Enhancement of fermentative hydrogen/ethanol production from cellulose using mixed anaerobic cultures

Batch tests were conducted to evaluate the enhancement of hydrogen/ethanol (EtOH) productivity using cow dung microflora to ferment α-cellulose and saccharification products (glucose and xylose). Hydrogen/ethanol production was evaluated based on hydrogen/ethanol yields (HY/EY) under 55 °C at various initial pH conditions (5.5–9.0). Our test results indicate that cow dung sludge is a good mixed natural-microflora seed source for producing biohydrogen/ethanol from cellulose and xylose. The heat-pretreatment, commonly used to produce hydrogen more efficiently from hexose, applied to mixed anaerobic cultures did not help cow dung culture convert cellulose and xylose into hydrogen/ethanol. Instead of heat-pretreatment, the mixed culture received enrichments cultivated at 55 °C for 4 days. Positive results were observed: hydrogen/ethanol production from fermenting cellulose and xylose was effectively enhanced at increases of 4.8 (ethanol) to 8 (hydrogen) and 2.4 (ethanol) to 15.6 (hydrogen) folds, respectively. In which, the ethanol concentration produced from xylose reached 4–4.4 g/L, an output comparable to that of using heat-treated sewage sludge and better than that (1.25–3 g/L) using pure cultures. Our test results show that for the enriched cultures the initial cultivation pH can affect hydrogen/ethanol production including HY, EY and liquid fermentation product concentration and distribution. These results were also concurred using a denaturing gradient gel electrophoresis analysis saying that both cultivation pH and substrate can affect the enriched cow dung culture microbial communities. The enriched cow dung culture had an optimal initial cultivation pH range of 7.6–8.0 with peak HY/EY values of 2.8 mmol-H₂/g-cellulose, 5.8 mmol-EtOH/g-cellulose, 0.3 mol-H₂/mol-xylose and 1 mol-EtOH/mol-xylose. However, a pH change of 0.5 units from the optimal values reduced hydrogen/ethanol production efficiency by 20%. Strategies based on the experimental results for optimal hydrogen/ethanol production from cellulose and xylose using cow dung microflora are proposed.     

Optimization of hydrogen production in a granule-based UASB reactor

Hydrogen production from sucrose in a granule-based upflow anaerobic sludge blanket (UASB) reactor was optimized through employing response surface methodology (RSM) with a central composite design in this study. The individual and interactive effects of influent sucrose concentration (S_in) and hydraulic retention time (HRT) on anaerobic hydrogen production were elucidated. Experimental results show that a maximum hydrogen yield of 1.62 mol-H₂/mol-hexose was obtained under the optimum conditions of S_in 14.5 g/L and an HRT 16.4 h. The hydrogen yield was individually dependent on S_in and HRT, while their interactive effect on the hydrogen yield was not significant. Throughout the experiments the hydrogen content fluctuated between 25.9% and 50.0%, but free of methane. Ethanol, acetate and butyrate were the main aqueous products and their yields all correlated well with S_in and HRT, indicating a mixed-type fermentation in this UASB reactor.     

Hydrogen and ethanol fermentation of various carbon sources by immobilized Escherichia coli (XL1-Blue)

In this study, hydrogen and ethanol production by a facultative anaerobic bacterium Escherichia coli XL1-Blue immobilized in calcium-alginate beads have been investigated. Batch fermentations were carried out at mesophilic temperature (35 °C) and an initial cultivation pH of 6.5. Firstly, the influence of biomass concentration in terms of dry cell weight (expressed in g DCW/L, range 0.2–1.0) was investigated using fructose (5 g/L) as a carbon source. The peak hydrogen yield (HY) of 1.17 mol-H₂/mol-fructose_utilized was obtained at an initial cell concentration of 0.4 g DCW/L. The hydrogen production potential of other simple carbon sources (glucose and xylose) was evaluated at this optimized cell concentration and peak HY values were attained as 0.96 mol-H₂/mol-glucose_utilized and 0.69 mol-H₂/mol-xylose_utilized, respectively. In addition, utilization of the beverage wastewater (BWW) showed the peak cumulative hydrogen production and ethanol concentration of 120 mL and 5.65 g/L, attained at the substrate concentration of 20 g_{(glucose equivalent)}/L. However, peak HY (1.65 mol-H₂/mol-_{glucose eqivalent utilized}) was observed at low substrate concentration of 5 g_{(glucose equivalent)}/L. The percentage of sugar utilization of BWW was ranged between 80 and 96.     

Improving hydrogen production from cassava starch by combination of dark and photo fermentation

The combination of dark and photo fermentation was studied with cassava starch as the substrate to increase the hydrogen yield and alleviate the environmental pollution. The different raw cassava starch concentrations of 10–25 g/l give different hydrogen yields in the dark fermentation inoculated with the mixed hydrogen-producing bacteria derived from the preheated activated sludge. The maximum hydrogen yield (HY) of 240.4 ml H₂/g starch is obtained at the starch concentration of 10 g/l and the maximum hydrogen production rate (HPR) of 84.4 ml H₂/l/h is obtained at the starch concentration of 25 g/l. When the cassava starch, which is gelatinized by heating or hydrolyzed with α-amylase and glucoamylase, is used as the substrate to produce hydrogen, the maximum HY respectively increases to 258.5 and 276.1 ml H₂/g starch, and the maximum HPR respectively increases to 172 and 262.4 ml H₂/l/h. Meanwhile, the lag time (λ) for hydrogen production decreases from 11 h to 8 h and 5 h respectively, and the fermentation duration decreases from 75–110 h to 44–68 h. The metabolite byproducts in the dark fermentation, which are mainly acetate and butyrate, are reused as the substrates in the photo fermentation inoculated with the Rhodopseudomonas palustris bacteria. The maximum HY and HPR are respectively 131.9 ml H₂/g starch and 16.4 ml H₂/l/h in the photo fermentation, and the highest utilization ratios of acetate and butyrate are respectively 89.3% and 98.5%. The maximum HY dramatically increases from 240.4 ml H₂/g starch only in the dark fermentation to 402.3 ml H₂/g starch in the combined dark and photo fermentation, while the energy conversion efficiency increases from 17.5–18.6% to 26.4–27.1% if only the heat value of cassava starch is considered as the input energy. When the input light energy in the photo fermentation is also taken into account, the whole energy conversion efficiency is 4.46–6.04%.     

Continuous biohydrogen production from liquid swine manure supplemented with glucose using an anaerobic sequencing batch reactor

Liquid swine manure supplemented with glucose (10 g/L) was used as substrate for hydrogen production using an anaerobic sequencing batch reactor at 37 ± 1 °C and pH 5.0 under different hydraulic retention times (HRTs). Decreasing HRT from 24 to 8 h caused an increasing hydrogen production rate from 0.05 to 0.15 L/h/L. Production rates of both total biogas and hydrogen were linearly correlated to HRT with R² being 0.993 and 0.997, respectively. The hydrogen yield ranged between 1.18 and 1.63 mol-H₂/mol glucose and the 12 h HRT was preferred for high production rate and efficient yield. For all the five HRTs examined, the glucose utilization efficiency was over 98%. The biogas mainly consisted of carbon dioxide and hydrogen (up to 43%) with no methane detected throughout the experiment. Ethanol and organic acids were the major aqueous metabolites produced during fermentation, with acetic acid accounting for 56–58%. The hydrogen yield was found to be related to the acetate/butyrate ratio.     

Effects of hydraulic retention time on anaerobic hydrogenation performance and microbial ecology of bioreactors fed with glucose–peptone and starch–peptone

This study evaluated anaerobic hydrogenation performance and microbial ecology in bioreactors operated at different hydraulic retention time (HRT) conditions and fed with glucose–peptone (GP) and starch–peptone (SP). The maximum hydrogen production rates for GP- and SP-fed bioreactors were found to be 1247 and 412 mmol-H₂/L/d at HRT of 2 and 3 h, respectively. At HRT > 8 h, hydrogen consumption due to peptone fermentation could occur and thus reduced hydrogen yield from carbohydrate fermentation. Results of cloning/sequencing and denaturant gradient gel electrophoresis (DGGE) indicated that Clostridium sporogenes and Clostridium celerecrescens were dominant hydrogen-producing bacteria in the GP-fed bioreactor, presumably due to their capability on protein hydrolysis. In the SP-fed bioreactor, Lactobacillus plantarum, Propionispira arboris, and Clostridium butyricum were found to be dominant populations, but the presence of P. arboris at HRT > 3 h might be responsible for a lower hydrogen yield from starch fermentation. As a result, optimizing HRT operation for bioreactors was considered an important asset in order to minimize hydrogen-consuming activities and thus maximize net hydrogen production. The limitation of simple parameters such as butyrate to acetate ratio (B/A ratio) in predicting hydrogen production was recognized in this study for bioreactors fed with multiple substrates. It is suggested that microbial ecology analysis, in addition to chemical analysis, should be performed when complex substrates and mixed cultures are used in hydrogen-producing bioreactors.     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.     

Seed inocula for biohydrogen production from biodiesel solid residues

This study aimed to optimize the hydrogen production from various seed sludges (two kinds of sewage sludges (S1, S2), cow dung (S3), granular sludge (S4) and effluent from condensed soluble molasses H₂ fermenter (S5)) and enhancement of hydrogen production via heat treatment for substrate and seed sludge by using the solid residues of biodiesel production (BDSR). Two batch assay tests were operated at a biodiesel solid residue concentration of 10 g/L, temperature of 55 °C and an initial cultivation pH of 8. The results showed that the peak hydrogen yield (HY) of 94.6 mL H₂/g volatile solid (VS) (4.1 mmolH₂/g VS) was obtained from S1 when substrate and seed sludge were both heat treated at 100 °C for 1 h. However, the peak hydrogen production rate (HPR) and specific hydrogen production rate (SHPR) of 1.48 L H₂/L-d and 0.30 L H₂/g VSS-d were obtained from S2 without any treatment. The heat treatment was found to increase the HY in both the cases of sewage sludges S1 and S2.The HY of 89.5 mL H₂/g VS (without treatment) was increased to 94.6 mL H₂/g VS and 82.6 mL H₂/g VS (without treatment) was increased to 85.7 mL H₂/g VS for S1 and S2. The soluble metabolic product (SMP) analysis showed that the fermentation followed mainly acetate–butyrate pathway with considerable production of ethanol. The total bioenergy production was calculated as 2.8 and 2.9 kJ/g VS for favorable hydrogen and ethanol production, respectively. The BDSR could be used as feedstock for dark fermentative hydrogen production.     

Isolation,characterization and optimization of photo-hydrogen production conditions by newly isolated Rhodobacter sphaeroides KKU-PS5

A purple non-sulfur (PNS) photosynthetic bacterium was isolated from an upflow anaerobic sludge blanket (UASB) bioreactor for methane production and was identified as Rhodobacter sphaeroides KKU-PS5 (GenBank Accession no. KC481702) by 16s rRNA gene sequence analysis. Strain KKU-PS5 could utilize glucose, xylose, fructose, arabinose, malate, succinate, acetate, butyrate, lactate and D-mannitol for growth and hydrogen production. Malate was a preferred carbon source while glutamate and Aji-L (waste from the process of crystallizing monosodium glutamate) were the preferred nitrogen sources. The ability to utilize Aji-L as a low-cost nitrogen supplement for photo-biohydrogen production by the strain KKU-PS5 is considered as its desirable characteristic. The threshold substrate concentration of malate was 30 mmol/L. The optimum conditions for hydrogen production from malate were an initial pH of 7.0, FeSO₄ concentration of 4 mg/L, temperature of 30 °C and light intensity of 6 klux. Under the optimum conditions, the maximum hydrogen production, the hydrogen yield (HY) and the hydrogen production rate (HPR) of 1330 mL-H₂/L, 3.80 mol-H₂/mol-malate, and 11.08 mL-H₂/L h, respectively, were achieved. Hydrogen production under a dark/light cycle led to a decreased HY and HPR in comparison to continuous illumination.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 was investigated in an up-flow anaerobic sludge blanket (UASB) reactor. The reactor was operated under non-sterile conditions at 40^○C and initial pH 8.0 at different hydraulic retention times (HRTs) (2–12 h) and glycerol concentrations (10–30 g/L). Decreasing the HRT led to an increase in hydrogen production rate (HPR) and hydrogen yield (HY). The highest HPR of 242.15 mmol H₂/L/d and HY of 44.27 mmol H₂/g glycerol consumed were achieved at 4 h HRT and glycerol concentrations of 30 and 10 g/L, respectively. The main soluble metabolite was 1,3-propanediol, which implies that Klebsiella sp. was dominant among other microorganisms. Fluorescence in situ hybridization (FISH) revealed that the microbial community was dominated by Klebsiella sp. with 56.96, 59.45, and 63.47% of total DAPI binding cells, at glycerol concentrations of 10, 20, and 30 g/L, respectively.     

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.     

Continuous biogenic hydrogen production from dilute acid pretreated algal hydrolysate using hybrid immobilized mixed consortia

This study investigated the bioconversion of dilute acid (2% H₂SO₄) pretreated red algae (Gelidium amansii) hydrolysate into H₂ by anaerobic fermentation in a continuous stirred tank reactor under mesophilic conditions using hybrid immobilized cells as microbial catalyst. Two different hydraulic retention times (HRT) of 24 h and 16 h with a feed concentration of 15 g/L hexose equivalent have been investigated over 85 days of operation to evaluate H₂ production performance and stability of the continuous system. The highest hydrogen production rate (HPR) and hydrogen yield (HY) of 2.7 L/L/d and 1.3 mol/mol substrate hexose_added was achieved at 24 h HRT, while further operation at 16 h HRT led to a significant drop in the hydrogen production with a HPR and HY values of 1.8 L/L/d and 0.7 mol/mol substrate hexose_added, respectively. The bacterial community analysis characterized by 454 pyrosequencing revealed that the changes in HRT significantly influence the composition of the dominant microflora. At longer HRT (24 h), the phyla Firmicutes was abundant over 98%, whereas at shorter HRT (16 h), Proteobacteria being the dominant populations with 84%. These outcomes suggested that controlling appropriate HRT is prerequisite for efficient hydrogen production.     

Simultaneous biohydrogen production and starch wastewater treatment in an acidogenic expanded granular sludge bed reactor by mixed culture for long-term operation

The biofilm-based expanded granular sludge bed (EGSB) reactor was developed to treat starch-containing wastewater and simultaneously recovery hydrogen by mixed microbial culture. Granular activated carbon (GAC) was used as the support media. Operating at the temperature of 30 °C for over 400 days (data not shown), the EGSB reactor presented high efficiency in hydrogen production and COD removal ability. The maximum hydrogen production rate (HPR) was found to be 1.64 L/L.d under the organic loading rate (OLR) of 1.0 g-starch/L.d, pH of 4.42 and HRT of 4 h. The hydrogen yield (HY) peaked at 0.11 L/g-COD, under the OLR of 0.5 g-starch/L.d, pH of 3.95 and HRT of 8 h. Hydrogen volume content was estimated to be 35–65% of the total biogas. The average COD removal rate was 31.1% under the OLR of 0.125 g-starch/L.d and HRT of 24 h. The main dissolved fermentation products were ethanol, acetate and butyrate. The average attached biofilm concentration was estimated to be 8.26 g/L, which favored hydrogen production and COD removal. It is speculated that the low pH operation in the present system would contribute significantly to lower the cost of alkaline amount required for pH control in the continuous operation, especially in the scale-up biohydrogen producing system. A model, built on the back propagation neural network (BPNN) theory and linear regression techniques, was developed for the simulation of EGSB system performance in the biodegradation of starch synthesis-based wastewater and simultaneous hydrogen production. The model well fitted the laboratory data, and could well simulate the removal of COD and the production of hydrogen in the EGSB reactor.     

Biohydrogen production in anaerobic fluidized bed reactors: Effect of support material and hydraulic retention time

This study evaluated two different support materials (polystyrene and expanded clay) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBRs contained either polystyrene (R1) or expanded clay (R2) as support materials were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C and a pH of approximately 5.5. The AFBRs were operated with a range of hydraulic retention times (HRTs) between 1 and 8 h. For R1 with an HRT of 2 h, the maximum hydrogen yield (HY) was 1.90 mol H₂ mol⁻¹ glucose, with 0.805 mg of biomass (as total volatile solids, or TVS) attached to each g of polystyrene. For R2 operated at an HRT of 2 h, the maximum HY was 2.59 mol H₂ mol⁻¹ glucose, with 1.100 mg of attached biomass (as TVS) g⁻¹ expanded clay. The highest hydrogen production rates (HPR) were 0.95 and 1.21 L h⁻¹ L⁻¹ for R1 and R2, respectively, using an HRT of 1 h. The H₂ content increased from 16–47% for R1 and from 22–51% for R2. No methane was detected in the biogas produced throughout the period of AFBR operation. These results show that the values of HY, HPR, H₂ content, and g of attached biomass g⁻¹ support material were all higher for AFBRs containing expanded clay than for reactors containing polystyrene.     

Non-sterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis

Bio-hydrogen production from food waste by anaerobic mixed cultures was conducted in a continuous stirred tank reactor (CSTR). The hydraulic retention time (HRT) was optimized in order to maximize hydrogen yield (HY) and hydrogen production rate (HPR). The maximum hydrogen content (38.6%), HPR (379 mL H₂/L. d) and HY (261 mL H₂/g-VS_added) were achieved at the optimum HRT of 60 h. The major soluble metabolite products were butyric and acetic acids which indicated a butyrate-acetate type fermentation. Operation of CSTR at HRT 60 h could select hydrogen producing bacteria and eliminate lactic acid bacteria and acetogenic bacteria. The microbial community analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the predominant hydrogen producer was Clostridium sp.     

Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage

This study investigated the effects of seed sludges, alkalinity and HRT on the thermophilic fermentative hydrogen production from cassava stillage. Five different kinds of sludges were used as inocula without any pretreatment. Though batch experiments showed that mesophilic anaerobic sludge was the best inoculum, the hydrogen yields with different seed sludges were quite similar in continuous experiments in the range of 82.9–92.3 ml H₂/gVS without significant differences which could be attributed to the establishment of Uncultured Thermoanaerobacteriaceae bacterium-dominant microbial communities in all reactors. It is indicated that results obtained from batch experiments are not consistent with those from continuous experiments and all the tested seed sludges are good sources for continuous thermophilic hydrogen production from cassava stillage. The influent alkalinity of 6 g NaHCO₃/L and HRT 24 h were optimal for hydrogen production with hydrogen yield of 76 ml H₂/gVS and hydrogen production rate of 3215 ml H₂/L/d. Butyrate was the predominant metabolite in all experiments. With the increase in alkalinity of more than 6 g/L, the concentration of VFA/ethanol increased while hydrogen yield decreased due to the higher concentration of acetate and propionate. The decrease in HRT resulted in the higher hydrogen production rate but lower hydrogen yield. Variation of hydrogen yields were quite correlated with butyrate/acetate (B/A) ratio with different influent alkalinities, however, butyrate was important parameter to justify the hydrogen yields with various HRTs.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.     

Hydrogen fermentation of different galactose–glucose compositions during various hydraulic retention times (HRTs)

This study investigated the effects of sugar composition and hydraulic retention time (HRT) on continuous hydrogen fermentation. Continuously-stirred tank reactors (CSTRs) were inoculated with heat-treated digester sludge and fed with 15 g/L of glucose, galactose and galactose: glucose mixture (8:2 ratio-simulating the hydrolysate composition of macroalgae) at HRTs of 6–24 h. Peak hydrogen production rate (HPR) and hydrogen yield (HY) of 4.49 L/L/d and 1.62 mol/mol glucose_added were attained while using glucose as feedstock at HRTs of 6 and 18 h, respectively. Meanwhile, galactose provided a peak HPR and HY of 2.35 L/L/d and 1.00 mol/mol galactose_added during the HRTs of 12 and 24 h, respectively. In case of mixed sugars (galactose 8: glucose 2) the production performances were inferior to the sole sugar conditions due to the low substrate utilization of less than 65%, which showed a maximal HPR and HY of 2.75 L/L/d and 0.48 mol/mol carbohydrate_added at the HRTs of 6 and 18 h, respectively.