Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using a novel strategy — shaking and extra-light supplementation approach

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H₂ production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000–8000 lux, markedly higher than that for standing-culture (4000–5000 lux). Under shaking and elevated illumination (7000–8000 lux), the culture was effective in promoting photo-H₂ production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000–5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H₂/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H₂ production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H₂ production efficiency.     

Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710

Photofermentative hydrogen production is influenced by several parameters, including feed composition, pH levels, temperature and light intensity. In this study, experimental results obtained from batch cultures of Rhodobacter capsulatus DSM 1710 were analyzed to locate the maximum levels for the rate and yield of hydrogen production with respect to temperature and light intensity. For this purpose, a 3^k general full factorial design was employed, using temperatures of 20, 30 and 38 °C and light intensities of 100, 200 and 340 W/m². ANOVA results confirmed that these two parameters significantly affect hydrogen production. Surface and contour plots of the regression models revealed a maximum hydrogen production rate of 0.566 mmol H₂/L/h at 27.5 °C and 287 W/m² and a maximum hydrogen yield of 0.326 mol H₂/mol substrate at 26.8 °C and 285 W/m². Validation experiments at the calculated optima supported these findings.     

Repeated-batch production of hydrogen using Rhodobacter sphaeroides S10

Photofermentation of acid hydrolyzed oil palm empty fruit bunch is reported for hydrogen production in repeated-batch fermentations using the bacterium Rhodobacter sphaeroides S10. Photofermentations were carried out at 35 °C at an incident light level of 10 klux. At specified times, different specified volumes of the culture broth were removed and replaced with an equal volume of the fresh medium. The initial mixed carbon (glucose, xylose, acetic acid) content in the medium of the repeated-batch reactors was adjusted to 20 mM. The kinetics of hydrogen production were evaluated in repeated-batch fermentations carried out in various ways: different volume exchange levels, different switch times from batch to repeated-batch operation, and different cycle times.     

Fermentative hydrogen production by diverse microflora

In this study, hydrogen production with activated sludge, a diverse bacterial source has been investigated and compared to microflora from anaerobic digester sludge, which is less diverse. Batch experiments were conducted at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The hydrogen production yields with activated sludge at 37 °C and 55 °C were 0.56 and 1.32 mol H₂/mol glucose consumed, respectively. While with anaerobically digested sludge hydrogen yield was 2.18 mol H₂/mol glucose consumed at 37 °C and 1.25 mol H₂/mol glucose consumed at 55 °C. The results of repeated batch experiments for 615 h resulted in average yields of 1.21 ± 0.62 and 1.40 ± 0.16 mol H₂/mol glucose consumed for activated sludge and anaerobic sludge, respectively. The hydrogen production with activated sludge was not stable during the repeated batches and the fluctuation in hydrogen production was attributed to formation of lactic acid as the predominant metabolite in some batches. The presence of lactic acid bacteria in microflora was confirmed by PCR-DGGE.     

Microarray analysis of high light intensity stress on hydrogen production metabolism of Rhodobacter capsulatus

Biohydrogen obtained from purple non sulfur bacteria (PNSB) is an environmentally friendly alternative for hydrogen production. PNSB can be employed in large scale outdoor photobioreactors to produce hydrogen by photofermentation with sunlight as the light source. In external environmental conditions, however, bacteria can experience stress due to high light intensities, which can inhibit or slow down hydrogen production. Previous studies with other PNSB showed varying responses to light intensities (above 4000 lux), in some cases improving, and in others adversely affecting hydrogen production.In this study, Rhodobacter capsulatus, a PNSB species that produce hydrogen efficiently from dark fermenter effluents containing acetate, was used to investigate the effects of high light intensity stress on the hydrogen production metabolism at the gene expression level. A microarray analysis was carried out using a custom-design Affymetrix GeneChip TR_RCH2a520699F. R. capsulatus DSM1710 was grown under a cyclic illumination of 2000 and 7000 lux (12 h light/12 h dark) in a hydrogen production medium having 30 mM acetate and 2 mM glutamate, and was exposed to a high light intensity (10,000 lux) for 1 h in the middle of a light period. The results reveal that photosynthetic reaction center genes were down-regulated in order to protect the photosynthetic membrane from damage. On the other hand, the expression of nitrogenase and electron transport system genes were enhanced by high light intensity. These results show that a high light intensity stress drives R. capsulatus to direct gene expression towards hydrogen production, which supports the hypothesis that hydrogen production is a way for the disposal of excess reducing equivalents to maintain the internal redox balance.     

Fermentative hydrogen production from acetate using Rhodobacter sphaeroides RV

The study of photosynthetic hydrogen production by using Rhodobacter sphaeroides RV from acetate was described. We investigated the effects of light source (fluorescent, halogen and tungsten lamps), light intensity (1200–6000 lux), inoculum quantity (OD₆₆₀ 0.212–OD₆₆₀ 1.082) and initial pH (4.0–10.0) on biohydrogen production. The results indicated that the hydrogen production for halogen and tungsten lamps was better than it for fluorescent lamp as light source. The best light intensity of hydrogen production was 3600 lux for tungsten lamp as light source. Inoculum quantity experiments indicated that the higher hydrogen production volume and hydrogen conversion rate were obtained at initial OD₆₆₀ of 0.931. The effect of initial pH on hydrogen production indicated that the maximum hydrogen yield reached to 653.2 mmol H₂/mol acetate at initial pH 7.0.     

Photo-fermentative hydrogen production performance of a newly isolated Rubrivivax gelatinosus YP03 strain with acid tolerance

Screening and excavating new photosynthetic bacteria with excellent hydrogen production performance is extremely important for improving the photo-fermentative hydrogen production. A new photosynthetic bacterium YP03 was isolated and identified to be Rubrivivax gelatinosus by morphological characterization and phylogenetic analysis. The effects of several key factors on hydrogen production performance were carried out. The results indicated that YP03 strain showed a preference for the carbon sources, and 5375 ± 398 mL/L of maximum hydrogen yield was obtained using butyrate medium. Meanwhile, YP03 strain could use several nitrogen sources to produce hydrogen, and glutamic acid was the optimum nitrogen source for hydrogen produced. Furthermore, YP03 exhibited better hydrogen production performance at initial pH 7.0, reaction temperature 33 °C and light intensity 5000 lux, and the maximum hydrogen production rate was 108.3 ± 12.4 mL/(Lh), which was relatively high compared with the previous reports by R. gelatinosus. Especially, the proper pH for hydrogen production by YP03 ranged from weak acid to neutral (6.5–7.0) and it still could produce hydrogen at pH 5.5 showing the characteristic of acid tolerance. It suggested that YP03 is a potential candidate for the integration of dark- and photo-fermentative hydrogen production. These findings contribute to our understanding of YP03 strain and provide a prospective photosynthetic bacterium for efficient hydrogen production in future research.     

Mathematical modeling of two-phase flow and transport in an immobilized-cell photobioreactor

A one-dimensional two-phase flow and transport model is presented for a packed bed photobioreactor with transparent gel granules containing immobilized photosynthetic bacterial cells. The inherently coupled two-phase flow and mass transport, along with the biochemical reactions occurring in the photobioreactor are taken into account. The source term in the species conservation equation of the substrate is derived from a local transport model for a single gel granule. Model predictions of the glucose consumption efficiency and hydrogen production rate are in good agreement with experimental data. The results show that the photoinhibition of immobilized cells appears at incident light intensities higher than 6000 lux. It is the most suitable for photo-hydrogen production under neutral conditions and 30 °C of the influent substrate solution. Moreover, a high influent substrate solution flow rate results in a large hydrogen production rate due to the improved substrate transport from the bulk solution to gel granules.     

Effect of physico-chemical parameters on biohydrogen production and growth characteristics by batch culture of Rhodobacter sphaeroides CIP 60.6

In this paper, Rhodobacter sphaeroides CIP 60.6 strain was newly used for the biohydrogen production in a perfectly shaken column photobioreactor, grown in batch culture under anaerobic and illumination conditions, to investigate the effects of some physico-chemical parameters in microbial hydrogen photofermentation. Luedeking–Piret model was considered for the data fitting to find out the mode of hydrogen generation and the relationship between the cell growth and hydrogen production. The results show that, both growth cells and resting cells can produce hydrogen at light intensities greater or equal to 2500 lux, however, at the weak intensities hydrogen is a metabolite associated to growth. Growth rate and hydrogen production rate increase with the increasing of light intensity. Moreover, hydrogen production rate become higher in stationary phase than that in logarithmic phase, with the enhancement of light intensity. Maximum hydrogen production rate obtained was 39.88 ± 0.14 ml/l/h, at the optimal conditions (4500–8500 lux). Modified Gompertz equation was applied for the data fitting to verify the accuracy and the agreement of the model with experimental results. It is revealed that, in the modified Gompertz equation, the lag time represents time for which hydrogen production becomes maximal, not the beginning time of hydrogen production. The stop of stirring reduced hydrogen production rate and created unstable hydrogen production in reactor. The pH ranges of 7.5 ± 0.1 were the favorable pH for hydrogen production.     

Extreme-thermophilic biohydrogen production by an anaerobic heat treated digested sewage sludge culture

Biohydrogen production process from glucose using extreme-thermophilic H₂-producing bacteria enriched from digested sewage sludge was investigated for five cycles of repeated batch experiment at 70 °C. Heat shock pretreatment was used for preparation of hydrogen-producing bacteria comparing to an untreated anaerobic digested sludge for their hydrogen production performance and responsible microbial community structures. The results showed that the heat shock pretreatment completely repressed methanogenic activity and gave the maximum hydrogen production yield of 355-488 ml H₂/g COD in the second cycle of repeated batch cultivation with more stable gas production during the cultivation when compared with control. Hydrogen production was accompanied by production of acetic acid. The average specific hydrogen in five cycles experiment ranged from 150 to 200 ml H₂/g VSS. PCR-DGGE profiling showed that the extreme-thermophilic culture predominant species were closely affiliated to Thermoanaerobacter pseudethanolicus.     

Photofermentive production of biohydrogen from oil palm waste hydrolysate

Oil palm empty fruit bunch (OPEFB) was hydrolyzed with dilute sulfuric acid (6% v/v; 8 mL acid per g dry OPEFB) at 120 °C for 15-min to release the fermentable sugars. The hydrolysate contained xylose (23.51 g/L), acetic acid (2.44 g/L) and glucose (1.80 g/L) as the major carbon components. This hydrolysate was used as the sole carbon source for photofermentive production of hydrogen using a newly identified photosynthetic bacterium Rhodobacter sphaeroides S10. A Plackett–Burman experimental design was used to examine the influence of the following on hydrogen production: yeast extract concentration, molybdenum concentration, magnesium concentration, EDTA concentration and iron concentration. These factors influenced hydrogen production in the following decreasing order: yeast extract concentration > molybdenum concentration > magnesium concentration > EDTA concentration > iron concentration. Under the conditions used (35 °C, 14.6 W/m² illumination, initial pH of 7.0), the optimal composition of the culture medium was (per L): mixed carbon in OPEFB hydrolysate 3.87 g, K₂HPO₄ 0.9 g, KH₂PO₄ 0.6 g, CaCl₂⋅2H₂O 75 mg, l-glutamic acid 795.6 mg, FeSO₄⋅7H₂O 11 mg, Na₂MoO₂⋅2H₂O 1.45 mg, MgSO₄⋅7H₂O 2.46 g, EDTA 0.02 g, yeast extract 0.3 g). With this medium, the lag period of hydrogen production was 7.65 h, the volumetric production rate was 22.4 mL H₂/L medium per hour and the specific hydrogen production rate was 7.0 mL H₂/g (xylose + glucose + acetic acid) per hour during a 90 h batch culture of the bacterium. Under optimal conditions the conversion efficiency of the mixed carbon substrate to hydrogen was nearly 29%.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H₂/mol glucose, 2.2 ± 0.2 mol H₂/mol xylose and 5.2 ± 0.2 mol H₂/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.     

Dark fermentative hydrogen production from xylose by a hot spring enrichment culture

Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H₂/mol xylose. In CSTR the highest H₂ yield and production rate at 45 °C were 1.97 mol H₂/mol xylose and 7.3 mmol H₂/h/L, respectively, and at 37 °C, 1.18 mol H₂/mol xylose and 1.7 mmol H₂/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H₂ production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.     

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.     

The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures

The effect of heat treatment at different temperatures on two types of inocula, activated sludge and anaerobically digested sludge, was investigated in batch cultures. Heat treatments were conducted at 65, 80 and 95 °C for 30 min. The untreated inocula produced less amount of hydrogen than the pretreated inocula, with lactic acid as the main metabolite. The maximum yields of 2.3 and 1.6 mol H₂/mol glucose were achieved for the 65 °C pretreated anaerobically digested and activated sludges, respectively. Approximately a 15% decrease in yield was observed with increasing pretreatment temperature from 65 to 95 °C concomitant with an increase in butyrate/acetate ratio from 1.5 to 2.4 for anaerobically digested sludge. The increase of pretreatment temperature of activated sludge to 95 °C suppressed the hydrogen production by lactic acid fermentation. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity.     

Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus

The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen-limited, anaerobic, photosynthetic culture conditions, using various carbon substrates. In the present study, the relationship between light intensity and hydrogen production has been modelled in order to predict both the rate of hydrogen production and the amount of hydrogen produced at a given time during batch cultures of R. capsulatus. The experimental data were obtained by investigating the effect of different light intensities (6000–50,000 lux) on hydrogen-producing cultures of R. capsulatus grown in a batch photobioreactor, using lactate as carbon and hydrogen source. The rate of hydrogen production increased with increasing light intensity in a manner that was described by a static Baly model, modified to include the square of the light intensity. In agreement with previous studies, the kinetics of substrate utilization and growth of R. capsulatus was represented by the classical Monod or Michaelis–Menten model. When combined with a dynamic Leudekong–Piret model, the amount of hydrogen produced as a function of time was effectively predicted. These results will be useful for the automatization and control of bioprocesses for the photoproduction of hydrogen.     

Effect of pH and sulfate concentration on hydrogen production using anaerobic mixed microflora

The effects of varying sulfate concentrations with pH on continuous fermentative hydrogen production were studied using anaerobic mixed cultures growing on a glucose substrate in a chemostat reactor. The maximum hydrogen production rate was 2.8 L/day at pH 5.5 and sulfate concentration of 3000 mg/L. Hydrogen production and residual sulfate level decreased with increasing the pH from 5.5 to 6.2. The volatile fatty acids (VFAs) and ethanol fractions in the effluent were in the order of butyric acid (HBu) > acetic acid (HAc) > ethanol > propionic acid (HPr). Fluorescence In Situ Hybridization (FISH) analysis revealed the presence of hydrogen producing bacteria (HPB) under all pH ranges while sulfate reducing bacteria (SRB) were present at pH 5.8 and 6.2. The inhibition in hydrogen production by SRB at pH 6.2 diminished entirely by lowering to pH 5.5, at which activity of SRB is substantially suppressed.     

Effect of extrinsic lactic acid on fermentative hydrogen production

In this paper we report the effect of extrinsic lactic acid on hydrogen production from a starch-containing medium by a mixed culture. Study of the effect of addition of four metabolites, namely ethanol, lactic acid, butyric acid and acetic acid illustrated that lactic acid had a positive effect on both the maximum hydrogen production and hydrogen production rate. The addition of 10 mM lactic acid to a batch containing starch increased the hydrogen production rate and hydrogen production yield from 4.31 to 8.23 mL/h and 5.70 to 9.08 mmol H₂/g starch, respectively. This enhancement in hydrogen production rate and yield was associated with a shift from acetic acid and ethanol formation to formation of butyric acid as the predominant metabolite. The increase in hydrogen production yield was attributed to the increase in the available residual NADH for hydrogen production. When lactic acid was used as the sole carbon source, no significant hydrogen production was observed.     

Catalytic activity and characterization of Fe–Zn–Mg–Al hydrotalcites in biohydrogen production

Four different M²⁺–Mg–Al hydrotalcite (HT) materials were investigated for their effect on biohydrogen enhancement, where M²⁺ is Fe and/or Zn. HTs were synthesized by the coprecipitation method and characterized by infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD). The effect of Fe–Zn–Mg–Al HTs dose (0–833 mg/L) on hydrogen production was investigated in batch tests using sucrose-fed anaerobic mixed culture at 37 °C. The best catalytic activity was observed on Mg–Al HT at 167 mg/L with the maximum hydrogen yield of 2.30 ± 0.37 mol H₂/mol sucrose, which was 44% higher than the control. The major metabolites detected in the test were acetic acid (3.6 g/L), butyric acid (4.1 g/L), and lactic acid (0.5 g/L). The basic properties of the different catalysts played an important role in stimulating or inhibiting the activity of hydrogen producing bacteria. Calcined Mg–Al HT did not promote biohydrogen production, suggesting that the catalytic enhancement was related to immobilization of bacteria in the electrostatically charged HT interlayers.     

Inhibitory effect of ethanol,acetic acid,propionic acid and butyric acid on fermentative hydrogen production

The inhibitory effect of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as substrate. The experimental results showed that, at 35 °C and initial pH 7.0, during the fermentative hydrogen production, the substrate degradation efficiency, hydrogen production potential, hydrogen yield and hydrogen production rate all trended to decrease with increasing added ethanol, acetic acid, propionic acid and butyric acid concentration from 0 to 300 mmol/L. The inhibitory effect of added ethanol on fermentative hydrogen production was smaller than those of added acetic acid, propionic acid and butyric acid. The modified Han–Levenspiel model could describe the inhibitory effects of added ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production rate in this study successfully. The modified Logistic model could describe the progress of cumulative hydrogen production.