Biohydrogen production via dark fermentation with mixed microbial cultures is a promising strategy for sustainable waste valorization and renewable energy generation. This study provides a comprehensive analysis of advancements and emerging trends in mixed-culture dark fermentation for biohydrogen production over the past two decades through a detailed bibliometric approach. A structured search query was developed, and relevant publications published between 2005 and 2025 were retrieved from the Web of Science Core Collection database. Bibliometric analysis and network visualization were conducted using the Bibliometrix package (RStudio) and VOSviewer software. The results reveal substantial scientific progress in this field, with an average annual growth rate of approximately 25.48%. Contributions originated from 83 countries, with China, India, and Brazil ranking among the leading contributors. In total, 1,177 institutions and 3,231 authors participated in this research domain, indicating the development of a broad and increasingly interconnected global network. The University of São Paulo was identified as the most productive institution. A strong and statistically significant positive correlation (r = 0.9295; p < 0.05) was observed between publication volume and citation impact, reflecting both quantitative expansion and increasing scholarly influence. Keyword analysis further indicates that current research primarily emphasizes process optimization, waste valorization strategies, and the integration of dark fermentation into sustainable and hybrid bioenergy systems. Overall, this study offers a structured overview that facilitates the identification of key research trends, influential contributors, and leading institutions in mixed-culture dark fermentation for biohydrogen production.
AzbarN, Çetinkaya DokgözFT, KeskinT, KorkmazKS, SyedHM. Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions. Int J Hydrog Energy, 2009; 34(17):7441–7447; doi: 10.1016/j.ijhydene.2009.04.032
2.
DawoodF, AndaM, ShafiullahGM. Hydrogen production for energy: An overview. Int J Hydrog Energy, 2020; 45(7):3847–3869; doi: 10.1016/j.ijhydene.2019.12.059
3.
HallenbeckPC. Fermentative hydrogen production: Principles, progress, and prognosis. Int J Hydrog Energy, 2009; 34(17):7379–7389; doi: 10.1016/j.ijhydene.2008.12.080
4.
Venkata MohanS, ChiranjeeviP, ChandrasekharK, BabuPS, SarkarO. Acidogenic Biohydrogen Production From Wastewater. In: Biohydrogen. Elsevier; 2019; pp. 279–320. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780444642035000113; doi: 10.1016/B978-0-444-64203-5.00011-3
5.
KumarG, ChoS-K, SivagurunathanP, et al.Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation. Int J Hydrog Energy, 2018; 43(43):19885–19901; doi: 10.1016/j.ijhydene.2018.09.040
6.
DzulkarnainELN, AuduJO, Wan DagangWRZ, Abdul-WahabMF. Microbiomes of biohydrogen production from dark fermentation of industrial wastes: Current trends, advanced tools and future outlook. Bioresour Bioprocess, 2022; 9(1):16; doi: 10.1186/s40643-022-00504-8
7.
Abo-HasheshM, WangR, HallenbeckPC. Metabolic engineering in dark fermentative hydrogen production; theory and practice. Bioresour Technol, 2011; 102(18):8414–8422; doi: 10.1016/j.biortech.2011.03.016
8.
MokhtaraniB, ZanganehJ, MoghtaderiB. A review on biohydrogen production through dark fermentation, process parameters and simulation. Energies (Basel), 2025; 18(5):1092; doi: 10.3390/en18051092
9.
MohanakrishnaG, SnehaNP, RafiSM, SarkarO. Dark fermentative hydrogen production: Potential of food waste as future energy needs. Sci Total Environ, 2023; 888:163801; doi: 10.1016/j.scitotenv.2023.163801
10.
WangS, ZhangT, BaoM, SuH, XuP. Microbial production of hydrogen by mixed culture technologies: A review. Biotechnol J, 2020; 15(1):e1900297; doi: 10.1002/biot.201900297
11.
Ergalİ, ZechE, HanišákováN, et al.Scale-up of dark fermentative biohydrogen production by artificial microbial co-cultures. Appl Microbiol, 2022; 2(1):215–226; doi: 10.3390/applmicrobiol2010015
12.
AlbuquerqueMM, SartorGdB, Martinez-BurgosWJ, et al.Biohydrogen produced via dark fermentation: A review. Methane, 2024; 3(3):500–532; doi: 10.3390/methane3030029
13.
JainR, PanwarNL, JainSK, et al.Bio-hydrogen production through dark fermentation: An overview. Biomass Conv Bioref, 2024; 14(12):12699–12724; doi: 10.1007/s13399-022-03282-7
14.
KeskinT, ArslanK, Nalakth AbubackarH, et al.Determining the effect of trace elements on biohydrogen production from fruit and vegetable wastes. Int J Hydrog Energy, 2018; 43(23):10666–10677; doi: 10.1016/j.ijhydene.2018.01.028
SinbuathongN, SillapacharoenkulB. Dark fermentation of starch factory wastewater with acid- and base-treated mixed microorganisms for biohydrogen production. Int J Hydrog Energy, 2021; 46(31):16622–16630; doi: 10.1016/j.ijhydene.2020.06.109
17.
SarangiPK, NandaS. Biohydrogen production through dark fermentation. Chem Eng & Technol, 2020; 43(4):601–612; doi: 10.1002/ceat.201900452
18.
JayachandranV, BasakN, De PhilippisR, AdessiA. Novel strategies towards efficient molecular biohydrogen production by dark fermentative mechanism: Present progress and future perspective. Bioprocess Biosyst Eng, 2022; 45(10):1595–1624; doi: 10.1007/s00449-022-02738-4
19.
EttadiliH, VuralC. Current global status of Candida auris an emerging multidrug-resistant fungal pathogen: Bibliometric analysis and network visualization. Braz J Microbiol, 2024; 55(1):391–402; doi: 10.1007/s42770-023-01239-0
20.
SilleroL, SganzerlaWG, Forster-CarneiroT, SoleraR, PerezM. A bibliometric analysis of the hydrogen production from dark fermentation. Int J Hydrog Energy, 2022; 47(64):27397–27420; doi: 10.1016/j.ijhydene.2022.06.083
21.
KiprotichA, NijejeE. Dark fermentative production of biohydrogen from organic biomass: A bibliometric analysis. Environmental and Earth Sciences, 2023; doi: 10.20944/preprints202303.0555.v1. Available from: https://www.preprints.org/manuscript/202303.0555/v1
22.
SridharA, PonnuchamyM, Senthil KumarP, KapoorA, XiaoL. Progress in the production of hydrogen energy from food waste: A bibliometric analysis. Int J Hydrog Energy, 2022; 47(62):26326–26354; doi: 10.1016/j.ijhydene.2021.09.258
23.
ZhangL, BaiY, SangJ, et al.Enhanced biohydrogen production through dark fermentation by humic acid: ınsights into microbial composition and functional genes. J Microbiol Biotechnol, 2025; 35:e2412071; doi: 10.4014/jmb.2412.12071
24.
MassetJ, CalusinskaM, HamiltonC, et al.Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp. Biotechnol Biofuels, 2012; 5(1):35; doi: 10.1186/1754-6834-5-35
25.
ParraR, Chicaiza-OrtizC, Herrera-FeijooRJ, et al.Advancements of biohydrogen production based on anaerobic digestion: Technologies, substrates, and future prospects. Sci, 2025; 7(2):52; doi: 10.3390/sci7020052
26.
GarcíaAB, CammarotaMC. Biohydrogen production from pretreated sludge and synthetic and real biodiesel wastewater by dark fermentation. Int J Energy Res, 2019; 43(4):1586–1596; doi: 10.1002/er.4376
27.
MedisettyVM, KumarR, AhmadiMH, et al.Overview on the current status of hydrogen energy research and development in India. Chem Eng & Technol, 2020; 43(4):613–624; doi: 10.1002/ceat.201900496
28.
DonatiS, BarbierI, García-SorianoDA, et al.Synthetic biology in Europe: Current community landscape and future perspectives. Biotechnol Notes, 2022; 3:54–61; doi: 10.1016/j.biotno.2022.07.003
29.
BertasiniD, BattistaF, RizzioliF, FrisonN, BolzonellaD. Decarbonization of the European natural gas grid using hydrogen and methane biologically produced from organic waste: A critical overview. Renew Energy, 2023; 206:386–396; doi: 10.1016/j.renene.2023.02.029
30.
BressaninJM, GuimarãesHR, ChagasMF, et al.Advanced technologies for electricity production in the sugarcane value chain are a strategic option in a carbon reward policy context. Energy Policy, 2021; 159:112637; doi: 10.1016/j.enpol.2021.112637
31.
Da Silva CésarA, Da Silva VerasT, MozerTS, Da Costa Rubim Messeder Dos SantosD, ConejeroMA. Hydrogen productive chain in Brazil: An analysis of the competitiveness’ drivers. J Clean Prod, 2019; 207:751–763; doi: 10.1016/j.jclepro.2018.09.157
32.
TauroR, GarcíaCA, SkutschM, MaseraO. The potential for sustainable biomass pellets in Mexico: An analysis of energy potential, logistic costs and market demand. Renew Sustain Energy Rev, 2018; 82:380–389; doi: 10.1016/j.rser.2017.09.036
33.
PoirierS, SteyerJP, BernetN, TrablyE. Mitigating the variability of hydrogen production in mixed culture through bioaugmentation with exogenous pure strains. Int J Hydrog Energy, 2020; 45(4):2617–2626; doi: 10.1016/j.ijhydene.2019.11.116
34.
Toledo-AlarcónJ, CabrolL, JeisonD, et al.Impact of the microbial inoculum source on pre-treatment efficiency for fermentative H2 production from glycerol. Int J Hydrog Energy, 2020; 45(3):1597–1607; doi: 10.1016/j.ijhydene.2019.11.113
35.
DauptainK, SchneiderA, NoguerM, et al.Impact of microbial inoculum storage on dark fermentative H2 production. Bioresour Technol, 2021; 319:124234; doi: 10.1016/j.biortech.2020.124234
36.
Braga NanL, TrablyE, Santa-CatalinaG, et al.Biomethanation processes: New insights on the effect of a high H2 partial pressure on microbial communities. Biotechnol Biofuels, 2020; 13(1):141; doi: 10.1186/s13068-020-01776-y
37.
RoslanE, MagdalenaJA, MohamedH, et al.Lactic acid fermentation of food waste as storage method prior to biohydrogen production: Effect of storage temperature on biohydrogen potential and microbial communities. Bioresour Technol, 2023; 378:128985; doi: 10.1016/j.biortech.2023.128985
38.
LacrouxJ, LlamasM, DauptainK, et al.Dark fermentation and microalgae cultivation coupled systems: Outlook and challenges. Sci Total Environ, 2023; 865:161136; doi: 10.1016/j.scitotenv.2022.161136
39.
LacrouxJ, MahieuxM, LlamasM, et al.Mixotrophic cultivation of microalgae-bacteria consortia enhances dark fermentation effluent treatment. Bioresour Technol, 2024; 414:131616; doi: 10.1016/j.biortech.2024.131616
40.
DauptainK, TrablyE, Santa-CatalinaG, CarrereH. Biomass acid pretreatment impacts on metabolic routes and bacterial composition of dark fermentation process. Waste Manag, 2024; 181:211–219; doi: 10.1016/j.wasman.2024.03.035
41.
PipereauK, TrablyE, Santa-CatalinaG, García-BernetD, CarrereH. Targeted pretreatment and inoculation strategies for horse manure fermentation: Impact on metabolites and microbial community composition. J Environ Manage, 2025; 387:125894; doi: 10.1016/j.jenvman.2025.125894
42.
Montiel-CoronaV, Palomo-BrionesR, Razo-FloresE. Continuous thermophilic hydrogen production from an enzymatic hydrolysate of agave bagasse: Inoculum origin, homoacetogenesis and microbial community analysis. Bioresour Technol, 2020; 306:123087; doi: 10.1016/j.biortech.2020.123087
43.
Palomo-BrionesR, Montoya-RosalesJDJ, Razo-FloresE. Advances towards the understanding of microbial communities in dark fermentation of enzymatic hydrolysates: Diversity, structure and hydrogen production performance. Int J Hydrog Energy, 2021; 46(54):27459–27472; doi: 10.1016/j.ijhydene.2021.06.016
44.
Núñez-ValenzuelaP, Palomo-BrionesR, CervantesFJ, Razo-FloresE. Humic substances improve the co-production of hydrogen and carboxylic acids by anaerobic mixed cultures. Int J Hydrog Energy, 2021; 46(65):32800–32808; doi: 10.1016/j.ijhydene.2021.07.128
45.
Fuentes-SantiagoV, Valdez-VazquezI, Vital-JácomeM, et al.Carbohydrates/acid ratios drives microbial communities and metabolic pathways during biohydrogen production from fermented agro-industrial wastewater. J Environ Chem Eng, 2023; 11(3):110302; doi: 10.1016/j.jece.2023.110302
46.
CardeñaR, Valencia-OjedaC, Chazaro-RuizLF, Razo-FloresE. Regulation of the dark fermentation products by electro-fermentation in reactors without membrane. Int J Hydrog Energy, 2024; 49:107–116; doi: 10.1016/j.ijhydene.2023.06.253
47.
Núñez-ValenzuelaP, Ontiveros-ValenciaA, René Rangel-MéndezJ, Nieto-DelgadoC, Razo-FloresE. Extractive fermentation as a strategy to increase the co-production of H2 and carboxylates in dark fermentation. Fuel, 2024; 362:130804; doi: 10.1016/j.fuel.2023.130804
48.
De Jesús Montoya-RosalesJ, Núñez-ValenzuelaP, Ontiveros-ValenciaA, et al.From syngas fermentation to chain elongation: The role of key microorganisms and multi-omics analysis. Bioenerg Res, 2023; 17(2):897–911; doi: 10.1007/s12155-023-10696-2
49.
GhimireA, FrunzoL, PirozziF, et al.A review on dark fermentative biohydrogen production from organic biomass: Process parameters and use of by-products. Appl Energy, 2015; 144:73–95; doi: 10.1016/j.apenergy.2015.01.045
50.
SaadyNMC. Homoacetogenesis during hydrogen production by mixed cultures dark fermentation: Unresolved challenge. Int J Hydrog Energy, 2013; 38(30):13172–13191; doi: 10.1016/j.ijhydene.2013.07.122
51.
KapdanIK, KargiF. Bio-hydrogen production from waste materials. Enzyme Microb Technol, 2006; 38(5):569–582; doi: 10.1016/j.enzmictec.2005.09.015
52.
DuboisM, GillesK, HamiltonJK, RebersPA, SmithF. A colorimetric method for the determination of sugars. Nature, 1951; 168(4265):167; doi: 10.1038/168167a0
53.
AlibardiL, CossuR. Effects of carbohydrate, protein and lipid content of organic waste on hydrogen production and fermentation products. Waste Manag, 2016; 47(Pt A):69–77; doi: 10.1016/j.wasman.2015.07.049
54.
KaparajuP, SerranoM, ThomsenAB, KongjanP, AngelidakiI. Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept. Bioresour Technol, 2009; 100(9):2562–2568; doi: 10.1016/j.biortech.2008.11.011
55.
AlibardiL, CossuR. Composition variability of the organic fraction of municipal solid waste and effects on hydrogen and methane production potentials. Waste Manag, 2015; 36:147–155; doi: 10.1016/j.wasman.2014.11.019
56.
ChengJ, LiH, DingL, et al.Improving hydrogen and methane co-generation in cascading dark fermentation and anaerobic digestion: The effect of magnetite nanoparticles on microbial electron transfer and syntrophism. Chem Eng J, 2020; 397:125394; doi: 10.1016/j.cej.2020.125394
57.
YangG, WangJ. Improving mechanisms of biohydrogen production from grass using zero-valent iron nanoparticles. Bioresour Technol, 2018; 266:413–420; doi: 10.1016/j.biortech.2018.07.004
58.
WangY, ZhaoJ, WangD, et al.Free nitrous acid promotes hydrogen production from dark fermentation of waste activated sludge. Water Res, 2018; 145:113–124; doi: 10.1016/j.watres.2018.08.011
59.
QuéméneurM, HamelinJ, BarakatA, et al.Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures. Int J Hydrog Energy, 2012; 37(4):3150–3159; doi: 10.1016/j.ijhydene.2011.11.033
60.
MillerGL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 1959; 31(3):426–428; doi: 10.1021/ac60147a030
61.
WangJ, YinY. Principle and application of different pretreatment methods for enriching hydrogen-producing bacteria from mixed cultures. Int J Hydrog Energy, 2017; 42(8):4804–4823; doi: 10.1016/j.ijhydene.2017.01.135
62.
AbubackarHN, KeskinT, ArslanK, et al.Effects of size and autoclavation of fruit and vegetable wastes on biohydrogen production by dark dry anaerobic fermentation under mesophilic condition. Int J Hydrog Energy, 2019; 44(33):17767–17780; doi: 10.1016/j.ijhydene.2019.05.106
63.
HuiY, WangM, GuoS, et al.Comprehensive review of development and applications of hydrogen energy technologies in China for carbon neutrality: Technology advances and challenges. Energy Convers Manag, 2024; 315:118776; doi: 10.1016/j.enconman.2024.118776
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
