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Concept: Dark fermentation


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


Feasibility of integrating Microbial electrolysis cell (MEC) process with dark-fermentation process for additional hydrogen recovery as well as substrate degradation was demonstrated in the present study. MEC was employed in order to utilize the residual organic fraction present in the acidogenic effluents of dark fermentation process as substrate for hydrogen production with input of small electric current. MEC was operated at volatile fatty acids (VFA) concentration of 3000mg/l under different poised potentials (0.2, 0.5, 0.6, 0.8 and 1.0V) using anaerobic consortia as biocatalyst. Maximum hydrogen production rate (HPR), cumulative hydrogen production (CHP) (0.53mmol/h and 3.6mmol), dehydrogenase activity (1.65μg/ml) and VFA utilization (49.8%) was recorded at 0.6V. Bio-electrochemical behavior of mixed consortia was evaluated using cyclic voltammetry and by Tafel slope analysis. Microbial diversity analysis using denaturing gradient gel electrophoresis confirmed the presence of γ-proteobacteria (50%), Bacilli (25%) and Clostridia (25%).

Concepts: Hydrogen, Electrochemistry, Electrolysis, Fermentation, Fermentative hydrogen production, Hydrogen production, Dark fermentation, Photofermentation


Dark fermentation is a bioprocess driven by anaerobic bacteria that can produce hydrogen (H2) from organic waste and wastewater. This review analyses a relevant number of recent studies that have investigated dark fermentative H2 production from wastewater using two different types of anaerobic biofilm reactors: anaerobic packed bed reactor (APBR) and anaerobic fluidized bed reactor (AFBR). The effect of various parameters, including temperature, pH, carrier material, inoculum pretreatment, hydraulic retention time, substrate type and concentration, on reactor performances was investigated by a critical discussion of the results published in the literature. Also, this review presents an in-depth study on the influence of the main operating parameters on the metabolic pathways. The aim of this review is to provide to researchers and practitioners in the field of H2 production key elements for the best operation of the reactors. Finally, some perspectives and technical challenges to improve H2 production were proposed.

Concepts: Enzyme, Fermentation, Environmental engineering, Fermentative hydrogen production, Hydrogen production, Dark fermentation, Photofermentation, Fluidized bed reactor


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


A new hydrogen-producing bacterium was isolated from the intestine of wild carp (Cyprinus carpio L.) of the Tarim River Basin. The isolate was identified as Klebsiella sp. based on 16S rDNA gene sequencing and examination of physiological and biochemical characteristics. The isolated strain, Klebsiella sp. WL1316, could effectively produce a high yield of hydrogen by using cotton stalk hydrolysate as substrate. The optimum fermentation conditions for hydrogen production were determined as follows: an initial sugar concentration of 40 g/L, a fermentation temperature of 37 °C and an initial pH value of 8.0. The scaled-up fermentation process was conducted in a 5-L fermenter using these parameters. Higher productivities with maximum daily hydrogen production of 937.0 ± 41.0 mL L-1 day-1, cumulative hydrogen production of 2908.5 ± 47.4 mL L-1, viable cell count of (20.2 ± 0.6) × 108 CFU mL-1and hydrogen yield of 1.44 ± 0.08 mol mol-1sugarconsumedwere obtained. The cumulative hydrogen production was predicted by the modified Gompertz equation with R2of 0.997, and values of Rmand P were 44.8 mL L-1 h-1and 3057.6 mL L-1, respectively. These results indicated that the strain Klebsiella sp. WL1316 resulted in a high hydrogen production rate (HPR) and good hydrogen production potential. Moreover, this strain exhibited higher values of maximum hydrogen yield and HPR than the reported pure cultures.

Concepts: Enzyme, Hydrogen, 16S ribosomal RNA, Fermentation, Fermentative hydrogen production, Dark fermentation, Tarim River, Tarim Basin


The present study aimed to increase the disintegration potential of marine macroalgae, (Ulva reticulata) through chemo mechanical pretreatment (CMP) in an energy efficient manner. By combining surfactant with disperser, the specific energy input was considerably reduced from 437.1 kJ/kg TS to 264.9 kJ/kg TS to achieve 10.7% liquefaction. A disperser rpm (10,000), pretreatment time (30 min) and tween 80 dosage (21.6 mg/L) were considered as an optimum for effective liquefaction of algal biomass. CMP was designated as an appropriate pretreatment resulting in a higher soluble organic release 1250 mg/L, respectively. Anaerobic fermentation results revealed that the volatile fatty acid (VFA) concentration was doubled (782 mg/L) in CMP when compared to mechanical pretreatment (MP) (345 mg/L). CMP pretreated algal biomass was considered as the suitable for biohydrogen production with highest H2 yield of about 63 mL H2/g COD than (MP) (45 mL H2/g COD) and control (10 mL H2/g COD).

Concepts: Carbon dioxide, Hydrogen, Omega-3 fatty acid, Thermodynamics, Anaerobic digestion, Biofuel, Algaculture, Dark fermentation


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