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

GAO Zixuan; CAO Wen; WEI Wenwen; GUO Liejin

doi:10.7652/xjtuxb202608008

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

更新时间：2026-07-28

- Construction and Hydrogen Production Characterization of Ammonium-Tolerant Photosynthetic Bacterial Mutants
- Journal of Xi'an Jiaotong University Vol. 60, Issue 8, Pages: 91-101(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：10.7652/xjtuxb202608008
  CLC： TK91
- Received：27 January 2026，
  
  Online First：16 April 2026，
  
  Published：10 August 2026
- 稿件说明：
移动端阅览
GAO Zixuan, CAO Wen, WEI Wenwen, et al. Construction and Hydrogen Production Characterization of Ammonium-Tolerant Photosynthetic Bacterial Mutants[J]. Journal of Xi'an Jiaotong University, 2026, 60(8): 91-101.
DOI：

GAO Zixuan, CAO Wen, WEI Wenwen, et al. Construction and Hydrogen Production Characterization of Ammonium-Tolerant Photosynthetic Bacterial Mutants[J]. Journal of Xi'an Jiaotong University, 2026, 60(8): 91-101. DOI： 10.7652/xjtuxb202608008.

摘要

为了缓解铵离子（

https://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=114120303&type=

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）对光发酵制氢的抑制作用，以荚膜红细菌（

Rhodobacter capsulatus

capsulatus

）SB1003为研究对象，通过基因工程构建耐铵突变株并评估其产氢性能。首先，采用重叠延伸聚合酶链式反应（SOE PCR）对固氮调控基因（

nifA

）进行改造，获得

nifA

1 N端缺失突变株ZX01、

nifA

2 N端缺失突变株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、不同浓度硫酸铵和谷氨酸钠为混合氮源的产氢液中，在光照、厌氧条件下进行光发酵产氢。通过实验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%时，ZX03的累计产氢量最高为（801.80±60.40）mL·L

-1

，较野生型提高22.7%。Gompertz动力学拟合表明，ZX01的产氢迟滞期最短为（7.99±3.41）h，表明其产氢启动更快。实时定量聚合酶链式反应PCR（qPCR）分析结果表明，在高铵条件（

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量浓度为8 mmol·L

-1

）下，ZX03中氮信号转导蛋白编码基因

glnB

1和

glnB

2的转录水平较野生型上调220倍，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，

nifA

1 N端缺失缩短产氢迟滞期，

nifA

2 N端缺失提升基础产氢能力；双缺失株耐铵性最优，且上调氮信号与电子传递相关基因表达，从而在高铵条件下实现最优产氢。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 (

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) on photo-fermentative hydrogen production，

Rhodobacter capsulatus

SB1003 was selected as the model organism. Ammonium-tolerant mutant strains were constructed via genetic engineering

and their hydrogen production performance was evaluated. The nitrogen fixation regulatory gene (

nifA

) was modified using overlap extension PCR (SOE-PCR)

generating three engineered strains:the

nifA

1 N-terminal deletion mutant ZX01

the

nifA

2 N-terminal deletion mutant ZX02

and the double-deletion mutant ZX03.These strains were inoculated into a hydrogen production medium with glucose as the carbon source and with different concentrations of ammonium sulfate and sodium glutamate as mixed nitrogen sources， and were cultivated under anaerobic and illuminated conditions for photo-fermentative hydrogen production. The hydrogen production performance

lag phase characteristics

and related gene expression profiles were comparatively analyzed under both ammonium-containing and ammonium-free conditions. The results show that at an ammonium nitrogen mass fraction of 20%

ZX03 achieves the highest cumulative hydrogen production of (801.80±60.40) mL·L

-1

representing a 22.7% increase compared with the wild-type strain. Gompertz kinetic modeling further shows that ZX01 exhibits the shortest lag phase (7.99±3.41) h

indicating significantly accelerated initiation of hydrogen production. Quantitative real-time polymerase chain reaction PCR (qPCR) analysis revealsthatunderhigh

ammoniumconditions (

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concentrationof8mmol·L

-1

)

the transcription levels of nitrogen signal transduction genes (

glnB

1 and

glnB

2) in ZX03 are upregulated by approximately 220-fold relative to the wild type. In addition

genes associated with electron transport

including

rnfB

and the flavodoxin-encoding gene

fldA

are also markedly upregulated

indicating enhanced metabolic electron flux. Overall

these findings demonstrate that N-terminal deletion of

nifA

1 primarily shortens the hydrogen production lag phase

whereas

nifA

2 N-terminal deletion enhances basal hydrogen production capability. Notably

the double-deletion mutant ZX03 exhibits the strongest ammonium tolerance and optimal hydrogen production performance under high ammonium stress by simultaneously upregulating nitrogen signaling and electron transport-related genes. This study provides a clear and effective molecular engineering strategy

as well as a theoretical foundation

for the development of robust

ammonium-tolerant

high-efficiency photo-fermentative hydrogen-producing strains.

关键词

Keywords

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