Photo-biological hydrogen production by a temperature-tolerant mutant of Rhodobacter capsulatus isolated by transposon mutagenesis
Graphical abstract
Introduction
The finite reserves of fossil fuels and environmental pollution worldwide, have led to the increasing interest in renewable and clean energy (Meky et al., 2020, Turhal et al., 2019). Hydrogen is a promising substitute for fossil fuels due to its high energy density and environmentally friendly generation (Abas et al., 2020, Chen and Chen, 2020). The global demand of hydrogen continues to increase, especially in the transportation sector (Wadjeam et al., 2019). Conventional hydrogen production depends on non-renewable resources, which is energy intensive, and goes against the global trend toward the less-C/higher-H content fuels (Zgonnik, 2020). Biological hydrogen production is considered to be an environment-friendly way due to the mild reaction conditions and non-pollution characteristics, and can be classified in the following categories: bio-photolysis, photo-fermentation and dark-fermentation (Verma et al., 2020). Among these approaches, efforts have been invested in photo-fermentation by photosynthetic bacteria because of (i) high production yield, (ii) high substrate conversion efficiency, and (iii) availability of a wide range of substrates to be used (Chen et al., 2019, Ghosh et al., 2017, Majidian et al., 2018).
The current production yield and rate by photo-fermentation cannot meet the demands of commercial bio-hydrogen. The theoretical yield for photo-fermentative hydrogen production is 12 mol-H2/mol-glucose, and 8.0 mol-H2/mol-glucose was estimated to be sufficient for an industrially sustainable process (Sharma et al., 2020). In this respect, many attempts have been paid on enhancing hydrogen yield and production rate by optimizing operational conditions, and manipulating related genes in bacteria. Up to now, hydrogen yield of 8.3 mol H2 from 1 mol starch-glucose equivalent algal biomass and even up to 9.0 mol-H2/mol-glucose in continuous mode have been reported, which is relatively above this benchmark (Abo-Hashesh et al., 2013, Kim et al., 2006a). However, the reaction conditions need to be strictly controlled. Substantial results on hydrogen yield have been reached so far, hence further attention is focused on the development of photo-fermentation process for practical purposes.
In the practical process, photo-fermentative hydrogen production is preferred to be carried out in the open-air environment rather than the incubators. Thus, seasonal variations in culture temperature are inevitable, especially the extremely high temperature when the bioreactors are exposed to sunlight in the summer. However, high temperature significantly hinders photo-fermentative hydrogen production performance and cell growth, even leading to cell death (Elreedy et al., 2019). The optimum culture temperature to produce hydrogen for most photosynthetic bacteria, such as Rhodovulum sulfidophilum (Cai and Wang, 2014), Rhodopseudomonas capsulata ATCC 23782 and ATCC 17013 (Stevens et al., 1984), Rhodobacter capsulatus DSM1710 (Sevinç et al., 2012), is about 30 °C. It has been reported that when temperature increases from 30 °C to 34 °C, photo-fermentative hydrogen yield and substrate conversion efficiency decrease by 33.43% and 33.28%, respectively (He et al., 2006). Therefore, it is necessary to control the culture temperature during the process. However, there are several issues associated with the implementation of temperature control, including increased operation complexity and cost. Given these negative effects, a possible way to address this challenge is to construct high temperature tolerant photosynthetic bacteria by genetic engineering.
Screening of high temperature tolerant mutant strains is of great importance for adaptation to high temperature stress with enhanced hydrogen production performance. The past decades have witnessed the rise of genetic engineering approaches that are designed for screening mutant strains. However, the majority of frequently-used isolation strategies are effective but time-consuming and costly. As the relationship between temperature shock and activity of the genes related to hydrogen production is yet not to be understood, it remains difficult to screen targeted strains by site-directed mutagenesis. Therefore, transposon technology is regarded as an excellent tool for bacterial genetic engineering for overcoming the mentioned disadvantages of isolation strategy, as well as creating mutant derivatives of species with less well-developed genetics. In parallel with the development of transposon technology, an aim of research is to enhance hydrogen yield via transposon mutagenesis (Cai and Wang, 2014, Wang et al., 2018b).
Most of the investigations have been focused on optimizing the basic parameters, including substrate selection, bioreactor design, fermentation condition optimization. (Niño-Navarro et al., 2020, Wang et al., 2019, Zhang et al., 2020b). Available literature on improving hydrogen production yield by screening high temperature resistance mutant strain is scarce (Gökçe et al., 2012). Moreover, only a few studies have attempted to improve hydrogen production performance by transposon mutagenesis. The aim of this study was to screen mutants of Rhodobacter capsulatus that are able to resist high temperature stress with enhanced hydrogen yield. Cornstalk pretreated with hydrochloric acid (HCl) was employed as the substrate to test the application potential of the mutant strain via energy conversion efficiency analysis.
Section snippets
Bacterial strains and cell culture
Rhodobacter capsulatus SB1003 (Yen and Marrs, 1976) was used as the wild type (WT) strain. R. capsulatus strains were cultured on modified MedA plates for 2 days (Feng et al., 2019), then single colonies were incubated in 50 mL sterile screw-cap tubes with 15 mL culture broth for 2 days aerobically. The sterile screw-cap tubes were shaken at 150 rpm in the dark. For basic growth, the temperature was set at 35 °C. Escherichia coli was grown in Luria-Bertani medium (LB) as described previously (
Characterization of the mutant strain MX01
It is reported that the biohydrogen production is associated with bacterial growth (Cai and Wang, 2012), thus the effect of transposon mutation on cell growth of the mutant strain MX01 under photo-fermentative condition should be investigated. According to our pre-tests, 30 °C is the optimum culture temperature for hydrogen production of the wild type strain Rhodobacter capsulatus SB1003. Therefore, the cell concentration of the temperature-tolerant strain MX01 during the photo-fermentation
Conclusions
The mutant strain R. capsulatus MX01 exhibited increased hydrogen yield comparing to the wild type strain R. capsulatus SB1003, at higher culture temperature and light intensity. The maximum hydrogen production yield from cornstalk was 3.64 ± 0.18 mol-H2/g-cornstalk and energy conversion efficiency of hydrogen was 10.6%, illustrating that the mutant strain MX01 may be an ideal candidate for producing hydrogen from agricultural wastes. This study preliminarily confirms that the mutation in MX01
CRediT authorship contribution statement
Xuan Wei: Conceptualization, Investigation, Writing - original draft. Jiali Feng: Writing - review & editing. Wen Cao: Writing - review & editing, Funding acquisition. Qing Li: Formal analysis. Liejin Guo: Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work is supported by the Basic Science Center Program for Ordered Energy Conversion of the National Natural Science Foundation of China (No. 51888103) and China Postdoctoral Science Foundation (No. 2019M653614). We especially appreciate Professor Fevzi Daldal for helpful discussion of the manuscript.
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