Construction of Ammonium-Tolerant Photosynthetic Bacterial Mutants and Characterization of Hydrogen Production

GAO Zixuan; CAO Wen; WEI Wenwen; GUO Liejin

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Construction of Ammonium-Tolerant Photosynthetic Bacterial Mutants and Characterization of Hydrogen Production

更新时间：2026-06-09

- Construction of Ammonium-Tolerant Photosynthetic Bacterial Mutants and Characterization of Hydrogen Production
- JOURNAL OF XI’AN JIAOTONG UNIVERSITY (2026)
- 作者机构：
  
  西安交通大学动力工程多相流国家重点实验室，710049，西安
- 作者简介：
- 基金信息：
- DOI：
  CLC： TK91
- Received：27 January 2026，
  
  Revised：2026-04-13，
  
  Accepted：14 April 2026，
- 稿件说明：
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GAO Zixuan, CAO Wen, WEI Wenwen, et al. Construction of Ammonium-Tolerant Photosynthetic Bacterial Mutants and Characterization of Hydrogen Production[J/OL]. JOURNAL OF XI’AN JIAOTONG UNIVERSITY, 2026.
DOI：

GAO Zixuan, CAO Wen, WEI Wenwen, et al. Construction of Ammonium-Tolerant Photosynthetic Bacterial Mutants and Characterization of Hydrogen Production[J/OL]. JOURNAL OF XI’AN JIAOTONG UNIVERSITY, 2026. DOI：

摘要

为了缓解铵离子（NH

）对光发酵制氢过程的抑制作用，研究以荚膜红细菌（

Rhodobacter capsulatus

R. capsulatus

） SB1003为研究对象。首先，采用重叠延伸聚合酶链式反应技术（SOE PCR）对固氮调控基因（

nifA

）进行定向改造，分别构建

nifA1

和

nifA2

基因N端缺失片段，并通过同源重组筛选获得

nifA1

缺失突变株ZX01、

nifA2

缺失突变株ZX02及双缺失突变株ZX03。然后，将上述菌株接种于以葡萄糖为碳源、以不同浓度的硫酸铵和谷氨酸钠为混合氮源的产氢液中，置于光照、厌氧条件下进行光发酵产氢，系统比较不同突变株在含铵和无铵条件下的产氢性能及相关基因表达水平。结果表明，在铵态氮质量分数为20%条件下，双缺失突变株ZX03的累计产氢量最高，达到801.80 ± 60.40 mL·L

-1

，较野生型

R. capsulatus

SB1003提高22.7%。基于Gompertz方程的动力学拟合结果显示，ZX01的产氢迟滞期最短（7.99 ± 3.41

h），表明

nifA1

N端缺失可显著加快产氢启动过程。实时定量聚合酶链式反应（qPCR）分析进一步表明，在高铵条件（NH

浓度为8 mmol·L

-1

）下，ZX03中氮信号转导蛋白编码基因

glnB1

和

glnB2

的转录水平较野生型上调约220倍，同时电子传递相关基因

rnfB

及黄素氧化还原蛋白编码基因

fldA

的表达亦显著增强。研究结果表明，

nifA1

N端缺失主要通过缩短产氢迟滞期提升菌株对铵胁迫的响应速率，而

nifA2

N端缺失则主要通过提高基础产氢能力增强耐铵性能；双缺失突变株ZX03协同整合两者优势，并通过上调氮信号转导及电子传递相关基因表达，在转录水平维持固氮系统活性，从而在高铵环境下实现最优产氢表现。该研究可为构建耐铵高效光发酵产氢工程菌株提供明确的分子改造策略与理论依据。

Abstract

To alleviate the inhibitory effect of ammonium ions (NH

) on photo-fermentative hydrogen production

this study employed the purple non-sulfur bacterium

Rhodobacter capsulatus

(

R. capsulatus

) strain SB1003 as the model organism. First

overlap extension polymerase chain reaction (SOE PCR) was used for targeted modification of the nitrogen fixation regulatory gene

nifA

. N-terminal deletion fragments of

nifA1

and

nifA2

were constructed separately

and through homologous recombination screening

the

nifA1

deletion mutant ZX01

the

nifA2

deletion mutant ZX02

and the double deletion mutant ZX03 were obtained. Subsequently

the above strains were inoculated into hydrogen-producing medium containing glucose as the carbon source and different concentrations of ammonium sulfate and sodium glutamate as mixed nitrogen sources

and were cultured under illuminated

anaerobic conditions for photo-fermentative hydrogen production. The hydrogen production performance and expression levels of relevant genes of the different mutants were systematically compared under ammonium-containing and ammonium-free conditions. The results showed that under the condition of 20% ammonium nitrogen mass fraction

the double mutant ZX03 achieved the highest cumulative hydrogen production of 801.80 ± 60.40 mL·L

-1

which was 22.7% highe

r than that of the wild-type

R. capsulatus

SB1003. Kinetic fitting based on the Gompertz equation revealed that ZX01 had the shortest hydrogen production lag phase (7.99 ± 3.41 h)

indicating that deletion of the N-terminus of

nifA1

significantly accelerates the initiation of hydrogen production. Quantitative real-time polymerase chain reaction (qPCR) analysis further demonstrated that under high ammonium conditions (8 mmol·L

-1

)

the transcript levels of the nitrogen signaling protein-encoding genes

glnB1

and

glnB2

in ZX03 were up-regulated by approximately 220-fold relative to the wild-type

while the expression of the electron transport-related gene

rnfB

and the flavodoxin-encoding gene

fldA

was also markedly enhanced. These findings indicate that deletion of the N-terminus of

nifA1

mainly improves the response rate of the strain to ammonium stress by shortening the lag phase

whereas deletion of the N-terminus of

nifA2

primarily enhances ammonium tolerance by elevating the basal hydrogen production capacity. The double mutant ZX03 synergistically integrates the advantages of both modifications and maintains nitrogenase system activity at the transcriptional level by up-regulating the expression of nitrogen signaling and electron transport-related genes

thereby achieving optimal hydrogen production performance under high ammonium conditions. This study provides a clear molecular engineering strategy and a theoretical foundation for constructing ammonium-tolerant

high-efficiency photo-fermentative hydrogen-producing strains.

关键词

Keywords

references

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