biohydrogen

The main reactions driving hydrogen formation involve the oxidation of substrates to obtain electrons. Then, these electrons are transferred to free protons to form molecular hydrogen. This proton reduction reaction is normally performed by an enzyme family known as hydrogenases.

In heterotrophic organisms, electrons are produced during the fermentation of sugars. Hydrogen gas is produced in many types of fermentation as a way to regenerate NAD⁺ from NADH. Electrons are transferred to ferredoxin, or can be directly accepted from NADH by a hydrogenase, producing H₂. Because of this most of the reactions start with glucose, which is converted to acetic acid.{{cite journal | last1 = Thauer | first1 = R. K. | year = 1998 | title = Biochemistry of Methanogenesis: a Tribute to Marjory Stephenson | journal = Microbiology | volume = 144 | pages = 2377–2406 | doi = 10.1099/00221287-144-9-2377 | pmid = 9782487 | doi-access = free }}

:C6H12O6 + 2 H2O -> 2 CH3COOH + 2 CO2 + 4 H2

A related reaction gives formate instead of carbon dioxide:

:C6H12O6 + 2 H2O -> 2 CH3COOH + 2 HCOOH + 2 H2

These reactions are exergonic by 216 and 209 kcal/mol, respectively.

It has been estimated that 99% of all organisms utilize or produce dihydrogen (H₂). Most of these species are microbes and their ability to use or produce H₂ as a metabolite arises from the expression of H₂ metalloenzymes known as hydrogenases.

{{cite journal |author1=Lubitz, Wolfgang |author-link1=Wolfgang Lubitz |author2=Ogata, Hideaki |author3=Rüdiger, Olaf |author4=Reijerse, Edward |year=2014 |title=Hydrogenases |journal=Chemical Reviews |volume=114 |issue=8 |pages=4081–148 |doi=10.1021/cr4005814 |pmid=24655035}}

Enzymes within this widely diverse family are commonly sub-classified into three different types based on the active site metal content: [FeFe]-hydrogenases (iron-iron), [NiFe]-hydrogenases (nickel-iron) hydrogenases, and [Fe]-hydrogenases (iron-only).{{Cite journal |last1=Vignais |first1=Paulette M. |last2=Billoud |first2=Bernard |date=2007-10-01 |title=Occurrence, Classification, and Biological Function of Hydrogenases: An Overview |url=https://pubs.acs.org/doi/10.1021/cr050196r |journal=Chemical Reviews |language=en |volume=107 |issue=10 |pages=4206–4272 |doi=10.1021/cr050196r |pmid=17927159 |issn=0009-2665}} Many organisms express these enzymes. Notable examples are members of the genera Clostridium, Desulfovibrio, Ralstonia or the pathogen Helicobacter, being most of them strict-anaerobes or facultative microorganisms. Other microorganisms such green algae also express highly active hydrogenases, as it is the case for members of the genera Chlamydomonas.File:ActiveSitesCorrected.pngDue to the extreme diversity of hydrogenase enzymes, on-going efforts are focused on screening for novel enzymes with improved features,{{Cite journal |last1=Land |first1=Henrik |last2=Ceccaldi |first2=Pierre |last3=Mészáros |first3=Lívia S. |last4=Lorenzi |first4=Marco |last5=Redman |first5=Holly J. |last6=Senger |first6=Moritz |last7=Stripp |first7=Sven T. |last8=Berggren |first8=Gustav |date=2019-11-06 |title=Discovery of novel [FeFe]-hydrogenases for biocatalytic H2-production |journal=Chemical Science |language=en |volume=10 |issue=43 |pages=9941–9948 |doi=10.1039/C9SC03717A |pmid=32055351 |pmc=6984386 |issn=2041-6539}}{{Cite journal |last1=Grinter |first1=Rhys |last2=Kropp |first2=Ashleigh |last3=Venugopal |first3=Hari |last4=Senger |first4=Moritz |last5=Badley |first5=Jack |last6=Cabotaje |first6=Princess R. |last7=Jia |first7=Ruyu |last8=Duan |first8=Zehui |last9=Huang |first9=Ping |last10=Stripp |first10=Sven T. |last11=Barlow |first11=Christopher K. |last12=Belousoff |first12=Matthew |last13=Shafaat |first13=Hannah S. |last14=Cook |first14=Gregory M. |last15=Schittenhelm |first15=Ralf B. |date=March 2023 |title=Structural basis for bacterial energy extraction from atmospheric hydrogen |journal=Nature |language=en |volume=615 |issue=7952 |pages=541–547 |doi=10.1038/s41586-023-05781-7 |pmid=36890228 |pmc=10017518 |bibcode=2023Natur.615..541G |issn=1476-4687}}{{Cite journal |last=Morra |first=Simone |date=2022 |title=Fantastic [FeFe]-Hydrogenases and Where to Find Them |journal=Frontiers in Microbiology |volume=13 |page=853626 |doi=10.3389/fmicb.2022.853626 |pmid=35308355 |pmc=8924675 |issn=1664-302X |doi-access=free }} as well as engineering already characterized hydrogenases to confer them more desirable characteristics.{{Cite journal |last1=Lu |first1=Yuan |last2=Koo |first2=Jamin |date=November 2019 |title=O2 sensitivity and H2 production activity of hydrogenases-A review |url=https://pubmed.ncbi.nlm.nih.gov/31403182/ |journal=Biotechnology and Bioengineering |volume=116 |issue=11 |pages=3124–3135 |doi=10.1002/bit.27136 |issn=1097-0290 |pmid=31403182|s2cid=199539477 }}

The biological hydrogen production with algae is a method of photobiological water splitting which is done in a closed photobioreactor based on the production of hydrogen as a solar fuel by algae.[http://labs.biology.ucsd.edu/schroeder/bggn227/2014%20Lectures/Mayfield/Gimpel%20COCB%202013.pdf 2013 - Gimpel JA, et al Advances in microalgae engineering and synthetic biology applications for biofuel production]{{cite journal|last1=Hemschemeier|first1=Anja|last2=Melis|first2=Anastasios|last3=Happe|first3=Thomas|title=Analytical approaches to photobiological hydrogen production in unicellular green algae|journal=Photosynthesis Research|volume=102|issue=2–3|year=2009|pages=523–540|issn=0166-8595|doi=10.1007/s11120-009-9415-5|pmid=19291418|pmc=2777220|bibcode=2009PhoRe.102..523H }} Algae produce hydrogen under certain conditions. In 2000 it was discovered that if C. reinhardtii algae are deprived of sulfur they will switch from the production of oxygen, as in normal photosynthesis, to the production of hydrogen.[https://www.wired.com/news/technology/0,70273-0.html Wired-Mutant Algae Is Hydrogen Factory] {{webarchive|url=https://web.archive.org/web/20060827033219/http://www.wired.com/news/technology/0%2C70273-0.html |date=August 27, 2006 }}{{cite web |url=http://www.science.org.au/nova/newscientist/111ns_002.htm |title=Further reading - New Scientist |accessdate=2009-03-11 |url-status=dead |archive-url=https://web.archive.org/web/20081031205453/http://www.science.org.au/nova/newscientist/111ns_002.htm |archive-date=2008-10-31 }}{{Cite journal|last1=Melis|first1=Anastasios|last2=Zhang|first2=Liping|last3=Forestier|first3=Marc|last4=Ghirardi|first4=Maria L.|last5=Seibert|first5=Michael|date=2000-01-01|title=Sustained Photobiological Hydrogen Gas Production upon Reversible Inactivation of Oxygen Evolution in the Green AlgaChlamydomonas reinhardtii|journal=Plant Physiology|language=en|volume=122|issue=1|pages=127–136|doi=10.1104/pp.122.1.127|issn=1532-2548|pmid=10631256|pmc=58851}}

Green algae express [FeFe] hydrogenases, being some of them considered the most efficient hydrogenases with turnover rates superior to 10⁴ s⁻¹. This remarkable catalytic efficiency is nonetheless shadowed by its extreme sensitivity to oxygen, being irreversibly inactivated by O_2. When the cells are deprived from sulfur, oxygen evolution stops due to photo-damage of photosystem II, in this state the cells start consuming O₂ and provide the ideal anaerobic environment for the native [FeFe] hydrogenases to catalyze H₂ production.

{{multiple image

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| footer = Algal cell-based bioreactors that can produce hydrogen

| image1 = Formation of Chlorella cell-based spheroids.webp

| alt1 = Formation of Chlorella cell-based spheroids

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| image2 = Schematic illustration showing the assembly, spatial organization and dual functionality of multicellular droplet-based living micro-reactors.webp

| alt2 = Schematic illustration showing the assembly, spatial organization and dual functionality of multicellular droplet-based living micro-reactors

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Photosynthesis in cyanobacteria and green algae splits water into hydrogen ions and electrons. The electrons are transported over ferredoxins.{{cite journal|last1=Peden|first1=E. A.|last2=Boehm|first2=M.|last3=Mulder|first3=D. W.|last4=Davis|first4=R.|last5=Old|first5=W. M.|last6=King|first6=P. W.|last7=Ghirardi|first7=M. L.|last8=Dubini|first8=A.|title=Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii|journal=Journal of Biological Chemistry|volume=288|issue=49|year=2013|pages=35192–35209|issn=0021-9258|doi=10.1074/jbc.M113.483727|pmid=24100040|pmc=3853270|doi-access=free}} Fe-Fe-hydrogenases (enzymes) combine them into hydrogen gas. In Chlamydomonas reinhardtii Photosystem II produces in direct conversion of sunlight 80% of the electrons that end up in the hydrogen gas.{{cite journal|last1=Volgusheva|first1=A.|last2=Styring|first2=S.|last3=Mamedov|first3=F.|title=Increased photosystem II stability promotes H2 production in sulfur-deprived Chlamydomonas reinhardtii|journal=Proceedings of the National Academy of Sciences|volume=110|issue=18|year=2013|pages=7223–7228|issn=0027-8424|doi=10.1073/pnas.1220645110|pmid=23589846|pmc=3645517|bibcode=2013PNAS..110.7223V|url=http://uu.diva-portal.org/smash/get/diva2:629400/FULLTEXT02|doi-access=free}}

In 2020 scientists reported the development of algal-cell based micro-emulsion for multicellular spheroid microbial reactors capable of producing hydrogen alongside either oxygen or CO₂ via photosynthesis in daylight under air. Enclosing the microreactors with synergistic bacteria was shown to increase levels of hydrogen production via reduction of O₂ concentrations.{{cite news |title=Research creates hydrogen-producing living droplets, paving way for alternative future energy source |url=https://phys.org/news/2020-11-hydrogen-producing-droplets-paving-alternative-future.html |access-date=9 December 2020 |work=phys.org |language=en}}{{cite journal |last1=Xu |first1=Zhijun |last2=Wang |first2=Shengliang |last3=Zhao |first3=Chunyu |last4=Li |first4=Shangsong |last5=Liu |first5=Xiaoman |last6=Wang |first6=Lei |last7=Li |first7=Mei |last8=Huang |first8=Xin |last9=Mann |first9=Stephen |title=Photosynthetic hydrogen production by droplet-based microbial micro-reactors under aerobic conditions |journal=Nature Communications |date=25 November 2020 |volume=11 |issue=1 |pages=5985 |doi=10.1038/s41467-020-19823-5 |pmid=33239636 |pmc=7689460 |bibcode=2020NatCo..11.5985X |url=|language=en |issn=2041-1723}} 50px Available under [https://creativecommons.org/licenses/by/4.0/ CC BY 4.0].

The chlorophyll (Chl) antenna size in green algae is minimized, or truncated, to maximize photobiological solar conversion efficiency and H₂ production. It has been shown that Light-harvesting complex photosystem II light-harvesting protein LHCBM9 promotes efficient light energy dissipation.{{cite journal |last1=Grewe |first1=S. |last2=Ballottari |first2=M. |last3=Alcocer |first3=M. |last4=D'Andrea |first4=C. |last5=Blifernez-Klassen |first5=O. |last6=Hankamer |first6=B. |last7=Mussgnug |first7=J. H. |last8=Bassi |first8=R. |last9=Kruse |first9=O. |year=2014 |title=Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii |journal=The Plant Cell |volume=26 |issue=4 |pages=1598–1611 |doi=10.1105/tpc.114.124198 |issn=1040-4651 |pmc=4036574 |pmid=24706511|bibcode=2014PlanC..26.1598G }} The truncated Chl antenna size minimizes absorption and wasteful dissipation of sunlight by individual cells, resulting in better light utilization efficiency and greater photosynthetic efficiency when the green alga are grown as a mass culture in bioreactors.{{cite journal|last1=Kirst|first1=H.|last2=Garcia-Cerdan|first2=J. G.|last3=Zurbriggen|first3=A.|last4=Ruehle|first4=T.|last5=Melis|first5=A.|title=Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene|journal=Plant Physiology|volume=160|issue=4|year=2012|pages=2251–2260|issn=0032-0889|doi=10.1104/pp.112.206672|pmid=23043081|pmc=3510145}}

With current reports for algae-based biohydrogen, it would take about 25,000 square kilometre algal farming to produce biohydrogen equivalent to the energy provided by gasoline in the US alone. This area represents approximately 10% of the area devoted to growing soya in the US.[https://www.newscientist.com/channel/earth/energy-fuels/mg18925401.600-growing-hydrogen-for-the-cars-of-tomorrow.html Growing hydrogen for the cars of tomorrow]

• Restriction of photosynthetic hydrogen production by accumulation of a proton gradient.
• Competitive inhibition of photosynthetic hydrogen production by carbon dioxide.
• Requirement for bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity.
• Competitive drainage of electrons by oxygen in algal hydrogen production.
• Economics must reach competitive price to other sources of energy and the economics are dependent on several parameters.
• A major technical obstacle is the efficiency in converting solar energy into chemical energy stored in molecular hydrogen.

Attempts are in progress to solve these problems via bioengineering.

Biological hydrogen production is also observed in nitrogen-fixing cyanobacteria. This microorganisms can grow forming filaments. Under nitrogen-limited conditions some cells can specialize and form heterocysts, which ensures an anaerobic intracellular space to ease N₂ fixation by the nitrogenase enzyme expressed also inside.

Under nitrogen-fixation conditions, the nitrogenase enzyme accepts electrons and consume ATP to break the triple dinitrogen bond and reduce it to ammonia.{{Cite web |date=2017-05-11 |title=5.15C: Nitrogen Fixation Mechanism |url=https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Boundless)/05%3A_Microbial_Metabolism/5.15%3A_Nitrogen_Fixation/5.15C%3A_Nitrogen_Fixation_Mechanism |access-date=2023-04-07 |website=Biology LibreTexts |language=en}} During the catalytic cycle of the nitrogenase enzyme, molecular hydrogen is also produced.

N2 + 8 H+ + 8NAD(P)H + 16 ATP-> 2 NH3 + H2 + 16 ADP + 16 Pi + 8 NAD(P)+

Nevertheless, since the production of H₂ is an important loss of energy for the cells, most of nitrogen fixing cyanobacteria also feature at least one uptake hydrogenase.{{Cite journal |last1=Tamagnini |first1=Paula |last2=Axelsson |first2=Rikard |last3=Lindberg |first3=Pia |last4=Oxelfelt |first4=Fredrik |last5=Wünschiers |first5=Röbbe |last6=Lindblad |first6=Peter |date=March 2002 |title=Hydrogenases and Hydrogen Metabolism of Cyanobacteria |journal=Microbiology and Molecular Biology Reviews |language=en |volume=66 |issue=1 |pages=1–20 |doi=10.1128/MMBR.66.1.1-20.2002 |issn=1092-2172 |pmc=120778 |pmid=11875125}} Uptake hydrogenases exhibit a catalytic bias towards oxygen oxidation, thus can assimilate the produced H₂ as a way to recover part of the energy invested during the nitrogen fixation process.

In 1933, Marjory Stephenson and her student Stickland reported that cell suspensions catalysed the reduction of methylene blue with H₂. Six years later, Hans Gaffron observed that the green photosynthetic alga Chlamydomonas reinhardtii, would sometimes produce hydrogen.[https://www.wired.com/news/technology/0,1282,54456,00.html Algae: Power Plant of the Future?] In the late 1990s Anastasios Melis discovered that deprivation of sulfur induces the alga to switch from the production of oxygen (normal photosynthesis) to the production of hydrogen. He found that the enzyme responsible for this reaction is hydrogenase, but that the hydrogenase lost this function in the presence of oxygen. Melis also discovered that depleting the amount of sulfur available to the algae interrupted their internal oxygen flow, allowing the hydrogenase an environment in which it can react, causing the algae to produce hydrogen.{{cite magazine |title=Multiplatform Pinup Girl |date=2002-04-01 |magazine=Wired |archive-url=https://web.archive.org/web/20121020205409/https://www.wired.com/wired/archive/10.04/mustread.html?pg=5 |archive-date=2012-10-20 |url-status=live |url=https://www.wired.com/wired/archive/10.04/mustread.html?pg=5}} Chlamydomonas moewusii is also a promising strain for the production of hydrogen.{{Cite journal|vauthors=Melis A, Happe T |year = 2001 | title = Hydrogen Production. Green Algae as a Source of Energy | journal = Plant Physiol. | volume = 127 | pages = 740–748 | doi = 10.1104/pp.010498 | pmid = 11706159 | issue = 3 | pmc = 1540156 }}{{cite journal|last1=Yang|first1=Shihui|last2=Guarnieri|first2=Michael T|last3=Smolinski|first3=Sharon| last4=Ghirardi|first4=Maria|last5=Pienkos|first5=Philip T|title=De novo transcriptomic analysis of hydrogen production in the green alga Chlamydomonas moewusii through RNA-Seq|journal=Biotechnology for Biofuels|volume=6|issue=1|year=2013|pages=118|issn=1754-6834|doi=10.1186/1754-6834-6-118|pmid=23971877| pmc=3846465 |doi-access=free |bibcode=2013BB......6..118Y }}

Competing for biohydrogen, at least for commercial applications, are many mature industrial processes. Steam reforming of natural gas - sometimes referred to as steam methane reforming (SMR) - is the most common method of producing bulk hydrogen at about 95% of the world production.P. Häussinger, R. Lohmüller, A. M. Watson, "Hydrogen, 2. Production" in Ullmann's Encyclopedia of Industrial Chemistry, 2012, Wiley-VCH, Weinheim. {{doi|10.1002/14356007.o13_o03}}{{cite journal |last=Ogden |first=J.M. |title=Prospects for building a hydrogen energy infrastructure |journal=Annual Review of Energy and the Environment |year=1999 |volume=24 |pages=227–279 |doi=10.1146/annurev.energy.24.1.227|doi-access=}}{{cite web|url= https://energy.gov/eere/fuelcells/hydrogen-production-natural-gas-reforming |title=Hydrogen Production: Natural Gas Reforming |publisher=Department of Energy|accessdate=6 April 2017}}

:CH4 + H2O <-> CO + 3 H2

• {{annotated link|Algaculture}}
• {{annotated link|Hydrogen production}}
• {{annotated link|Hydrogenase}}
• {{annotated link|Photohydrogen}}
• {{annotated link|Timeline of hydrogen technologies}}

• [http://www1.eere.energy.gov/hydrogenandfuelcells/production/pdfs/photobiological.pdf DOE - A Prospectus for Biological Production of Hydrogen]
• [http://www.fao.org/docrep/w7241e/w7241e0g.htm FAO]
• [http://www.hydrogen.energy.gov/pdfs/review06/pdp_11_melis.pdf Maximizing Light Utilization Efficiency and Hydrogen Production in Microalgal Cultures] {{Webarchive|url=https://web.archive.org/web/20131019033352/http://www.hydrogen.energy.gov/pdfs/review06/pdp_11_melis.pdf |date=2013-10-19 }}
• [http://www.futurefarmers.com/survey/algae.php DIY Algae/Hydrogen Bioreactor 2004]
• [https://web.archive.org/web/20170809024416/https://www1.eere.energy.gov/hydrogenandfuelcells/pdfs/30535f.pdf EERE-CYCLIC PHOTOBIOLOGICAL ALGAL H2-PRODUCTION]

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