOB是一类以H₂为能量来源的化能自养微生物，广泛分布于土壤、淡水、海洋等环境中，分属假单胞菌门(Pseudomonadota)、放线菌门(Actinomycetota)等，常见属包括氢噬菌属、贪铜菌属(Cupriavidus)等^[4]。这类微生物凭借自身的氢代谢以及碳、氮固定能力(具体代谢过程如图1所示)在生态修复中发挥重要作用。

HOB利用H₂代谢过程中的关键功能酶为氢化酶，该酶通过氧化H₂产生质子和电子，为CO₂固定提供还原当量，同时将电子传递至呼吸链，以ATP形式回收能量；根据活性中心组成，氢化酶可分为[NiFe]氢化酶、[FeFe]氢化酶和[Fe]氢化酶，大多数HOB含有定位于细胞膜或细胞质的[NiFe]氢化酶；多数菌株携带膜结合氢化酶(membrane-bound hydrogenase, MBH)，部分菌株含有可溶性氢化酶(soluble hydrogenase, SH)，少数菌株同时具备2类氢化酶；MBH通过小亚基疏水C端固定于细胞膜，借助膜结合细胞色素与呼吸链相连并完成电子传递；SH由4个异源亚基与2个辅助亚基组成，可将H₂氧化产生的电子传递给烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADP⁺)，将其还原为还原型烟酰胺腺嘌呤二核苷酸磷酸(reduced nicotinamide adenine dinucleotide phosphate, NADPH)，作为还原当量参与卡尔文(Calvin-Benson-Bassham, CBB)循环或通过呼吸链生成ATP^[11]。

在CO₂固定层面，HOB主要依托CBB循环完成，该过程包含羧化、还原和再生3个阶段，其中羧化阶段由核酮糖-1,5-二磷酸羧化酶/加氧酶(ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO)催化，捕获环境中的CO₂并与核酮糖-1,5-二磷酸(ribulose-1,5-bisphosphate, RuBP)结合，生成3-磷酸甘油酸(3-phosphoglycerate, 3-PGA)；还原阶段微生物利用H₂氧化产生的能量与还原力，将3-PGA转化为甘油醛-3-磷酸(glyceraldehyde-3-phosphate, G3P)；再生阶段部分G3P重新参与循环以合成RuBP，保障CBB循环持续完成CO₂固定过程^[12]。

部分HOB还携带固氮酶，固氮过程所需的ATP和NAD(P)H同样来自H₂氧化，但该过程需要消耗更多H₂以满足N≡N键断裂的能量需求，固氮生成的氨可与三羧酸循环(tricarboxylic acid cycle, TCA)的中间产物结合，作为氨基酸与蛋白质合成的氮源^[4]。当环境中氧或氮匮乏时，HOB可将代谢中间产物转化为聚羟基丁酸酯(polyhydroxybutyrate, PHB)，储存多余的能量和碳源以应对环境胁迫；当环境中氧、氮供应恢复后，PHB可重新分解进入代谢通路生成目标产物^[4,13]。这些独特的代谢特性赋予HOB极强的环境适应性和多元应用潜力，部分HOB除固氮能力外，同时具备有机物降解能力^[11]，可进一步提升其环境耐受能力。此外，HOB可适配好氧-缺氧交替的生态环境，为溶解氧与硝态氮浓度较高的污水处理厂实现温室气体减排提供可能，已有研究表明HOB可耦合H₂S进行培养，驱动污泥转化为SCP^[14]。

2 代表性HOB

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2.1 黄色杆菌

自养黄色杆菌(Xanthobacter autotrophicus)是一类具有代表性的HOB，1976年首次被分离得到，属于革兰氏阴性、好氧至微需氧的无芽孢杆状细菌，可在不同碳源(CO₂、甲醇、乙醇、正丙醇、正丁醇及多种有机酸)条件下调控自身代谢状态，实现自养和异养生长^[15-18]。该菌株具备极强的代谢可调性和环境适应性，对其开展系统研究不仅有助于解析微生物的环境适应机制，还能为资源利用和污染环境修复提供新的技术方案。

X. autotrophicus可利用多种单碳化合物和低浓度有机酸作为碳源，灵活的代谢特性使其在不同生境中均具备极强的生存能力。例如，将其置于最高9 T的静态磁场中时，磁场可提升细胞膜通透性，降低钠钾ATP酶与钙镁ATP酶活性，引发胞内代谢扰动与ATP水平下降，触发细胞应激反应，通过主动缩小细胞体积适配极端环境^[19]。进一步构建电化学水分解与X. autotrophicus耦合的生物-无机混合系统，依托电化学水分解提供还原当量，通过电极施加环境刺激并通入N₂和CO₂，蛋白质组学与代谢组学分析显示，该菌株内499种蛋白上调、380种蛋白下调，20种代谢物上调、4种代谢物下调^[17]。此外，构建X. autotrophicus与吸光材料、半胱氨酸组合的微生物-半导体杂交体系，依托吸光材料提供硫化镉量子点，结果发现727种蛋白与29种代谢物丰度显著升高，53种蛋白与27种代谢物丰度显著降低^[20]。综上所述，该菌株灵活的代谢活性和环境调控特性，为解析其环境适应机制与全局调控网络奠定了重要基础。

依托X. autotrophicus的灵活代谢特性，可将其应用于环境修复领域，实现甲苯和卤代脂肪族化合物的高效去除。已有研究从甲苯污染淡水溪流的岩石表面生物膜中分离得到2株甲苯降解菌株TiO1和TiO2，其16S rRNA基因序列与X. autotrophicus完全一致，脂肪酸组分分析也将其归类为X. autotrophicus；2株菌的甲苯降解速率分别达到(3.8±0.5) μmol/mg和(28.3±2.2) μmol/mg^[21]。此外，卤代烷烃脱卤酶可降解卤代脂肪族化合物，如1,2-二氯乙烷(1,2-dichloroethane, DCE)。自养X. autotrophicus GJ10所产的卤代烷烃脱卤酶A (haloalkane dehalogenase A, DhlA)可将DCE转化为毒性更低的醇类物质，目前已应用于DCE污染地下水的净化工程^[22]。在蛋白质与氨基酸生产方面，通过优化发酵条件(温度30 ℃、pH 7.0、接种量10%)，X. autotrophicus合成L-苏氨酸的产率可达12.14%^[23]。将固氮型可变黄杆菌(Xanthobacter variabilis) NFM-97与异养菌申氏菌(Shinella sp.) NM101进行共培养，可使前者菌体数量提升(11.4±3.5)倍，蛋白含量提高(24±2)%，必需氨基酸占比提升(28±11)%^[24]。同时该菌株还可通过发酵大量合成玉米黄质，这是一类广泛应用于食品领域的类胡萝卜素^[25]。综上所述，深入挖掘X. autotrophicus的代谢潜力，有望为我国资源短缺与环境修复领域的问题解决提供新的技术支撑。

2.2 钩虫贪铜菌

钩虫贪铜菌(Cupriavidus necator)，曾用名富养罗尔斯通氏菌(Ralstonia eutropha)，是HOB的代表性模式菌株之一，属于革兰氏阴性、中温、无芽孢的兼性化能自养细菌^[26]，分离自自然土壤环境，可利用多种碳源，既能以H₂为电子供体、CO₂为碳源开展化能自养生长，厌氧条件下又具备完整的反硝化代谢通路^[27-28]。其代谢途径独特且多元，不仅可通过固碳过程削减温室气体排放，还能通过代谢调控合成多种高价值生物材料与蛋白质，在环境修复和资源化利用领域展现出巨大应用潜力。

目前，针对C. necator的代谢特性及生长过程中的资源分配已有较多研究。研究者对C. necator中电子传递机制进行了解析，明确细胞色素c氧化酶是核心位点，亚硝酸还原酶为次要位点，外膜蛋白A可能参与中介体运输，这些发现为优化生物电化学系统提供了依据^[29]。同时，研究者构建了以硝酸盐为末端电子受体的生长动力学模型，发现自养条件下菌体生长速率极低；当硝酸盐为限制性底物时，最大比生长速率(0.509 d^-1)显著高于亚硝酸盐限制时的0.195 d^-1，模型预测值与实验数据高度吻合，可有效支撑生长速率的深入探究^[30]。与此同时，关于C. necator蛋白质与酶的资源分配研究表明，该菌虽能表达大量蛋白质，但其中约43%仅作为储备资源以应对环境变化，其蛋白质利用的核心规律是按需分配、重点保障，在自养和异养模式下分配不同的酶发挥作用^[31-32]，上述结果为后续菌株改造和生产效率提升提供了参考依据。

关于C. necator的应用，因其具备合成多种生物材料的潜力，尤其是在聚羟基脂肪酸酯(polyhydroxyalkanoate, PHA)的生物回收方面，为生物材料科学提供了新方向。研究表明，C. necator H16中含有15种PhaA同工酶；通过生物信息学序列分析及单基因与多基因缺失突变株的构建，确认PhaA、BktB和H16-A0170是该菌参与PHB合成的关键β-酮硫解酶；该菌还可生成不溶于水的PHB颗粒、以甲酸为原料生产巴豆酸，以及合成日常所需的氨基酸^[27,33-35]。在营养受限条件下，该菌可大量积累PHA (最高可达细胞干重的90%)；而通过调节培养条件，又能将PHA含量降至最低，使75%的干物质转化为生物质用于蛋白质生产。早在20世纪60年代，该菌就被提议作为空间站的蛋白质来源^[25]。2002年的研究进一步表明，C. necator可在无机盐培养基中进行高密度发酵，干细胞密度达到150-182 g/L；在氧限制条件下，该菌不产有机酸，而是合成PHB以平衡多余还原力，且不抑制自身生长，蛋白质含量高，适用于蛋白质生产；例如，选用合适的phaP启动子，将有机磷水解酶置于其下，通过双亲本接合将单拷贝整合到染色体上，可获得生物量达99.1 g/L的菌体密度^[36]，充分证明该菌可用于高效合成蛋白质。此外，C. necator在环境应用方面也展现出潜力。实验证实，该菌含有3-羟基丙酸(3-hydroxypropanoate, 3-HP)的代谢途径，能够以3-HP为唯一碳源生长。通过生物信息学与转录组分析鉴定出3个推定的甲基丙二酸半醛脱氢酶基因(mmsA1、mmsA2、mmsA3)及1个推定的脱氢酶基因，其中3种脱氢酶均参与3-HP的降解^[37]。这表明该菌富含多种降解基因，可用于部分有机物的生物降解，从而实现环境的净化和改善。综上所述，对C. necator的持续研究与开发将推动其在环境修复、生物材料及生物化工等领域的广泛应用，更好地服务于人类需求。

2.3 沼泽红假单胞菌

沼泽红假单胞菌(Rhodopseudomonas palustris)是一种紫色非硫细菌，广泛分布于污泥、土壤、水生沉积物、碱性水体、稻草、枯枝落叶及富营养化池塘等多种环境中；在光照条件下，该菌既能利用光能、以CO₂为碳源生长，也可直接利用有机物作为碳源；同时，它还能从有机化合物中获取碳源与能量，或从无机化合物中获得能量^[38-39]。R. palustris具有独特特性，可进行光合自养和光合异养生长，同时具备代谢多样性。这种灵活的代谢模式使其兼具固碳与有机物降解等多重功能，在高价值产物合成和环境污染物处理方面展现出良好的应用潜力。

针对R. palustris的CO₂固定机制与浓缩机制，近期研究已取得重要进展。通过微生物电合成技术与无标记定量蛋白质组学联用，揭示了电位对CO₂固定的调控机制：利用电位调控含NADH结合结构域的蛋白，激活泛醌和萜类-醌生物合成通路，为电子传递提供支撑，从而提高CO₂固定效率；更负的阴极电位(-0.8 V)可增强补光相关蛋白的富集，进一步提升电子摄取与CO₂固定的协同效率^[40]。此外，关于CO₂浓缩机制的研究表明，该菌能以甲酸为唯一碳源，通过色素合成、膜结构及黄素调控的多方面协同实现CO₂浓缩；细菌叶绿素和类胡萝卜素合成过程中产生CO₂并消耗质子，形成低透性的HCO₃^-碱性环境；同时，细菌的类囊体膜可降低CO₂扩散速率，减少流失；通过突变RibBX提高黄素可用性，增强甲酸脱氢酶活性，使CO₂合成速率提升，满足CBB循环需求，从而更高效地实现CO₂浓缩^[41]，上述发现为进一步揭示其碳代谢机制提供了依据。与此同时，该菌独特的碳源利用规律也被揭示：R. palustris代谢途径多样，可利用多种有机酸，但无法单独以乳酸为唯一碳源；然而，当乳酸与乙酸、琥珀酸、甘油等底物共存时可被快速共利用；在多种碳源同时存在的情况下，该菌优先利用乙酸，同时抑制甘油的利用，表现出显著的底物特异性代谢特征^[42]。

关于R. palustris的应用，目前主要聚焦于污染物处理。研究表明，从沼泽红假单胞菌中筛选并鉴定出可水解γ-戊内酯酶Rpa3624，用于该类污染物的处理^[42]。利用沼泽红假单胞菌对褐煤进行微生物改性，可通过调控表面化学特性提升对Fe²⁺的吸附能力，其最佳吸附条件为褐煤投放量1 g、初始Fe²⁺浓度65 mg/L、pH 4.0、温度45 ℃，此时最大吸附容量可达13.10 mg^[43]，表明该菌可用于天然褐煤的改性处理。此外，R. palustris能在无替代碳源和固定氮源的条件下彻底降解偶氮染料甲基红，且降解过程中光合作用活性和电子传递速率均提升^[44]，显示出其在偶氮染料生物处理方面的良好应用前景。

除污染物处理外，R. palustris还可用于生产生物质及清洁能源。研究表明，菌株CGA009能利用木质素降解产物合成PHA，但PHA颗粒形成是实现高效生物塑料生产的限制因素；将C. necator H16的phaP1基因克隆至载体pBBR1-MCS2并转化至CGA009中，可成功表达phaP1基因，使沼泽红假单胞菌在有氧条件下也能实现生物塑料合成^[38]。另外，向无氧光合自养菌R. palustris TIE-1中导入2种正丁醇合成表达盒，并敲除固氮、PHB合成及糖原合成途径后，在以3-羟基丁酸酯为碳源、NH₄⁺为氮源的光合异养条件下，正丁醇产量可达(4.98±0.87) mg/L^[45]。通过改造R. palustris的固氮酶可提高产氢效率，该菌含有固氮酶相关蛋白(Nif、Vnf、Anf)，通常以Nif为主，Vnf和Anf仅在缺氮条件下少量表达；给Vnf和Anf匹配强启动子强制其表达，可使菌体在非生长状态下仍能持续产氢，从而实现工业连续产氢，为清洁能源生产提供新途径^[46]。深入探究R. palustris有助于解决当前的环境与资源问题。

综上所述，X. autotrophicus、C. necator和R. palustris均可从土壤中分离获得，但R. palustris的分布范围更广，这可能与其属于光合异养菌、代谢谱宽泛、环境适应性极强有关。将3种代表性菌株的电子供体来源、碳代谢能力、氮代谢能力、代谢调控、关键代谢产物、环境适应性与应用潜力进行对比(表1)。三者代谢机制的研究侧重点不同，X. autotrophicus的研究多集中于代谢功能与环境适应能力，可针对不同物理化学刺激(电磁场、电化学水解等)产生强适应性与代谢调控响应；C. necator的研究多聚焦于不同代谢模式下的资源分配及电子传递机制，适配高密度合成代谢(如PHA、SCP、PHB)及复杂环境下的高效自养-异养切换过程；R. palustris具备特有的光能利用机制(光合生长、叶绿素合成)和光依赖型代谢调控模式，相关研究多围绕碳利用规律展开，涵盖CO₂浓缩与固定过程。在应用层面，三者均具备环境修复能力，但具体方向各有差异。X. autotrophicus偏向工业污染物处理，重点针对化工类污染场景；C. necator本质上更适配工业生产菌株定位，可在生产过程中同步实现污染物降解；R. palustris的环境修复覆盖范围更广，相关研究成果也更多集中于环境治理领域。在高价值产物合成方面，X. autotrophicus合成的生物质产物多偏向食品与营养类方向；C. necator是合成生物塑料的优势菌株，还可生产巴豆酸、SCP等化学品，更适配大规模工业化生物质生产场景；R. palustris依托光合驱动过程，清洁能源属性突出，可利用木质素及各类废弃物实现低碳生产。

3 HOB设计改造进展

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HOB自身在遗传操作体系、代谢调控网络、固碳效率与气体传质等层面仍存在诸多局限，上述问题成为制约其工业化生产落地的核心瓶颈^[4,13]。针对上述技术障碍，研究者围绕遗传工具箱构建、基因组编辑、代谢通路重构、多组学解析以及微生物-材料耦合等方向开发了一系列适配HOB底盘的菌株改造策略(表2)。这些方法为氢能自养底盘细胞的机制解析与定向改造奠定了技术基础，也为后续其工业化开发利用提供了明确的研究思路。

3.1 使能技术开发

自养底盘细胞的工程化改造高度依赖成熟的技术工具体系，从遗传元件挖掘、高效编辑方法建立到系统生物学层面的全局设计，共同构成了HOB定向改造的使能技术支撑体系。本节从遗传工具箱构建、基因组编辑技术、系统生物学辅助设计3个维度系统梳理当前HOB改造领域的核心技术方法。

3.1.1 遗传工具箱构建与表达元件优化

建立稳定可用的遗传操作系统，是对氢能自养底盘细胞进行工程化改造的基础。由于HOB种类繁多、遗传背景复杂，长期以来缺乏可靠的标准化基因工程工具，因此构建适配宿主特性的专属遗传工具箱具有重要的研究价值。现有研究通常从复制起点、拷贝数、选择性标记、启动子和终止子等多个功能模块入手，逐步完善HOB的标准化表达元件体系。

在复制子选择过程中，广宿主范围质粒常被用于筛选适用于不同菌株的骨架元件，需针对不同细菌进行系统评估并择优选择。对于拷贝数的选择，低和中等拷贝数复制子可起到平衡基因表达与代谢负荷的作用，而更高的拷贝数可能在提升宿主表达量的同时增加宿主代谢负担；梯度拷贝数的复制子可实现对基因表达宽范围的调控，复制子是外源基因稳定复制的核心元件，针对HOB不同表达需求，目前已筛选得到梯度拷贝数的复制子元件，实现了表达强度的精准匹配；在模式氢能固碳菌株X. autotrophicus中，pBBR1复制子可实现约100 copies/cell的高拷贝表达，RK2复制子为约6 copies/cell的低拷贝表达，pSa复制子为约10 copies/cell的中等拷贝表达，拷贝数动态调控范围超过15倍；该梯度体系可根据目标基因功能灵活选择：高拷贝复制子用于RuBisCO、氢化酶等关键限速酶的过表达，低拷贝复制子用于代谢平衡相关基因的微调，有效降低了外源基因表达对底盘细胞自养生长的代谢负荷^[18]。此外，选择性标记和调控元件的优化同样关键。不同的抗生素筛选标记在不同菌株中的抑制效果与表达效果存在差异，需选择适配的选择性标记以提升筛选效率。经定量表征的启动子与终止子为基因表达的精准可控提供了基础。针对HOB中基因表达可控性差的问题，研究人员系统表征了以下元件：组成型启动子库在X. autotrophicus中的表达强度覆盖超过10倍的动态范围，其中最强启动子的转录活性可达弱启动子的12倍以上，可满足不同强度的本底表达需求；IPTG诱导型启动子系统的诱导倍数超过100倍，可实现从“低表达”到“高表达”的快速切换，避免组成型过表达对细胞生长的抑制；且高效终止子的转录终止效率超过95%，可有效降低表达泄漏，进一步提升了基因表达的可控性^[18]。

针对HOB长期缺乏通用多片段组装工具的问题，Soltysiak等^[47]开发了XanthoMoClo模块化克隆工具包，实现了多基因通路的标准化构建；该工具基于Golden Gate组装策略，≥5个片段的多基因组装阳性克隆成功率可达90%以上，相较于传统分步克隆方法，组装周期从数周缩短至3-5 d，组装效率提升超过70%；该工具包可适配21种Xanthobacter与Roseixanthobacter属菌株，通用性相较于现有零散工具提升了近3倍，为氢能驱动固碳底盘细胞的规模化改造提供了标准化操作平台。

综上所述，梯度化表达元件与标准化克隆工具的构建，为HOB底盘的精准基因调控与多通路组装提供了核心技术支撑。不同拷贝数复制子的搭配策略可在强化关键限速酶表达的同时精准控制代谢负荷，避免过表达对自养生长的非特异性抑制；模块化克隆体系则大幅缩短了多基因通路的构建周期，为HOB底盘的规模化工程改造扫清了操作层面的障碍。

3.1.2 基因组编辑与定点代谢改造

在遗传工具箱建立的基础上，菌株的深度定向改造高度依赖高效基因组编辑技术。对于氢能自养底盘细胞而言，基因组编辑不仅可实现目标基因的精准敲除、插入和替换，还可用于全局重构代谢网络，强化核心功能通路、弱化竞争性支路(图2)。目前常用的技术策略包括CRISPR-Cas系列技术、同源重组介导的基因操作、电转化导入体系、靶基因定向敲除等。依托上述方法，研究者可对菌株核心代谢节点开展定点改造，实现代谢功能的定向重编程。

针对碳固定模块，通过同源重组技术敲除CBB操纵子相关基因或C. necator的大质粒，证实菌株携带的2个CBB操纵子均处于活跃状态，且对碳固定过程的贡献几乎相等；类似的敲除实验表明，尽管SH和MBH同属供能氢酶模块并支持自养生长，但SH发挥了更核心的作用；引入携带内源性GroES/EL伴护系统和RbcX的异源蓝藻RuBisCO后，工程菌株自养生长72 h后的OD₆₀₀提升了89.15%，证明活性更高的蓝藻RuBisCO在C. necator中展现出优于原有CBB途径的功能；同时，通过调节MBH和SH的表达优化氢化酶性能，可使C. necator H16的OD₆₀₀在72 h内进一步升高93.4%^[48]，其主要工业产物PHB的自养产率也同步提高99.71%。

针对能量供给模块，利用电转化导入介导的定点修饰技术，将光诱导强启动子pucBa (Ppuc)插入R. palustris中Vnf、Anf替代固氮酶操纵子的上游区域，Ppuc介导的Vnf/Anf转录水平较野生型提升1 000-4 000倍；固氮酶表达量的提升直接改善菌株产氢性能，在非生长氮饥饿条件下培养212 h时，Vnf:puc/Anf:puc菌株的产氢速率较野生型提升超5倍，为氢能驱动固碳过程提供了充足的能量与还原力^[46]；同时该电转化体系将R. palustris标记缺失菌株的构建时间从传统接合转化的24 d缩短至12 d，转化效率达(1.79-3.57)×10³ CFU/μg质粒，大幅提升了该菌的遗传改造效率^[46]。

针对菌株生长优化，通过敲除负调控转录基因可显著改善C. necator H16在甲酸底物下的自养生长性能，为氢能固碳底盘细胞的高密度培养奠定基础^[54]。基因敲除技术是削减竞争性代谢流、实现目标产物定向合成的关键手段^[32]，通过敲除氢能驱动固碳底盘细胞的竞争性代谢支路基因，可将碳流、电子流定向引导至目标产物合成通路，同时还能扩展菌株底物利用谱，提升C. necator H16等模式菌株在自养、异养、混合营养3种生长模式下的环境适应性，该策略已成功应用于C. necator H16的L-异亮氨酸、L-缬氨酸及PHA的定向合成^[32]。除上述应用外，相关研究还建立了CRISPR-Cas系统，实现多位点同步改造与高效多基因编辑，为氢能自养底盘代谢工程、CO₂固碳优化提供了核心工具^[55-57]。该系统在模式产甲烷古菌海沼甲烷球菌(Methanococcus maripaludis)中表现出优异的编辑性能；CRISPR-Cas9系统可同步删除3个基因组位点上的13个基因，对单基因、双基因及9 kb大片段的敲除效率分别达100%、75%和100%，且在高达55个染色体拷贝的多倍体基因组中仍能实现全拷贝精准编辑^[55]；通过标准化启动子/终止子表达或10 nt内切核糖核酸酶切割序列介导的共转录加工2种策略，进一步将多基因同步编辑效率提升至80%-100%，其中共转录策略可使90%以上的染色体拷贝完成编辑^[56]。CRISPR-Cas12a系统则凭借其AT富集的PAM序列优势，在该菌中实现了高达95%的基因敲除阳性率，成功替换8.9 kb的鞭毛操纵子，并支持长达5 kb片段的插入编辑，其多向导RNA加工能力进一步简化了多位点改造的质粒构建流程^[57]。此外，衍生的CRISPR-Cas9系统可实现90%以上的基因表达抑制，而四环素诱导型CRISPR系统能对必需基因实现可控抑制(诱导后表达量仅为对照的2%)，为固碳关键基因的功能解析提供了精准工具^[56]。

除精准基因编辑外，物理诱变作为非转基因改造手段，可在不引入外源基因的前提下快速获得高性能菌株，其中常压室温等离子体(atmospheric and room temperature plasma, ARTP)诱变在HOB改造中展现出显著优势(图3)；ARTP诱变具有操作安全、突变率高、宿主范围广等特点，可通过等离子体诱导基因组单核苷酸多态性、小片段插入缺失与染色体重排，实现代谢网络的全局重编程；相较于紫外诱变、化学诱变等传统方法，ARTP对C. necator的正向突变率更高，致死率可控，且无化学试剂残留与光复活等缺陷；基于ARTP的代谢重布线策略，可快速强化HOB的H₂利用、CO₂固定、中心碳代谢与蛋白合成能力，为氢能自养底盘的快速迭代提供了合规、高效的新路径^[49]。

这些定点改造策略的优势在于方向明确、操控性强，能够围绕目标进行针对性优化。然而，HOB的代谢网络复杂，单一基因的编辑可能无法达到理想效果，需要在代谢通路重构层面协同使用。HOB底盘设计需将精准编辑与全局诱变结合，兼顾改造的定向性与性能迭代的高效性。CRISPR系统的多位点编辑能力可一次性重构多条代谢支路，实现碳流与电子流的协同调控；ARTP诱变则适合快速获得生长与抗逆性能提升的突变株，为理性设计提供优良的底盘出发菌株。

3.1.3 组学解析与系统生物学辅助设计

由于氢能自养底盘的代谢调控高度复杂，组学分析和系统生物学方法成为指导工程改造的重要辅助手段。通过转录组学、蛋白质组学、代谢组学以及多组学联合分析，研究者可从全局层面揭示菌株在不同环境条件、不同营养模式和不同工程背景下的代谢响应规律。

针对C. necator的多组学研究显示，其蛋白质组覆盖度达81.0% (5 357种蛋白质来自6 614个注释基因)，其中43%的蛋白质质量(以细胞干重计)为非利用或功能未知类型，包括大量未参与核心代谢的转运蛋白、转录因子及假定蛋白^[28]。通过资源平衡分析建模与蛋白质组数据耦合，将代谢酶按利用率分为低(<33%，710个)、中(33%-66%，153个)、高(>66%，149个) 3类，发现高利用率酶的蛋白质丰度显著更高(P<0.001)，且在不同营养条件下的表达变异性更低，其中CBB循环关键酶RuBisCO在异养条件下仍维持3%的蛋白质组占比^[28]。在X. autotrophicus的生物电化学耦合体系中，整合蛋白质组与代谢组分析发现，电化学水分解条件下菌株代谢发生显著重编程：在鉴定的2 570种蛋白质中，499种呈显著上调(fold change>1.5, P<0.05)，380种显著下调(fold change<0.66, P<0.05)；87种代谢物中20种上调、4种下调，其中固氮酶相关蛋白(NifB、NifE、NifN等)表达量平均提升1.8倍，碳固定产物3-PGA和乙酰辅酶A的丰度分别增加1.6倍和1.4倍，直接佐证电化学环境对固氮-固碳协同代谢的强化效应^[57]。同时，ATP合酶亚基表达上调2.1倍，电子传递关键蛋白中2/3亚型显著富集，揭示能量代谢与电子传递的适配机制，为生物电化学系统的参数优化提供了直接靶点^[57]。

针对ARTP诱变获得的C. necator突变株R3的多组学解析进一步揭示了自养生长强化的分子机制。全基因组重测序验证表明，突变株仅存在赖氨酸tRNA连接酶(lysine-tRNA ligase, lysS)的单一同义突变，该突变通过表观转录调控使lysS转录水平上调1.75倍，提升胞内游离赖氨酸含量；转录组与代谢组联合分析显示，突变株ABC转运系统、硫代谢通路、TCA循环与糖异生关键基因显著上调，其中半胱氨酸合成模块(cysP、cysE、cysK)表达上调，L-半胱氨酸添加可显著提升野生型菌株生长；中心碳代谢中，甘油醛-3-磷酸脱氢酶高表达是驱动生长加速的关键因子，过表达该基因可显著提升H16自养生长性能；多组学数据明确了硫代谢、能量代谢与翻译调控协同驱动固碳产蛋白的核心机制，为C. necator的理性改造提供了关键靶点^[49]。

综上所述，组学解析可量化菌株代谢网络的资源分配模式、关键酶调控规律及环境响应机制，系统生物学建模则能整合多维度数据，为代谢工程改造提供精准靶点，预测改造后的代谢响应，减少试错成本，提升HOB底盘工程改造的成功率。

3.2 代谢通路重构与强化策略

对HOB的代谢通路进行重构是氢能自养底盘细胞改造的核心路线之一，其目标在于通过重构碳流、电子流和能量流提高菌株的固碳效率和目标产物合成能力。对于多数HOB而言，CBB循环是主要的CO₂固定途径，而RuBisCO则是该途径中的关键限速酶，同时也需要氢化酶的参与。因此，围绕RuBisCO和氢化酶开展优化(图4)是提升自养性能的重要策略。

3.2.1 固碳代谢模块优化

生物固碳是HOB物质代谢的核心，以CBB循环为核心的固碳通路负责将CO₂转化为有机碳骨架，其效率直接决定碳转化速率与产物合成潜力。已有研究证明，通过更换原有菌株中的RuBisCO酶并搭配分子伴侣协同表达可显著提高菌株的生长效率和碳固定能力，因此在C. necator H16中引入蓝细菌Synechococcus sp. PCC 7002的异源RuBisCO，并结合菌株内源GroES/EL分子伴侣系统及RbcX辅助蛋白后，工程菌株在自养条件下培养72 h的OD₆₀₀较野生型提升89.15%，生长效率与碳固定能力得到显著提升；若同时结合氢化酶表达调控进行组合改造，该菌株自养培养96 h的OD₆₀₀可进一步提升93.4%，其核心固碳产物PHB的自养产率更可提升99.71%，从产物合成层面进一步佐证碳固定能力的高效提升^[48]。同时在CBB循环中氢化酶也发挥着重要作用，其氧化H₂并产生能量提供还原力，氢化酶系统也是工程改造的重要对象^[59]。通过过表达氢化酶，并更换适配的强启动子可实现高效的碳固定和充足的能量供给，提升菌株的生长速度和目标产物的产量^[48]。

提高胞内CO₂浓度可提高RuBisCO的催化效率，R. palustris的CO₂浓缩机制研究表明，该菌能以甲酸为唯一碳源，通过色素合成、膜结构及黄素调控的多方面协同实现CO₂浓缩，在细菌叶绿素和类胡萝卜素合成过程中产生CO₂并消耗质子，从而形成低透性的HCO₃^-碱性环境；同时，细菌的类囊体膜可降低CO₂扩散速率，增强甲酸脱氢酶活性，使CO₂合成速率提高，满足CBB循环需求，从而更高效地实现CO₂固定^[41]。

3.2.2 能量代谢模块优化

近年来，除传统遗传工程外，微生物-材料耦合与生物电化学策略也逐渐成为氢能自养底盘细胞改造的重要研究方向。这类方法无需改变菌株基因组，而是通过引入新的功能组分，比如半导体材料、纳米颗粒、电极以及其他功能介质，重构电子传递方式与局部微环境，实现菌株代谢性能的增强。

目前已有研究证明，在微生物-半导体杂交系统中，将半导体材料作为电子传递媒介，可实现量子点与微生物之间光诱导电荷的快速转移，从而提高CO₂和N₂的固定效率^[20]；纳米材料与微生物的协同作用为氢能驱动固碳技术提供了高效解决方案，具体表现为量子点/纳米颗粒介导的电荷转移强化、微生物代谢调控及产物合成效率提升。在X. autotrophicus与CdTe量子点构建的微生物-半导体杂化体系中，实现了光驱动下CO₂和N₂的高效共固定(图5)，其CO₂固定内部量子效率达(47.2±7.3)%，N₂固定内部量子效率为(7.1±1.1)%，接近生化途径化学计量学限定的理论极限(46.1%和6.9%)；且电荷转移速率常数k_ET达2.1×10⁸ s^-1，远高于辐射复合速率1.2×10⁷ s^-1和非辐射复合速率8×10⁶ s^-1，确保了光生电子向微生物的高效传递；蛋白质组学和代谢组学分析证实，量子点可调控微生物代谢，固氮酶相关蛋白(NifB、NifE等)显著上调，氧化磷酸化下游复合体IV和ATP合酶表达下调，减少ATP消耗以优化固碳固氮的电子分配效率^[20]。

此外，将微生物与纳米材料相结合，无论是纳米载体还是功能纳米颗粒，都可通过诱导温和应激反应、改变细胞膜通透性或增强电子传递能力实现菌株生长性能与目标产物积累量的同步提升^[60-61]。HOB可作为底盘合成功能性纳米材料，如异源表达金属硫蛋白的重组R. palustris/pSCMT，其CdSe量子点合成量与量子产率均较野生型提升3倍以上，产物分散性与生物相容性优异，拓展了HOB在生物纳米材料领域的应用场景^[60]。Fe₃O₄纳米颗粒对Rhodopseudomonas faecalis PA2的培养强化进一步拓展了固碳应用场景，0.1%浓度为最优添加量，可使细菌生物量、蛋白质、脂质、类胡萝卜素产量显著提升，蛋白质含量达(63.78±0.25)%，覆盖所有必需氨基酸及ω-3/ω-6脂肪酸，同时促进废食用油降解率达65%，诱导PHB积累和抗氧化活性提升，同步实现废弃物降解与储能物质积累，且代谢组学与毒理验证也证实了该体系的生物安全性^[61]。这类混合系统的价值不仅在于提高单一代谢指标，更在于拓展了微生物改造的边界，使其能够在更复杂的能源输入和环境条件下实现高效转化。

电化学强化策略的核心价值在于以电能驱动生物固碳过程，重构能量与碳素的协同转化路径。通过电解水原位供给H₂与O₂，该系统将可再生能源(电能)转化为微生物可直接利用的化学能(H₂)，为混合HOB菌群提供持续、可控的能量与电子供应，突破传统气体发酵中H₂/O₂传质限制与成本瓶颈；在能量供给优化的基础上，菌群通过CBB循环实现CO₂的高效固定，而调控氮、氧供给则进一步引导碳流定向分配，氧限制与氮-氧双重限制条件下PHB含量峰值达55.65%，实现了能量驱动的碳流精准重定向；气升式反应器设计协同增强了气液传质效率，无需机械搅拌，适配电化学耦合体系规模化推广；该策略以低成本的混合菌群替代纯菌株，以电化学原位供气替代传统高压供气，以能量-碳素协同调控替代单一培养基优化，为氢能驱动的CO₂固碳与高值产物合成提供了规模化、低成本、可落地的全新范式^[62]。

综上所述，氢能自养底盘细胞工程改造已经形成较为完整的研究方法体系：从遗传工具箱的建立发掘细胞自身的遗传特性，再对其中主要的调控基因进行基因组编辑和定点改造，实现对菌株的精准调控；之后对代谢通路进行重构和优化，使细胞朝着预设方向定向发展，实现菌株代谢方向的精准调控；进一步通过组学分析和系统生物学辅助设计，检验改造是否按既定方向推进并解析代谢过程中的资源消耗路径，实现产物生成效率的提升；以上方法均属于菌株内部基因层面的改造，最后通过微生物-材料耦合的方式实现从外部对微生物代谢过程的调控。这些方法之间相互配合，共同推动氢能固碳自养微生物从基础研究走向应用开发，为其工业化生产奠定基础。

3.3 HOB应用进展与前景

HOB固定CO₂后的应用方向主要涵盖生物材料合成、清洁能源生产以及高密度生物量生产三大类(表3)。基于技术经济分析(techno-economic analysis, TEA)与生命周期评价(life cycle assessment, LCA)的成熟框架，HOB固碳体系在环境与经济维度均展现出可观前景。以C. necator R3生产SCP为例，LCA结果表明其净全球变暖潜值为-1.84 kg CO₂-eq/kg SCP，实现了碳负排放，该优势源于直接固碳、替代豆粕生产及副产物利用的综合环境效益^[49]。在具体应用路径上，CO₂转化为L-乳酸的经济性主要受碳转化得率、发酵强度及电力成本影响，碳交易与菌体资源化可进一步拓宽盈利空间^[68]；而基于有机废弃物共消化的多产品策略中，将沼气升级为生物压缩天然气或合成PHB可显著改善经济性，PHB路线在特定条件下甚至可实现负成本运营^[69]。HOB以氢为能量供体、CO₂为唯一碳源的天然低碳代谢特性，使其适配多元低碳应用场景，商业化潜力突出。面向未来，突破H₂传质瓶颈与提升固碳核心效率将是压缩生产成本、放大环境效益的关键，有望加速该技术从中试研究向工业化规模的实质性落地。

4 挑战与发展方向

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4.1 H₂ 传质与安全问题

HOB的自养生长依赖H₂作为能量和电子供体，但H₂的物理特性和工业应用要求带来了双重难题。一方面，H₂传质效率低，H₂在水溶液中的溶解度极低(20 ℃、101 kPa下仅为1.6 mg/L)，传统搅拌式生物反应器的气液界面传质效率低，导致培养液中可利用的溶解态H₂浓度远低于HOB代谢需求，大量H₂未被利用即逸出，降低了氢能转化效率，造成能源浪费；另一方面，H₂的易燃易爆特性带来严重的安全隐患，H₂的爆炸极限为4.0%-75.6% (体积分数)，在密闭培养体系中易与空气形成爆炸性混合物，且H₂分子量小，易通过反应器密封缝隙泄漏，增加了操作风险；此外，规模化培养中需要持续的H₂供给，其储存、运输及反应器内的动态补料过程均需严格的安全管控。

针对传质问题，目前已有新型构型反应器可实现传质强化，例如中空纤维膜生物反应器^[70]，H₂由中空纤维膜内腔渗透溶入液相，气液两相物理分隔，H₂溶解浓度可达传统鼓泡反应器的1.59-2.30倍，既提升H₂溶解效率又隔绝气相H₂与空气大范围混合，降低燃爆风险；针对安全问题可采用原位电解耦合就地供氢模式：反应器外接电解水装置，利用光伏/风电电能原位裂解水产H₂，H₂直接通入发酵液，省去外购H₂储运环节，从源头规避长途运输H₂泄漏爆炸隐患，是当前微生物电化学固碳的主流工艺。

4.2 氢化酶与活性氧问题

HOB的MBH和SH多数对O₂高度敏感，O₂会与H₂竞争氢化酶的活性位点，甚至导致酶的不可逆失活，而开放或半开放的培养体系中不可避免的溶氧会显著抑制氢化酶活性，使电子传递链的电子通量大幅降低；同时，部分HOB的氢化酶表达量低、催化活性弱，无法为CO₂固定和物质合成提供充足的电子，限制了电子传递效率。

氢能驱动固碳的核心是实现氢能的能量转化与CO₂的物质合成的高效耦合，但目前HOB的这一耦合过程存在显著失衡。一方面，能量代谢与碳固定的耦合匹配性差，HOB的电子传递链中部分电子会通过呼吸链的分支途径以产热形式散失，而非用于ATP合成，导致氢能的能量转化效率低；同时，CBB循环的关键限速酶RuBisCO的催化效率低、对CO₂的特异性差，易发生加氧反应，造成碳固定的能量损耗，进一步加剧能量-碳固定的耦合失衡。另一方面，碳流的定向分配调控难度大，HOB的代谢网络具有高度灵活性，碳流可在自养固碳、异养代谢、储能物质合成(如PHB)之间自由切换，人工改造后虽能定向引导碳流向目标产物，但仍存在大量碳流通过竞争性代谢支路流失的问题；且当氢能供给不足时，细胞会优先将有限的碳源用于自身生长，而非目标产物合成，导致产物产率和碳转化效率难以提升。

微生物-电化学耦合是强化氢能驱动固碳的重要策略，但其原位电化学培养体系中不可避免的活性氧(reactive oxygen species, ROS)产生成为制约细胞存活和代谢效率的新难题。在电化学水分解产H₂的过程中，电极表面会因电子泄漏产生超氧阴离子、过氧化氢、羟基自由基等ROS，这些ROS会对细菌细胞造成多重氧化损伤：(1)攻击细胞膜的磷脂双分子层，导致细胞膜通透性改变、结构破坏；(2)氧化损伤DNA、RNA等遗传物质，引发基因突变，导致固碳关键酶(如RuBisCO)、氢化酶的表达异常；(3)氧化失活细胞内的功能性蛋白，尤其是含金属辅基的氢化酶、RuBisCO，直接抑制电子传递和碳固定过程。

4.3 未来发展方向

未来氢能驱动固碳底盘细胞的研究与开发，将以提升氢能-碳固定-产物合成的耦合效率为核心，融合合成生物学、生物反应器工程、生物电化学、材料科学等多学科技术，从多维度推进技术突破与产业化落地：一方面将精准优化气液传质体系、开发氢能安全高效利用策略，定向改造电子传递与还原力代谢模块实现能量-物质代谢的精准耦合，同时构建电化学培养的ROS防御体系并优化原位微环境调控，开发跨属种的高通量遗传操作工具，结合多组学与系统生物学实现代谢网络的系统重构和精准调控，还会通过定向基因编辑与自适应实验室进化相结合筛选高性能菌株；另一方面将开发低成本的培养基质与高密度规模化培养工艺，建立涵盖生态与产品层面的全链条安全评估体系，推进中试放大研究并开发一体化工业工艺，同时跨学科融合构建微生物-纳米材料、微生物-半导体智能耦合体系，结合人工智能实现培养过程的智能调控，进一步拓展技术在工业CO₂资源化利用、水体土壤碳修复、绿色生物制造等多元应用场景，最终实现碳循环利用与绿色制造的深度融合，推动该技术从实验室研究向工业化规模化应用转化。

5 结论

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HOB凭借氢能氧化供能、CBB循环固碳的核心代谢特征，以及自养/异养/混合营养的灵活生长模式，在环境保护与资源再生领域展现出突出应用潜力。以X. autotrophicus、C. necator、R. palustris为代表的菌株兼具高效固碳、固氮与高值产物合成能力，可实现从CO₂、H₂等廉价无机原料直接合成PHA、生物燃料、高值精细化学品等多种产品，兼具碳减排与资源增值的双重效益，为氢能驱动固碳生物制造体系的构建筑牢了生物底盘基础。依托遗传工具箱构建、基因组编辑、代谢通路重构及微生物-材料耦合等工程化策略，通过RuBisCO定向进化、氢化酶活性优化等精准改造，可持续提升其固碳效率、能量转化效率与目标产物合成水平。未来需进一步突破氢能传质、能量-碳代谢耦合等技术瓶颈，完善菌株遗传操作工具与工业化培养工艺，探索多菌株协同固碳、光合-化能自养耦合等新型体系，同时做好全链条环境安全评估。综上所述，HOB有望成为破解全球气候与资源难题、助力“双碳”目标实现与绿色生物制造发展的核心生物载体，推动碳循环利用与可持续生物工业的深度融合。

基金

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中国科学院战略性先导科技专项(XDC0120300)

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2026年第66卷第9期

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doi: 10.13343/j.cnki.wsxb.20260357

接收时间：2026-04-30
首发时间：2026-09-09
出版时间：2026-09-04

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收稿日期：2026-04-30
录用日期：2026-07-07

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the Strategic Priority Research Program of Chinese Academy of Sciences(XDC0120300)

中国科学院战略性先导科技专项(XDC0120300)

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^1.中国科学院天津工业生物技术研究所，天津

^2.中国科学院大学，北京

^3.南开大学生命科学学院，天津

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Parameter	Xanthobacter autotrophicus	Cupriavidus necator	Rhodopseudomonas palustris	References
Energy source	CO₂, methanol, ethanol,N-butanol, organic acids, etc.	Chemolithoautotrophic growth using H₂ as electron donor, also capable of utilizing various heterotrophic carbon sources in combination with nitrate metabolism	Photoautotrophy (CO₂ as carbon source) or photoheterotrophy (obtaining energy and carbon from organic compounds)	[15-18,26-28,38-39]
Carbon metabolism	Carbon fixation capability with flexible metabolism under different electron donors and carbon sources	Chemolithoautotrophic growth using CO₂ as carbon source; metabolizes nitrate and nitrite to optimize growth rate	Strong carbon fixation capability; metabolizes organic acids and lignin degradation products	[15-18,26-28,40-41]
Nitrogen metabolism	Nitrogen fixation capability (potential for agricultural microbial modification)	Denitrification metabolic capacity (reduces nitrate to dinitrogen under anaerobic conditions)	Nitrogenase-related proteins; possesses photosynthetic nitrogen fixation capability applicable for nitrogen production optimization	[24,28,46]
Metabolic regulation	Environmental stresses such as magnetic fields, electric fields, and semiconductor materials; actively regulates membrane permeability to survive extreme conditions	High maximum specific growth rate under nitrate limitation; allocates protein resources on demand to adapt to environmental changes	High metabolic flexibility under illumination (can regulate photosynthetic electron transfer rate); thylakoid membrane structure limits CO₂ diffusion	[17,19-20,30-32,41]
Key metabolites	Carotenoids (pigments)	PHA/PHB or SCP; balance reducing power accumulation under anaerobic conditions	PHA production under optimized conditions;N-butanol synthesis and high-efficiency H₂ production	[25,36,38,45]
Environmental applications & potential	Degradation of environmental pollutants such as toluene and DCE (closely related to environmental remediation)	High-density fermentation of SCP; applicable for treating 3-HP and heavy metal contamination	Degradation of azo dyes (e.g., methyl red); enhances Fe²⁺ adsorption capacity through modified lignite	[21-22,25,36-37,43-44]

Parameter

Xanthobacter autotrophicus

Cupriavidus necator

Rhodopseudomonas palustris

References

Energy source

CO₂, methanol, ethanol,N-butanol, organic acids, etc.

Chemolithoautotrophic growth using H₂ as electron donor, also capable of utilizing various heterotrophic carbon sources in combination with nitrate metabolism

Photoautotrophy (CO₂ as carbon source) or photoheterotrophy (obtaining energy and carbon from organic compounds)

[15-18,26-28,38-39]

Carbon metabolism

Carbon fixation capability with flexible metabolism under different electron donors and carbon sources

Chemolithoautotrophic growth using CO₂ as carbon source; metabolizes nitrate and nitrite to optimize growth rate

Strong carbon fixation capability; metabolizes organic acids and lignin degradation products

[15-18,26-28,40-41]

Nitrogen metabolism

Nitrogen fixation capability (potential for agricultural microbial modification)

Denitrification metabolic capacity (reduces nitrate to dinitrogen under anaerobic conditions)

Nitrogenase-related proteins; possesses photosynthetic nitrogen fixation capability applicable for nitrogen production optimization

[24,28,46]

Metabolic regulation

Environmental stresses such as magnetic fields, electric fields, and semiconductor materials; actively regulates membrane permeability to survive extreme conditions

High maximum specific growth rate under nitrate limitation; allocates protein resources on demand to adapt to environmental changes

High metabolic flexibility under illumination (can regulate photosynthetic electron transfer rate); thylakoid membrane structure limits CO₂ diffusion

[17,19-20,30-32,41]

Key metabolites

Carotenoids (pigments)

PHA/PHB or SCP; balance reducing power accumulation under anaerobic conditions

PHA production under optimized conditions;N-butanol synthesis and high-efficiency H₂ production

[25,36,38,45]

Environmental applications & potential

Degradation of environmental pollutants such as toluene and DCE (closely related to environmental remediation)

High-density fermentation of SCP; applicable for treating 3-HP and heavy metal contamination

Degradation of azo dyes (e.g., methyl red); enhances Fe²⁺ adsorption capacity through modified lignite

[21-22,25,36-37,43-44]

Strains	Growth mode	Key enzymes	Engineering strategies	Synthetic products	References
X. autotrophicus	Chemolithoautotrophic	Hydrogenase, dehalogenase	Construction of gradient-copy number replicons and strong/weak promoter libraries; XanthoMoClo modular cloning; coupling of carbon fixation and nitrogen fixation; functional optimization of dehalogenase; CdTe quantum dot-microbe coupling; expansion of substrate spectrum	Degradation of chloroalkane/haloacid pollutants; biofertilizers (nitrogen-fixing inoculants); plant growth regulators; high-value pigments	[15,18,47]
C. necator	Facultative autotrophic	[NiFe]^- hydrogenase, RuBisCO	Knockout of CBB operons and competitive branches; co-expression of heterologous RuBisCO chaperones; expression regulation of hydrogenases (MBH/SH); carbon flux redirection; global reprogramming via ARTP mutagenesis; precise engineering of multi-omics identified targets	Bioplastics; high-value chemicals; biofuels; SCP	[26-27,32,48-49]
Hydrogenovibrio marinus	Obligate chemolithoautotrophic	Multiple RuBisCO isoforms	Synergistic expression of multiple RuBisCO isoforms; construction of salt-tolerant chassis; regulation of metabolic flux distribution; engineering optimization of promoter elements; enhancement of carbon fixation modules	Bioplastics; high-value solutes; biofuels	[50]
Acidithiobacillus ferrooxidans	Acidophilic chemolithoautotrophic	Hydrogenase, sulfur oxidase	Activity enhancement of hydrogenase and sulfur oxidase; engineering of extreme acid-base and heavy metal resistance; optimization of energy metabolic pathways; development of genetic tools; improvement of oxidative stress tolerance	Biohydrometallurgy; heavy metal removal from acid mine drainage; magnetic nanomaterials; barium sulfate crystals	[51]
Paracoccus denitrificans	Mixotrophic	Formate dehydrogenase, RuBisCO	Enhancement of heterotrophic nitrification-aerobic denitrification (HN-AD); engineering of formate dehydrogenase; optimization of pollutant degradation pathways; engineering of restriction-modification systems; synergistic regulation of carbon and nitrogen metabolism	Environmental governance; bioremediation; degradation of sulfonamide antibiotics and polycyclic aromatic hydrocarbons; carotenoids; PHB	[46]
Rhodococcus opacus	Dual chemolithoautotrophic and heterotrophic	NAD⁺-reducing hydrogenase, RuBisCO	Reconstruction of photosynthetic metabolic pathways; precise coupling of hydrogenase and carbon metabolism; knockout of competitive metabolic branches; functional enhancement of whole-cell biocatalysis; balanced regulation of carbon fixation and energy storage metabolism	Biofuels; PHA; fine chemicals	[52]

Strains

Growth mode

Key enzymes

Engineering strategies

Synthetic products

References

X. autotrophicus

Chemolithoautotrophic

Hydrogenase, dehalogenase

Construction of gradient-copy number replicons and strong/weak promoter libraries; XanthoMoClo modular cloning; coupling of carbon fixation and nitrogen fixation; functional optimization of dehalogenase; CdTe quantum dot-microbe coupling; expansion of substrate spectrum

Degradation of chloroalkane/haloacid pollutants; biofertilizers (nitrogen-fixing inoculants); plant growth regulators; high-value pigments

[15,18,47]

C. necator

Facultative autotrophic

[NiFe]^- hydrogenase, RuBisCO

Knockout of CBB operons and competitive branches; co-expression of heterologous RuBisCO chaperones; expression regulation of hydrogenases (MBH/SH); carbon flux redirection; global reprogramming via ARTP mutagenesis; precise engineering of multi-omics identified targets

Bioplastics; high-value chemicals; biofuels; SCP

[26-27,32,48-49]

Hydrogenovibrio marinus

Obligate chemolithoautotrophic

Multiple RuBisCO isoforms

Synergistic expression of multiple RuBisCO isoforms; construction of salt-tolerant chassis; regulation of metabolic flux distribution; engineering optimization of promoter elements; enhancement of carbon fixation modules

Bioplastics; high-value solutes; biofuels

[50]

Acidithiobacillus ferrooxidans

Acidophilic chemolithoautotrophic

Hydrogenase, sulfur oxidase

Activity enhancement of hydrogenase and sulfur oxidase; engineering of extreme acid-base and heavy metal resistance; optimization of energy metabolic pathways; development of genetic tools; improvement of oxidative stress tolerance

Biohydrometallurgy; heavy metal removal from acid mine drainage; magnetic nanomaterials; barium sulfate crystals

[51]

Paracoccus denitrificans

Mixotrophic

Formate dehydrogenase, RuBisCO

Enhancement of heterotrophic nitrification-aerobic denitrification (HN-AD); engineering of formate dehydrogenase; optimization of pollutant degradation pathways; engineering of restriction-modification systems; synergistic regulation of carbon and nitrogen metabolism

Environmental governance; bioremediation; degradation of sulfonamide antibiotics and polycyclic aromatic hydrocarbons; carotenoids; PHB

[46]

Rhodococcus opacus

Dual chemolithoautotrophic and heterotrophic

NAD⁺-reducing hydrogenase, RuBisCO

Reconstruction of photosynthetic metabolic pathways; precise coupling of hydrogenase and carbon metabolism; knockout of competitive metabolic branches; functional enhancement of whole-cell biocatalysis; balanced regulation of carbon fixation and energy storage metabolism

Biofuels; PHA;

fine chemicals

[52]

Product	Strains	Fermentation conditions	Cultivation period	Titer	Process limits & mitigations	References
PHA	Cupriavidus eutrophusB-10646	Autotrophic cultivation with H₂:O₂:CO₂ (7:2:1) in the recycled-gas closed-circuit culture system	80 h	36 g/L	Limit: low cell density in chemostat, high mass transfer requirements for H₂/O₂ Mitigation: two-stage batch with nitrogen limitation achieved high cell density and polymer content; gas mixture recirculation improved substrate utilization	[63]
PHB	C. necator DSM 545	Cultivation with formic acid and acetic acid in fed-batch fermentations	63 h	58.5 g/L	Limit: O₂ transfer limits growth Mitigation: acetic acid is directly converted to acetyl-CoA; linear growth phase extends biomass accumulation	[64]
PHB	C. necator DSM 545	Cultivation with waste	48 h	66 g/L	Limit: exponential feeding alone caused over or under feeding due to parameter inaccuracies Mitigation: novel three-stage strategy maintained substrate within optimal range	[65]
L-threonine	Brevibacterium flavum	Cultivation with glucose, peptone, beef extract, and yeast extract in batch fermenters	44 h	12.14% (yield, g/100 mL)	Limit: pH drops during fermentation due to metabolic byproducts; high pH control increases ammonia cost Mitigation: optimal conditions determined via orthogonal design; maintaining pH at 6.0-7.0 is recommended to save ammonia costs with acceptable yield	[23]
SCP	X. variabilis NFM-97 and Shinella sp. NM-101	Cultivation with H₂:O₂:CO₂:N₂(10:2:10:78) in sealed bottles	72 h	0.6 g/L	Limit: energy-expensive N₂ fixation reduces biomass yield compared to NH₄⁺-based HOB Mitigation: coculture with heterotroph enhanced growth and protein quality via metabolic cooperation	[24]
N-butanol	R. palustris TIE-1	Cultivation in batch cultures under photoheterotrophic or photoautotrophic conditions	10 d	4.98 mg/L	Limit: deleting acetyl-CoA consuming pathways diverted carbon to acetone (byproduct), lowering N-butanol yield Mitigation: deleting the electron-consuming N-fixation pathway created a more reduced intracellular environment, enhancing N-butanol production and carbon conversion efficiency	[45]
H₂	R. palustris CGA009	Cultivation with glycerol and glutamate in batch bioreactors under anaerobic phototrophic conditions	Up to 212 h	~16 mL/h	Limit: overexpression of alternative nitrogenases caused a metabolic burden Mitigation: applying non-growing conditions reduced competition from cell division	[46]
SCP	HOB community	Autotrophic cultivation with H₂:O₂:CO₂(65:20:15)	90 d	24 g/L	Limit: Low productivity (0.08 g/L/h) due to predatory Bdellovibrio (33% abundance) Mitigation: high dilution rate selected for fast-growing Sulfuricurvum spp. (97% abundance), achieving stable, high-rate SCP production	[66]
SCP	NF-HOB community (92% Xanthobacter)	Autotrophic cultivation with H₂:O₂:CO₂:N₂(59:19:7:15-50:25:8:17)in airlift reactor	19.2 d	2.3 g/L	Limit: O₂>2% inhibits nitrogenase in start-up; low N₂ fixation efficiency wastes nitrogen gas Mitigation: two-stage gas supply reduced start-up lag from 20 to 4 days; CO₂ conversion efficiency>25%	[2]
SCP	C. necator H16	Autotrophic cultivation with H₂:O₂:CO₂:N₂(70:10:12:8) in a custom 300 L pressurized deep-jet bioreactor	35 h	72 g/L	Limit: heterotrophic inoculum caused 10 h lag phase; foaming at high cell densities Mitigation: enabling modified strains to outperform the wild-type with higher rates at later time points	[67]
SCP	C. necator R3	Autotrophic cultivation with H₂:O₂:CO₂(7:1:1-7:2:1) in fed-batch fermentations	5 d	13.8 g/L	Limit: low CO₂ fixation efficiency limits SCP productivity Mitigation: multi-omics revealed key upregulations; synonymous mutation in lysine-tRNA ligase enhances translation; process optimization maximized performance	[49]

Product

Strains

Fermentation conditions

Cultivation period

Titer

Process limits & mitigations

References

PHA

Cupriavidus eutrophusB-10646

Autotrophic cultivation with H₂:O₂:CO₂ (7:2:1) in the recycled-gas

closed-circuit culture system

80 h

36 g/L

Limit: low cell density in chemostat, high mass transfer requirements for H₂/O₂

Mitigation: two-stage batch with nitrogen limitation achieved high cell density and polymer content; gas mixture recirculation improved substrate utilization

[63]

PHB

C. necator DSM 545

Cultivation with formic acid and acetic acid in fed-batch fermentations

63 h

58.5 g/L

Limit: O₂ transfer limits growth

Mitigation: acetic acid is directly converted to acetyl-CoA; linear growth phase extends biomass accumulation

[64]

PHB

C. necator DSM 545

Cultivation with waste

48 h

66 g/L

Limit: exponential feeding alone caused over or under feeding due to parameter inaccuracies

Mitigation: novel three-stage strategy maintained substrate within optimal range

[65]

L-threonine

Brevibacterium flavum

Cultivation with glucose, peptone, beef extract, and yeast extract in batch fermenters

44 h

12.14% (yield, g/100 mL)

Limit: pH drops during fermentation due to metabolic byproducts; high pH control increases ammonia cost

Mitigation: optimal conditions determined via orthogonal design; maintaining pH at 6.0-7.0 is recommended to save ammonia costs with acceptable yield

[23]

SCP

X. variabilis NFM-97 and Shinella sp. NM-101

Cultivation with H₂:O₂:CO₂:N₂(10:2:10:78) in sealed bottles

72 h

0.6 g/L

Limit: energy-expensive N₂ fixation reduces biomass yield compared to NH₄⁺-based HOB

Mitigation: coculture with heterotroph enhanced growth and protein quality via metabolic cooperation

[24]

N-butanol

R. palustris TIE-1

Cultivation in batch cultures under photoheterotrophic or photoautotrophic conditions

10 d

4.98 mg/L

Limit: deleting acetyl-CoA consuming pathways diverted carbon to acetone (byproduct), lowering N-butanol yield

Mitigation: deleting the electron-consuming N-fixation pathway created a more reduced intracellular environment, enhancing N-butanol production and carbon conversion efficiency

[45]

H₂

R. palustris CGA009

Cultivation with glycerol and glutamate in batch bioreactors under anaerobic phototrophic conditions

Up to 212 h

~16 mL/h

Limit: overexpression of alternative nitrogenases caused a metabolic burden

Mitigation: applying non-growing conditions reduced competition from cell division

[46]

SCP

HOB community

Autotrophic cultivation with H₂:O₂:CO₂(65:20:15)

90 d

24 g/L

Limit: Low productivity (0.08 g/L/h) due to predatory Bdellovibrio (33% abundance)

Mitigation: high dilution rate selected for fast-growing Sulfuricurvum spp. (97% abundance), achieving stable, high-rate SCP production

[66]

SCP

NF-HOB community

(92% Xanthobacter)

Autotrophic cultivation with H₂:O₂:CO₂:N₂(59:19:7:15-50:25:8:17)in airlift reactor

19.2 d

2.3 g/L

Limit: O₂>2% inhibits nitrogenase in start-up; low N₂ fixation efficiency wastes nitrogen gas

Mitigation: two-stage gas supply reduced start-up lag from 20 to 4 days; CO₂ conversion efficiency>25%

[2]

SCP

C. necator H16

Autotrophic cultivation with H₂:O₂:CO₂:N₂(70:10:12:8) in a custom 300 L pressurized deep-jet bioreactor

35 h

72 g/L

Limit: heterotrophic inoculum caused 10 h lag phase; foaming at high cell densities

Mitigation: enabling modified strains to outperform the wild-type with higher rates at later time points

[67]

SCP

C. necator R3

Autotrophic cultivation with H₂:O₂:CO₂(7:1:1-7:2:1) in fed-batch fermentations

5 d

13.8 g/L

Limit: low CO₂ fixation efficiency limits SCP productivity

Mitigation: multi-omics revealed key upregulations; synonymous mutation in lysine-tRNA ligase enhances translation; process optimization maximized performance

[49]