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In lignocellulose-to-hydrogen bioconversion, reducing the concentration of chemical agents in pretreatment is of great interest. In this study, rice straw (RS) pretreated at reduced NaOH and urea (NU) concentrations was evaluated. Results showed that the composition of RS exhibited excellent pretreatment performance at a reduced concentration of NU. When the concentration of NaOH was decreased to 3 wt% in combination with 6 wt% urea, the lignin was reduced by 59.52% with a cellulose and hemicellulose loss of less than 17%. Moreover, extending the pretreatment time at a low concentration of NU could effectively promote the biodegradability of RS. Upon fermentation by Thermoanaerobacterium thermosaccharolyticum M18 for H2 production, the H2 production increased up to 213.06 mL/g with a substrate treated by 3 wt% NaOH/6 wt% urea at low solid loading for 15 d, which was 16.31% higher than the counterpart subjected to a 7 wt% NaOH/12 wt% urea pretreatment. The present results suggest the NU pretreatment can be carried out at low concentrations to improve the conversion of RS into bio-H2 production.

To enhance photo-fermentative hydrogen production (PFHP), biofilm reactor (BR) was employed as an ideal strategy with optimization on key factors of acetate concentration and carriers in this work. Optimal conditions for hydrogen production were acetate concentration of 4 g/L and carriers (silicon sheet) of 10 cm × 1 cm at amount of 1 piece. Biofilm formed on silicon sheet strongly improved hydrogen production compared with control reactor (CR). Cumulative hydrogen volume was enhanced about 20% from 2850 ± 130 mL/L of CR to 3349 ± 153 mL/L of BR and hydrogen yield was increased 20% from 2.61 ± 0.13 mol H2/mol acetate of CR to 3.06 ± 0.15 mol H2/mol acetate of BR at 4 g/L acetate. Protein and deoxyribonucleic acid (DNA) were important components to form the biofilm and they occupied 90% of extracellular polymeric substances (EPS). In particular, DNA, nearly 50% content of EPS, likely indicated a substantial contribution to biofilm formation and bacterial communication. Moreover, it suggested biofilm could regulate free cells to decline EPS secretion for improved hydrogen production. This work indicates BR could be a promising and economic strategy to enhance hydrogen production by photo-fermentation.

A photo-fermentative bacterium for hydrogen production, Rhodopseudomonas sp. nov. strain A7, was isolated from sludge in a running bioreactor. The strain A7 has a flagellum and capsule. The hydrogen production performance exhibited by the strain A7 under several key parameters was investigated in batch culture. The maximum hydrogen yield achieved was160 ml-H2/60 ml-vessel using 60 mmol/l acetate, 15 mmol/l glutamate at a pH 7.0 and an inoculum time of 14 h. This study determined that the strain A7 can utilize glucose and, especially, fructose and sucrose for cell growth and hydrogen production. The hydrogen yield obtained with these substrates reached 33, 30 and 43 ml-H2/60 ml-vessel, respectively. These results imply that the strain A7 possesses a wide range of substrate utilization capabilities and is a potential candidate for photo-hydrogen production.

Photo-fermentative H2 production by mixed bacteria and pure bacterium Rhodopseudomonas faecalis RLD-53 using single and mixed substrate as carbon source was investigated in batch culture. Experimental results showed that 60 mmol/L acetate was the optimal concentration for mixed bacterial H 2 production and maximum cumulative H2 volume was 2468 ± 123 mL H2/L-culture. It was also found that propionate or butyrate was a key factor for enhancing H2 production in mixed substrate system. Photo-H2 production can be greatly promoted when proper concentration of propionate and butyrate were added into acetate medium as mixed substrate and a higher H2 yield of 2931 ± 146 mL H2/L-culture was obtained. In addition, it was worth noting that when the strain RLD-53 was added into mixed bacteria with different concentration ratios, H2 yield did not yet increase. Interestingly, H2 production capacity gradually decreased with ratio of strain RLD-53 to mixed bacteria from 8:0 to 4:4, and then gradually increased from 4:4 to 0:8. This implied that the competition relationship between strain RLD-53 and mixed bacteria in substrate utilization strongly influenced their H2 production.

The applications of photo-fermentative bacteria (PFB) for continuous hydrogen production are generally subjected to a serious biomass washout from photobioreactor, resulting from poor flocculation of PFB. In this study, through reducing the absolute ζ-potentials of PFB, Ca 2+ greatly decreased total interaction energy barrier of PFB based on DLVO theory, thus promoted the bioflocculation of Rhodopseudomonas faecalis RLD-53. Average floc size of PFB increased with the Ca 2+ concentration, and reached maximum 30.07 μm at 4 mmol/l. Consequently, biomass retention capacity of photobioreactor significantly enhanced after 30 min settling with half working volume discharge. In the continuous photo-fermentative sequencing batch reactor, compared with the free cell culture, bioflocculation reached a higher steady-state hydrogen production rate of 879 ml H 2 /l/d and hydrogen yield of 2.64 mol H 2 /mol acetate, respectively. Therefore, bioflocculation promoted by calcium ion was an effective strategy for retaining PFB in photobioreactor to produce hydrogen continuously.

In this work, the kinetic characterization of hydrogen production by the photofermentative bacteria Rhodopseudomonas faecalis RLD-53 was investigated at different growth phase. During entire fermentation, 89.30% of total biomass was accumulated in exponential growth phase, while hydrogen yield was only 1.82 mol H 2 /mol acetate at the expense of 51.25% substrate. In the stationary phase, biomass synthesis was minimal (7.51%), and 38.17% of the substrate was directly converted into hydrogen. As a result, hydrogen (59.19%) was mainly produced in stationary phase with highest hydrogen yield of 3.67 mol H 2 /mol acetate. Consequently, bacteria in stationary phase were most effective for hydrogen production. Based on these findings, a novel membrane photobioreactor was developed to retain bacteria during stationary phase in reactor through membrane separation. Maximum rate (32.82 ml/l/h) and yield (3.27 mol H 2 /mol acetate) of hydrogen production were achieved using membrane photobioreactor under the continuous operation. Therefore, using bacteria in stationary phase as hydrogen producer can offer considerable benefits for enhancing photo-hydrogen production.

A novel photo-fermentative sequencing batch reactor (PFSBR) process assisted by activated carbon fibers (ACFs) was used to continuously produce hydrogen gas by Rhodopseudomonas faecalis RLD-53. Feasibility of continuous hydrogen production in PFSBR operation at different hydraulic retention times (HRTs) (48, 96, 144 and 192 h) and influent acetate concentrations (20, 40, 60 and 80 mmol/l) was investigated. The rate and yield of hydrogen production increased with HRTs from 48 to 144 h, and then decreased with the HRTs from 144 to 192 h. Regulation of the proper influent acetate concentration (60 mmol/l) not only increased hydrogen production by PFSBR, but also maintained quality of the effluent with high substrate removal efficiency (97.70%). Free R . faecalis RLD-53 was adsorbed on the surface of ACFs, initially isolated cells, then monolayer, and finally mature biofilm with three dimensional multilayers structures. The PFSBR reached a maximum hydrogen yield (3.12 mol H 2 /mol acetate), and achieved a steady state when mature biofilm developed on ACFs. Therefore, photo-fermentative sequencing batch reactor was a promising process for continuous photo-fermentative hydrogen production.

This study investigated the effect of EDTA concentration in medium on the growth, hydrogen production and nitrogenase activity of Rhodopseudomonas faecalis RLD-53 by batch cultures. Experimental results indicated that bacterial growth and hydrogen production were strongly inhibited with EDTA concentration increasing to 0.6–0.7 g/L. However, the lag time of hydrogen production and the trends of biomass at EDTA concentration of 0–0.5 g/L were similar. The maximum cumulative hydrogen volume of 3325 ml H 2 /L culture, hydrogen production rate of 27.6 ml H 2 /L/h and hydrogen yield of 2.97 mol H 2 /mol acetate were obtained when EDTA concentration was at 0.3 g/L, and the maximum OD 660 attained 3.83. And the nitrogenase activity also reached a maximum value of 1331.9 μl-C 2 H 4 /h/mg dry weight in the medium containing Fe 2+ and EDTA. These results showed that a proper concentration of EDTA can promote the availability of iron, thereby further enhancing the activity of nitrogenase and photo-hydrogen production.

In this work, a novel bio-carrier was used to immobilize photo-fermentative bacteria for hydrogen production. The results showed that the bacterial immobilization and hydrogen production were strongly affected by particle size, amount of bio-carrier and light intensity. Controlling the proper size of bio-carrier not only prevented light shading effect from each other, but also made solid carriers better fluidized during operation. The scanning electronic microscopy revealed that the biofilm formed by photo-fermentative bacteria on the surface of bio-carrier enhanced hydrogen production. Because of bio-carrier fluidization, each immobilized bacterium could receive light energy and produce hydrogen. With the optimal particle size (2 × 2 mm), amount (3% weight to volume ratio) and light intensity (6000 lux), the maximum hydrogen yield of 3.24 mol H2/mol acetate and production rate of 36.06 ml/l/h were obtained in continuous operation stage. Bio-carrier was an effective solid carrier to immobilize photo-fermentative bacteria for improving hydrogen production.

Clostridium butyricum and Rhodopseudomonas faecalis RLD-53 were employed to produce hydrogen in mixed culture with glucose as sole substrate. Due to the great difference on growth rate and acid-resistant capacity between photo-fermentative bacteria and dark-fermentative bacteria, directly mixed culture of the two kinds of bacteria in different ratio was studied in this work. Hydrogen yield, volatile acids, pH and biomass in different periods were evaluated. Acetic acid and butyric acid produced by C. butyricum were dominant terminal fermentation products, and they were effective substrates for photo-fermentative bacteria. The cooperation was formed in a way like food chain. But compared to the production rate of volatile acids produced by C. butyricum , the utilization rate by photo-fermentative bacteria was far slower. The results demonstrated that the growth of photo-fermentative bacteria was limited when pH decreased sharply. The best ratio of C. butyricum to R. faecalis RLD-53 was 1:600. The maximum yield of hydrogen reached 122.4 ml-H 2 /vessel and hydrogen production rate was 0.5 ml-H 2 /ml-culture/day.

This study evaluated hydrogen production by co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53 with different control strategies. To enhance cooperation of dark and photo-fermentation bacteria during hydrogen production process, the glucose concentration, phosphate buffer concentration and initial pH were controlled at 6 g/l, 50 mmol/l and 7.5, respectively. The maximum yield and rate of hydrogen production were 3.10 mol H 2 /mol glucose and 17.2 mmol H 2 /l/h, respectively. Ethanol from E. harbinense B49 in acetate medium can enhance hydrogen production by R. faecalis RLD-53 except the ratio of ethanol to acetate ( R E/A ) among 0.8 to 1.0. Control of the proper phosphate buffer concentration (50 mmol/l) not only increased acetic acid production by E. harbinense B49, but also maintained stable pH of co-culture system. Therefore, the results showed that co-culture of E. harbinense B49 and immobilized R. faecalis RLD-53 was a promising way of converting glucose into hydrogen.

In this work, the effects of different parameters on co-culture hydrogen production using Clostridium Butyricum and immobilized Rhodopseudomonas faecalis RLD-53 were investigated. The maximum hydrogen yield of 4.134 mol H 2 /mol glucose was obtained using 6 g/l glucose, 50 mmol/l phosphate buffer, initial pH of 7.5, the ratio of dark to photo bacteria of 1:10 and light intensity of 8000 lux. The maximum hydrogen production rate was 33.85 ml H 2 /l/h. Phosphate buffer concentration was the most important parameter influencing hydrogen production in this co-culture. The ratio of acetate to butyrate increased from 0.74 to 1.82 in the soluble metabolites from C. butyricum with phosphate buffer concentration of 10–50 mmol/l. Experimental results could be of great significance for further pilot studies of co-culture hydrogen production.

In the present study, the effect of Ni2+ (0–10 μmol/l), Fe2+ (0–200 μmol/l) and Mg2+ (0–15 mmol/l) concentration on photo-hydrogen production from acetate was investigated by batch culture. Results showed that under a proper concentration range, Ni2+ was able to enhance the hydrogen production rate and the hydrogen yield; Fe2+ was able to increase the hydrogen yield, and hydrogen production rate was enhanced only when the culturing time was 24–72 h. Ni2+ and Fe2+ at a higher concentration inhibited cell growth. When Ni2+ and Fe2+ concentrations were 4 μmol/l and 80 μmol/l, respectively, maximal hydrogen yield of 2.87 and 2.78 mol H2/mol acetate was obtained when batch culturing at 35 °C with initial pH 7.0. Mg2+ did not significantly affect hydrogen production and hydrogen yield which maintained at about 2.45 mol H2/mol acetate, but it was favorable to cell growth.