Opportunities toward hydrogen production biotechnologies

doi:10.1016/j.copbio.2020.03.002

Current Opinion in Biotechnology

Volume 62, April 2020, Pages 248-255

https://doi.org/10.1016/j.copbio.2020.03.002 Get rights and content

Highlights

•
Maintaining low pH₂, pO₂, and pCO₂ can be highly beneficial for H₂ production.
•
Complex [FeFe]-hydrogenases can be mutated for oxygen tolerance.
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Increasing ATP and electron flux supports high photoautotrophic growth rates and light tolerance.
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Large heat reservoirs and nocturnal heat radiation can improve H₂ and O₂ storage efficiencies.
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Recent advances and insights suggest economic feasibility for biohydrogen technologies.

Hydrogen is already a major commodity and process intermediate for fertilizer production, petroleum processing, and chemical synthesis. It also offers unrealized potential for energy storage. While biological production offers an expandable and sustainable source, enthusiasm has been dampened by slow research progress. Also, the very low cost of natural gas (the major current hydrogen source) imposes severe economic challenges. This discussion describes process, metabolic, and protein engineering opportunities toward cost-effective biohydrogen production. Recent progress in hydrogenase engineering and photosynthetic bacterial research now suggests a favorable risk versus reward opportunity. Although the risks are still significant, successful technologies would provide important components in an integrated energy portfolio that enables global sustainability.

Graphical abstract

Introduction

In 2003, President George W. Bush announced a major R&D initiative toward adoption of hydrogen fuel for powering vehicles and generating electricity (CNN.com/inside politics, 2/6/03; https://georgewbush-whitehouse.archives.gov/infocus/technology/economic_policy200404/chap2.html). Unfortunately, the scientific, technological, and economic hurdles proved daunting. One major limitation, still in force, is the paucity of cost-effective and sustainable technologies for hydrogen production. Nascent biological technologies apparently provided attractive opportunities, and many development projects were funded. However, results were disappointing. This overview is offered to briefly assess past efforts toward cost-effective biohydrogen production, but, more importantly, to use fundamental scientific and engineering insights to suggest an effective path forward.

In the U.S., most of the hydrogen used industrially is produced from natural gas (afdc.energy.gov/fuels/hydrogen_production.html). While this technology releases large quantities of CO₂, it also sets a high bar for biohydrogen economic performance since current wholesale natural gas prices are about 20% of wholesale gasoline prices based on energy content. Consequently, initial large scale implementation of biohydrogen technologies will most likely require specific local incentives as well as exceptional metabolic and process engineering. This overview will concentrate on two such opportunities: 1) geographically distributed conversion of crop wastes (hydrolyzed cellulosic biomass) into hydrogen for local production of ammonia fertilizer; thereby lowering agricultural carbon release footprints; and 2) solar production and storage of hydrogen (and oxygen) for centralized electricity generation to mitigate renewable energy intermittencies.

The basic metabolic systems are diagrammed in Figure 1. Both approaches offer relatively direct and potentially efficient utilization of reducing equivalents, a characteristic essential for cost-effective hydrogen production.

Section snippets

Learning from previous biohydrogen R&D

This article is not intended as a thorough review of previous biohydrogen process development, but a brief chronological summary helps to define challenges and opportunities.

Exploiting further [FeFe]-hydrogenase mutagenesis

Functional comparison of over 300 singly or doubly mutated CpIs [10^••] failed to reveal any correlation between the effects of electron transfer pathway mutations on activity versus effects on oxygen tolerance. This observation suggests a strong probability for discovering more fully oxygen tolerant mutants with high activities. The new production and evaluation procedures can now be implemented for screening thousands of mutants in 96-well plate formats. To reduce electrical impedance, random

Conclusions

Rejuvenated expectations toward economical, large scale biohydrogen technologies are now supported by: a) opportunities to reinvent photosynthetic systems, b) highly integrated process designs, c) improved host organisms, and d) advances in [FeFe]-hydrogenase functionality. While development and deployment decisions must still recognize that Permian Basin natural gas is currently a liability (i.e. methane derived H₂ is very inexpensive); global concerns are paramount. We absolutely must address

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

The author would like to thank Professor Chris Walsh and Dr Richard Sassoon for encouragement and helpful discussions.

This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors.

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Cited by (12)

Using directed evolution to improve hydrogen production in chimeric hydrogenases from algal species
2024, Enzyme and Microbial Technology
Algae generate hydrogen from sunlight and water utilizing high-energy electrons generated during photosynthesis. The amount of hydrogen produced in heterologous expression of the wild-type hydrogenase is currently insufficient for industrial applications. One approach to improve hydrogen yields is through directed evolution of the DNA of the native hydrogenase. Here, we created 113 chimeric algal hydrogenase gene variants derived from combining segments of three parent hydrogenases, two from Chlamydomonas reinhardtii (CrHydA1 and CrHydA2) and one from Scenedesmus obliquus (HydA1). To generate chimeras, there were seven segments into which each of the parent hydrogenase genes was divided and recombined in a variety of combinations. The chimeric and parental hydrogenase sequences were cloned for heterologous expression in Escherichia coli, and 40 of the resultant enzymes expressed were assayed for H₂ production. Chimeric clones that resulted in equal or greater production obtained with the cloned CrHydA1 parent hydrogenase were those comprised of CrHydA1 sequence in segments #1, 2, 3, and/or 4. These best-performing chimeras all contained one common region, segment #2, the part of the sequence known to contain important amino acids involved in proton transfer or hydrogen cluster coordination. The amino acid sequence distances among all chimeric clones to that of the CrHydA1 parent were determined, and the relationship between sequence distances and experimentally-derived H₂ production was evaluated. An additional model determined the correlation between electrostatic potential energy surface area ratios and H₂ production. The model yielded several algal mutants with predicted hydrogen productions in a range of two to three times that of the wild-type hydrogenase. The mutant data and the model can now be used to predict which specific mutant sequences may result in even higher hydrogen yields. Overall, results provide more precise details in planning future directed evolution to functionally improve algal hydrogenases.
[FeFe]-hydrogenases: Structure, mechanism, and metallocluster biosynthesis
2023, Comprehensive Inorganic Chemistry III, Third Edition
[FeFe]-hydrogenases catalyzes the reversible conversion of hydrogen into protons and electrons with astonishing efficiency. The reaction occurs at the H-cluster, which is unique in biology. It consists of an organometallic [2Fe]-subcluster bound to a classical [4Fe4S] cluster by a single cysteine bridging ligand. This book chapter will describe the structure and mechanism of [FeFe]-hydrogenases and focus on recent developments on [FeFe]-hydrogenase maturation, leading to the assembly of the [2Fe]-subcluster, which have revealed a remarkably complex pattern of mostly novel biochemical reactions. This chemical process depends on the concerted action of three metalloproteins. Two radical-SAM enzymes HydE and HydG and a scaffold iron-sulfur containing protein, HydF.
Current strategies and future perspectives in biological hydrogen production: A review
2022, Renewable and Sustainable Energy Reviews
Citation Excerpt :
Also, the cost imposed on storing biohydrogen is similar to that of conventional hydrogen storage. Although the production of intermediate byproducts and CO2 emission is unavoidable, and the separation of hydrogen from the gas mixture is a key drawback, the recent biotechnological advancements in biohydrogen production are promising on an economic scale [201]. The feasibility of economical biohydrogen production depends on various factors.
Biohydrogen is a green and eco-friendly energy carrier with the potential to reduce our dependency on fossil fuels. Renewable biohydrogen production from waste biomass sources is potentially cheap; however, large-scale commercial production has not yet been achieved. Problems that need to be tackled include identifying industrially competent microorganisms, and appropriate bioreactor designs enabling novel hybrid methods such as integration of dark fermentation with electro-fermentation and utilization of microbial organisms doped with semiconducting nanomaterials for enhanced production. This review focuses on the production of hydrogen by biological methods, highlighting various fermentation processes, the role of enzymes, and different pretreatment methods. The waste materials used are briefly summarized, and current strategies in biological hydrogen production, including biomimetic and synthetic biology approaches, are assessed for their economic feasibility and their potential to contribute to net zero carbon emission. The lignocellulosic waste and the dynamic membrane bioreactor are the best suitable biomass and bioreactor, respectively for biohydrogen production. The integrated method of dark fermentation and electro-fermentation yields 41% higher hydrogen compared with dark fermentation alone. Finally, this review points out that significant efforts focusing on the development of hybrid fermentation technologies along with the development of novel engineered strains are needed for the commercial-scale production of biohydrogen in the future.
Effects of potential inhibitors present in dilute acid-pretreated corn stover on fermentative hydrogen production by Escherichia coli
2022, International Journal of Hydrogen Energy
Citation Excerpt :
Although various microorganisms produce H2 as an anaerobic fermentation product under environmentally friendly mild conditions, the productivity needs to be improved to establish an economically feasible industrial process. For this purpose, there has been a substantial focus on the genetic engineering of H2-producing microorganisms [2–5]. Escherichia coli is the best characterized microorganism in terms of physiology and genetics, and there have been extensive advancements in the genetic engineering tools for this model bacterium.
This study reports the inhibitory compounds present in dilute sulfuric acid-pretreated corn stover for fermentative hydrogen production by Escherichia coli. The E. coli W3110-derived strain deficient in lactate and succinate production pathways was used. When dilute acid-treated corn stover liquor containing 175 mM acetate, 21 mM furfural, and 2.6 mM 5-hydroxymethylfurfural was added at fourfold dilution to the xylose-containing culture medium, the microbial growth and hydrogen production were almost completely inhibited. The addition of acetate, furfural, or 5-hydroxymethylfurfural, at up to 200, 80, or 40 mM, respectively, to the xylose- or glucose-containing medium led to dose-dependent inhibition of fermentative hydrogen production. No synergistic inhibition by furfural with acetate or 5-hydroxymethylfurfural at concentrations based on the corn stover hydrolysate was observed. Altogether, these results suggest that the inhibition of the E. coli-based hydrogen production by the corn stover hydrolysate is primarily attributed to acetate under the conditions used.
Interplay between hydrogen production and photosynthesis in a green alga expressing an active photosystem I-hydrogenase chimera
2022, International Journal of Hydrogen Energy
Citation Excerpt :
Photosynthetic biohydrogen is one of the venues for a potentially inexpensive carbon-neutral fuel that relies on solar-driven production by living organisms [2–4]. Unfortunately, progress towards achieving a cost-effective biological H2 production technology has so far been lackluster [5]. More radical approaches are, therefore, required to make significant progress.
We have previously created and expressed a chimeric polypeptide joining the PsaC subunit of Photosystem I (PSI) to the HydA2 hydrogenase of Chlamydomonas reinhardtii and demonstrated that it assembles into the PSI complex and feeds electrons directly to the hydrogenase domain, allowing for prolonged photobiological hydrogen production. Here we describe a new PSI-hydrogenase chimera using HydA1, the more abundant and physiologically active endogenous hydrogenase of this alga. When the PsaC-HydA1 polypeptide was expressed in a C. reinhardtii strain lacking endogenous hydrogenases, it was assembled into active PSI-HydA1 complexes that were accumulated at a level ∼75% that of PSI, which is ∼5 times higher than the PSI-HydA2 chimera. Hydrogen production by the chimera could be restored after complete inactivation by oxygen without requiring new synthesis of PSI or the PsaC-HydA1 polypeptide, demonstrating that the complex could be repaired in vivo. The PSI-HydA1 chimera reduces ferredoxin in vivo to such an extent that it can drive the Calvin-Benson-Bassham cycle, leading to high O₂ production rates, and eventually resulting in inactivation of the hydrogenase; use of media that drastically diminished CO₂ fixation and an O₂-scavenging material allowed H₂ production for at least 4 days.
A cytosolic ferredoxin-independent hydrogenase possibly mediates hydrogen uptake in Trichomonas vaginalis
2022, Current Biology
Citation Excerpt :
Hydrogenases, enzymes of high interest in diverse fields, including biofuel and renewable energy,1,2 pathogenesis,3 ecology, and biogeochemistry,4 are evolutionarily ancient metalloproteins that catalyze the rapid interconversion between electrons, protons, and molecular hydrogen (i.e., they are involved both in hydrogen evolution and its utilization as a source of reducing power).
Trichomonads, represented by the highly prevalent sexually transmitted human parasite Trichomonas vaginalis, are anaerobic eukaryotes with hydrogenosomes in the place of the standard mitochondria. Hydrogenosomes form indispensable FeS-clusters, synthesize ATP, and release molecular hydrogen as a waste product. Hydrogen formation is catalyzed by [FeFe] hydrogenase, the hallmark enzyme of all hydrogenosomes found in various eukaryotic anaerobes. Eukaryotic hydrogenases were originally thought to be exclusively localized within organelles, but today few eukaryotic anaerobes are known that possess hydrogenase in their cytosol. We identified a thus-far unknown hydrogenase in T. vaginalis cytosol that cannot use ferredoxin as a redox partner but can use cytochrome b5 as an electron acceptor. Trichomonads overexpressing the cytosolic hydrogenase, while maintaining the carbon flux through hydrogenosomes, show decreased excretion of hydrogen and increased excretion of methylated alcohols, suggesting that the cytosolic hydrogenase uses the hydrogen gas as a source of reducing power for the reactions occurring in the cytoplasm and thus accounts for the overall redox balance. This is the first evidence of hydrogen uptake in a eukaryote, although further work is needed to confirm it. Assembly of the catalytic center of [FeFe] hydrogenases (H-cluster) requires the activity of three dedicated maturases, and these proteins in T. vaginalis are exclusively localized in hydrogenosomes, where they participate in the maturation of organellar hydrogenases. Despite the different subcellular localization of cytosolic hydrogenase and maturases, the H-cluster is present in the cytosolic enzyme, suggesting the existence of an alternative mechanism of H-cluster assembly.

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View full text

Opportunities toward hydrogen production biotechnologies

Highlights

Graphical abstract

Introduction

Section snippets

Learning from previous biohydrogen R&D

Exploiting further [FeFe]-hydrogenase mutagenesis

Conclusions

Conflict of interest statement

References and recommended reading

Acknowledgements

Planta

Int J Hydrogen Energy

Appl Biochem Biotechnol

J Biol Chem

PLoS One

PNAS

mBio

Enzymatic production of hydrogen

Nature

Bio-hydrogen production by different operational modes of dark and photo-fermentation: an overview

Int J Hydrogen Energy

The role of carbon dioxide in light-activated hydrogen production by Chlamydomonas reinhardtii

Photosynth Res

High-yield hydrogen production from biomass by in vitro metabolic engineering: mixed sugars co-utilization and kinetic modeling

PNAS