Regulation of hydrogen photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers and reducers

doi:10.1016/j.apenergy.2014.06.019

Applied Energy

Volume 131, 15 October 2014, Pages 20-25

https://doi.org/10.1016/j.apenergy.2014.06.019 Get rights and content

Highlights

•
Reducers dithiothreitol (DTT) and dithionite inhibited growth but stimulated H₂ production by R. sphaeroides.
•
Oxidizer ferricyanide inhibited both cell growth and H₂ production.
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These effects have a concentration-dependent manner.
•
Dicyclohexylcarbodiimide-inhibited ATPase activity increased in the presence of DTT.
•
A role of the F_oF₁-ATPase in redox sensing under photo-fermentation and H₂ production is proposed.

Abstract

Photo-fermentative production of hydrogen (H₂) by purple bacterium Rhodobacter sphaeroides MDC6521 from Armenian mineral springs and its regulation by external reducers and oxidizers have been investigated. Reducers such as dl-dithiothreitol (DTT) and dithionite suppress bacterial growth but enhance H₂ yield of R. sphaeroides, whereas oxidizer ferricyanide inhibits both processes. The effect of DTT on DCCD-inhibited F_oF₁-ATPase activity of R. sphaeroides membrane vesicles has been analyzed too. DTT increases the DCCD-inhibited ATPase activity. Thus, more negative values of E_h by addition of DTT might regulate F_oF₁-ATPase activity. The participation of the ATPase in redox sensing under photo-fermentative H₂ production is suggested. This enzyme might be a target, having a significant role in these processes of purple bacteria.

Introduction

Hydrogen (H₂) is considered as a renewable and environmentally friendly fuel, which burning chemically or electrochemically generates large amounts of energy [1], [2], [3]. Nowadays the biological production of H₂ is considered as a perspective area of biotechnology, which offers the potential production of usable H₂ from a variety of resources [4], [5], [6], [7]. Photosynthetic organisms such as green algae, cyanobacteria and purple bacteria can produce H₂ using the sunlight as energy source [2], [6], [7], [8]. The photo-fermentation process is one of the many approaches to generate H₂ by microorganisms [3], [5], [6], [7].

Photosynthetic purple bacteria are able to grow in anaerobic conditions under illumination and to perform a photo-fermentation of various organic compounds with H₂ production, which is released by means of redox processes [9], [10], [11], [12]. Under photoheterotrophic conditions, purple bacteria use various organic carbon sources for generating NADH by oxidation to CO₂, electrons, and protons in the tricarboxylic acid cycle (TCA) [2], [3], [6], [11]. Via the photosynthetic electron transport chain protons are pumped through the membrane with development proton motive force, which is used by the F_oF₁-ATPase to generate ATP. In nitrogen-limited conditions, nitrogenase catalyses conversion of protons to H₂ in an ATP-dependent manner.

The bacterial ability to realize redox processes depends on the redox state of the medium or its redox potential (E_h), which itself depends on rate of redox reactions [13], [14], [15]. Bacterial anaerobic growth is coupled with decrease of E_h from positive to negative value, which describes transfer of electrons within bacterial membrane and formation of proton motive force [16], [17].

E_h of the medium is very significant parameter, which can be determined as the ability of a biological system to oxidize or reduce different substrates [13]. Positive and negative mV values show the reduced and oxidized states of biological systems. The value of E_h spreads from +300 mV for aerobic bacteria to −400 mV for anaerobic bacteria [13], [18]. It is known, that positive values of E_h suppress the growth of the anaerobic bacteria [13], [19]. Therefore the reduced medium and negative values of E_h are required for bacterial growth. During photo-fermentation of organic carbon sources the E_h of Rhodobacter sphaeroides growth medium decreased to negative values up to ∼−600 mV [10], [11], [20]. Such low value of E_h is correlated to H₂ production by bacteria, because for the 2H⁺ → H₂ reaction was observed E_h equal to −420 mV [13], [15], [21]. According to the Nernst equation bacterial medium E_h depends on the reduced and oxidized products of fermentation, as well as on pH [11], [14].

It is known, that H₂ production by R. sphaeroides is coupled to [Mo–Fe]-nitrogenase, which not only participates in nitrogen fixation, but also in reduction of H⁺ to H₂. R. sphaeroides contains also [Ni–Fe]-hydrogenase, which is responsible to H₂ uptake under anaerobic photo-fermentative conditions [2], [3], [4], [5], [6], [22]. H₂ production by nitrogenase is irreversible process, requiring large amount of ATP, which is produced by the proton F_oF₁-ATPase, the main membrane-associated enzyme of bioenergetics relevance [6], [16], [17], [23]. In the literature are available some data about the actual E_h necessary for H₂ production, but there are a few data about the effect of E_h on the nitrogenase activity [21], [24], [25]. Hallenbeck has shown that a low E_h must be maintained for nitrogenase activity and effective nitrogen fixation in vitro and in vivo [25].

The important role of E_h was demonstrated for Escherichia coli, lactic acid and other bacteria growing under anaerobic conditions [18], [19], [26], [27]. It was shown that bacterial growth is affected by different redox reagents, which can change the E_h of growth medium or have direct effects on the cell surface and various intracellular processes. Reducer such as dl-dithiothreitol (DTT) stimulates growth of Enterococcus hirae, but suppresses growth of E. coli [27], [28].

In this paper, novel data about effects of various external reducers (DTT, dithionite) and oxidizer (ferricyanide) on photo-fermentative H₂ production by purple non-sulfur bacterium R. sphaeroides str. MDC6521 grown in batch culture under anaerobic conditions and illumination are presented. The effect of redox reagents on H₂ production ability of R. sphaeroides has not been reported yet. A role of the F_oF₁-ATPase in redox sensing during photo-fermentation is proposed. This study will be helpful for revealing the regulatory pathways of bacterial redox sensing, and for optimization the conditions for efficient H₂ production by R. sphaeroides.

Section snippets

Bacterial strain and growth conditions

In the present work we used R. sphaeroides strain MDC6521 (Microbial Depository Center, Armenia, WDCM803), which was isolated from Arzni mineral waters in Armenian mountains [10], [17], [20]. The bacterium was grown in batch culture anaerobically at 30 ± 2 °C and pH 7.5 ± 0.1 under illumination with a halogen lamp with a light intensity of ∼36 W/m² in Ormerod medium with carbon source – succinate (30 mM) and nitrogen source – yeast extract (0.2%) as described previously [11], [17]. The growth of

Redox potential kinetics during R. sphaeroides growth in the presence of external reducers and oxidizer

During photo-fermentation purple bacterium R. sphaeroides utilize different organic carbon and nitrogen sources and produce various by-products such as H₂ which may affect the E_h of the medium [10], [11], [17].

Oxidizer ferricyanide and reducers DTT and dithionite affect the E_h in a different manner. DTT maintaining negative values of the E_h at the level of −200 ± 20 mV delays bacterial growth and enhances drop in E_h (Fig. 1a). The addition of DTT into the growth medium leads to decrease of E_h in a

Conclusions

The kinetics of E_h and photo-fermentative H₂ production during R. sphaeroides anaerobic growth in batch culture with external redox reagents has been first studied. Redox reagents have been used for maintain of positive and negative E_h values.

Bacterial growth is suppressed in the redox medium, but the reductive conditions increase H₂ yield by R. sphaeroides. The principal reason of H₂ photoproduction by purple non-sulfur bacteria might be a reduction of photosynthetic electron transfer chain.

Acknowledgment

This study was supported by Research Grant from the Ministry of Education and Science of Armenia (13-1F002).

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Regulation of hydrogen photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers and reducers

Highlights

Abstract

Introduction

Section snippets

Bacterial strain and growth conditions

Redox potential kinetics during R. sphaeroides growth in the presence of external reducers and oxidizer

Conclusions

Acknowledgment

Appl Energy

Bioresour Technol

Appl Energy

Appl Energy

Appl Energy

Int J Hydrogen Energy

Int J Hydrogen Energy

Bioresour Technol

Biomass Bioenergy

Int J Hydrogen Energy

Bioresour Technol

Arch Biochem Biophys

Bioelectrochemistry

Biochem Biophys Res Commun

Int J Hydrogen Energy

Biochem Biophys Res Commun

J Biol Chem

Microbiological methods of hydrogen generation