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Concept: Biohydrogen


Biohydrogen production (BHP) can be achieved by direct or indirect biophotolysis, photo-fermentation and dark fermentation, whereof only the latter does not require the input of light energy. Our motivation to compile this review was to quantify and comprehensively report strains and process performance of dark fermentative BHP. This review summarizes the work done on pure and defined co-culture dark fermentative BHP since the year 1901. Qualitative growth characteristics and quantitative normalized results of H2 production for more than 2000 conditions are presented in a normalized and therefore comparable format to the scientific community.Statistically based evidence shows that thermophilic strains comprise high substrate conversion efficiency, but mesophilic strains achieve high volumetric productivity. Moreover, microbes of Thermoanaerobacterales (Family III) have to be preferred when aiming to achieve high substrate conversion efficiency in comparison to the families Clostridiaceae and Enterobacteriaceae. The limited number of results available on dark fermentative BHP from fed-batch cultivations indicates the yet underestimated potential of this bioprocessing application. A Design of Experiments strategy should be preferred for efficient bioprocess development and optimization of BHP aiming at improving medium, cultivation conditions and revealing inhibitory effects. This will enable comparing and optimizing strains and processes independent of initial conditions and scale.

Concepts: Anaerobic digestion, Motivation, Fermentation, Fermentative hydrogen production, Dark fermentation, Biohydrogen, Photofermentation


Apart from being applied as an energy carrier, hydrogen is in increasing demand as a commodity. Currently, the majority of hydrogen (H2) is produced from fossil fuels, but from an environmental perspective, sustainable H2 production should be considered. One of the possible ways of hydrogen production is through fermentation, in particular, at elevated temperature, i.e. thermophilic biohydrogen production. This short review recapitulates the current status in thermophilic biohydrogen production through fermentation of commercially viable substrates produced from readily available renewable resources, such as agricultural residues. The route to commercially viable biohydrogen production is a multidisciplinary enterprise. Microbiological studies have pointed out certain desirable physiological characteristics in H2-producing microorganisms. More process-oriented research has identified best applicable reactor types and cultivation conditions. Techno-economic and life cycle analyses have identified key process bottlenecks with respect to economic feasibility and its environmental impact. The review has further identified current limitations and gaps in the knowledge, and also deliberates directions for future research and development of thermophilic biohydrogen production.

Concepts: Agriculture, Hydrogen, Research, Anaerobic digestion, Fermentative hydrogen production, Hydrogen production, Dark fermentation, Biohydrogen


Hythane (H2+CH4) has attracted growing attention due to its versatile advantages as, for instance vehicle fuel. Biohythane consisting of biohydrogen and biomethane via two-stage fermentation is a potential high-value solution for the valorization of waste biomass resources and probably an alternative to the fossil based hythane. However, the significance and application potential of biohythane have not yet been fully recognized. This review focuses on the progress of biohydrogen and subsequent biomethane fermentation in terms of substrate, microbial consortium, reactor configuration, as well as the H2/CH4 ratio from the perspective of the feasibility of biohythane production in the past ten years. The current paper also covers how controls of the microbial consortium and bioprocess, system integration influence the biohythane productivity. Challenges and perspectives on biohythane technology will finally be addressed. This review provides a state-of-the-art technological insight into biohythane production by two-stage dark fermentation from biomass.

Concepts: Petroleum, Anaerobic digestion, Technology, Biofuel, Fermentative hydrogen production, Dark fermentation, Biohydrogen, Photofermentation


Silver nanoparticles were added into anaerobic batch reactors to enhance acidogenesis and fermentative hydrogen production simultaneously. The effects of silver nanoparticles concentration (0-200nmolL(-1)) and inorganic nitrogen concentration (0-4.125gL(-1)) on cell growth and hydrogen production were investigated using glucose-fed mixed bacteria dominated by Clostridium butyricum. The tests with silver nanoparticles exhibited much higher H2 yields than the blank, and the maximum hydrogen yield (2.48mol/molglucose) was obtained at the silver concentration of 20nmolL(-1). Presence of silver nanoparticles reduced the yield of ethanol, but increased the yield of acetic acid. The high silver nanoparticles had higher cell biomass production rate. Further study using the alkaline pretreated culture as inoculum was carried out to verify the positive effect of silver nanoparticles on H2 production. Results demonstrated that silver nanoparticles could not only increase the hydrogen yield, but reduce the lag phase for hydrogen production simultaneously.

Concepts: Oxygen, Hydrogen, Ethanol, Base, Fermentative hydrogen production, Hydrogen production, Dark fermentation, Biohydrogen


Cyanobacteria provide all components for sunlight driven biohydrogen production. Their bidirectional NiFe-hydrogenase is resistant against low levels of oxygen with a preference for hydrogen evolution. However, until now it was unclear if its catalytic efficiency can keep pace with the photosynthetic electron transfer rate. We identified NikKLMQO (sll0381-sll0385) as a nickel transporter, which is required for hydrogen production. ICP-MS measurements were used to quantify hydrogenase molecules per cell. We found 400 to 2000 hydrogenase molecules per cell depending on the conditions. In-vivo turnover frequencies of the enzyme ranged from 62 H2/s in the wild type to 120 H2/s in a mutant during photohydrogen production. These frequencies are above maximum in-vivo photosynthetic electron transfer rates of 47 e-/s (equivalent to 24 H2/s). They are also above those of existing in-vitro systems working with unlimited electron supply and show that in-vivo photohydrogen production is limited by electron delivery to the enzyme.

Concepts: Cyanobacteria, Oxygen, Bacteria, Hydrogen, Redox, Electrolysis, Hydrogen production, Biohydrogen


This study assessed the impact of swine manure (SM) dilution ratio on the microalgal biomass cultivation and further tested for biohydrogen production efficiency from the mixed microalgal biomass. At first, various solid/liquid (S/L) ratio of the SM ranged from 2.5 to 10 g/L was prepared as a nutrient medium for the algal biomass cultivation without addition of the external nutrient sources over a period of 18 d. The peak biomass concentration of 2.57 ± 0.03 g/L was obtained under the initial S/L loading rates of 5 g/L. Further, the cultivated biomass was subjected to two-step (ultrasonication + enzymatic) pretreatment and evaluated for biohydrogen production potential. Results showed that the variable amount of hydrogen production was observed with different S/L ratio of the SM. The peak hydrogen yield of 116 ± 6 mL/g TSaddedwas observed at the 5 g/L grown SM mixed algal biomass.

Concepts: Agriculture, Hydrogen, Anaerobic digestion, Biofuel, Hydrogen production, Dark fermentation, Biohydrogen, Cultivation


Extensive effort is being made to explore renewable energy in replacing fossil fuels. Biohydrogen is a promising future fuel because of its clean and high energy content. A challenging issue in establishing hydrogen economy is sustainability. Biohydrogen has the potential for renewable biofuel, and could replace current hydrogen production through fossil fuel thermo-chemical processes. A promising source of biohydrogen is conversion from algal biomass, which is abundant, clean and renewable. Unlike other well-developed biofuels such as bioethanol and biodiesel, production of hydrogen from algal biomass is still in the early stage of development. There are a variety of technologies for algal hydrogen production, and some laboratory- and pilot-scale systems have demonstrated a good potential for full-scale implementation. This work presents an elucidation on development in biohydrogen encompassing biological pathways, bioreactor designs and operation and techno-economic evaluation. Challenges and prospects of biohydrogen production are also outlined.

Concepts: Hydrogen, Petroleum, Biofuel, Fossil fuel, Renewable energy, Coal, Internal combustion engine, Biohydrogen


Microalgae are simple chlorophyll containing organisms, they have high photosynthetic efficiency and can synthesize and accumulate large quantities of carbohydrate biomass. They can be cultivated in fresh water, seawater and wastewater. They have been used as feedstock for producing biodiesel, bioethanol and biogas. The production of these biofuels can be integrated with CO2mitigation, wastewater treatment, and the production of high-value chemicals. Biohydrogen from microalgae is renewable. Microalgae have several advantages compared to terrestrial plants, such as higher growth rate with superior CO2fixation capacity; they do not need arable land to grow; they do not contain lignin. In this review, the biology of microalgae and the chemical composition of microalgae were briefly introduced, the advantages and disadvantages of hydrogen production from microalgae were discussed, and the pretreatment of microalgal biomass and the fermentative hydrogen production from microalgal biomass pretreated by different methods (including physical, chemical, biological and combined methods) were summarized and evaluated. For the production of biohydrogen from microalgae, the economic feasibility remains the most important aspect to consider. Several technological and economic issues must be addressed to achieve success on a commercial scale.

Concepts: Photosynthesis, Agriculture, Water, Anaerobic digestion, Biofuel, Fermentative hydrogen production, Hydrogen production, Biohydrogen


The generation of biohydrogen as source of biofuel/bioenergy from the wide variety of biomass has gathered a substantial quantum of research efforts in several aspects. One of the major thrusts in this field has been the pursuit of technically sound and effective methods and/or approaches towards significant improvement in the bioconversion efficiency and enhanced biohydrogen yields. In this perspective, the present contribution showcases the views formulated based on the latest advances reported in dark fermentative biohydrogen production (DHFP), which is considered as the most feasible route for commercialization of biohydrogen. The potential prospects and future research avenues are also presented.

Concepts: Present, Hydrogen, Future, Anaerobic digestion, Fermentative hydrogen production, Hydrogen production, Dark fermentation, Biohydrogen


Pilot tests of sequential dark and photo fermentation H2 production were for the first time conducted in a 11 m3 reactor (3 m3 for dark and 8 m3 for photo compartments). A combined solar and light-emitting diode illumination system and a thermal controlling system was installed and tested. With dark fermentation unit maintained at pH 4.5 and 35 °C and photo fermentation unit at pH 7.0 and 30 °C, the overall biogas production rate using hydrolyzed corn stover as substrate reached 87.8 ± 3.8 m3/d with 68% H2 content, contributed by dark unit at 7.5 m3-H2/m3-d and by photo unit at 4.7 m3/m3-d. Large variation was noted for H2 production rate in different compartments of the tested units, revealing the adverse effects of poor mixing, washout, and other inhomogeneity associated with large reactor operations.

Concepts: Enzyme, Fermentation, Microbial fuel cell, Fermentative hydrogen production, Hydrogen production, Dark fermentation, Biohydrogen, Photofermentation