Photohydrogen production using purple nonsulfur bacteria with hydrogen fermentation reactor effluent

Hydrogen production from waste using photosynthetic bacteria is an attractive methodology. A combination of purple nonsulfur photosynthetic bacteria and anaerobic bacteria is ideal for the efficient conversion of wastewater into hydrogen. In this paper, photohydrogen production using effluent from different hydrogen fermentation reactors was carried out using two strains of photosynthetic purple nonsulfur bacteria. The results indicated that the effluent from the hydrogen fermentation reactors could be used directly for photohydrogen production without aeration or dilution pretreatment. Effluent from the carbohydrate fed hydrogen fermentation reactors is more suitable for photohydrogen production than effluent from a peptone fed reactor. Among the initial dark hydrogen fermentation stage effluents from the three carbohydrate fed reactors (CSTR, ASBR, UASB), CSTR effluent was the most suitable for photohydrogen production.     

Influence of sulfate-reducing bacteria,sulfide and molybdate on hydrogen photoproduction by purple nonsulfur bacteria

The interaction between bacterial species is of great importance for H₂ production using microbial consortia or non-sterile conditions. Sulfate reducing bacteria were found in anaerobic starch-hydrolyzing consortium and their inhibitory effect on the following H₂ photoproduction by purple nonsulfur bacteria was shown. This inhibition was clearly demonstrated in the mixed culture of Rhodobacter sphaeroides and Desulfomicrobium baculatum using the synthetic medium. This effect was conditioned by sulfide production rather than H₂ consumption or competition for organic substrate. Actually, the addition of equivalent sulfide concentration brought about the similar effects: inhibition of H₂ production without growth inhibition, cells aggregation, and the increase of carbohydrate content as an alternative way of expenditure of organic acids. In the long-term experiments the average sulfide concentration of about 0.3 mM was detrimental while in short-terms the H₂ production was not inhibited even at 3.2 mM. The protective effect of molybdates against sulfate reducers and sulfide was discussed.     

New tolerant strains of purple nonsulfur bacteria for hydrogen production in a two-stage integrated system

In this study we described the isolation of eight new strains of purple non-sulfur bacteria resistant to salinity ≥30 g L⁻¹ and high concentration of VFAs (200 mM). These strains were characterized by their general physiological properties and the occurrence of hupSL genes. Some correlation was observed between the rate of H₂ photoproduction, the absence of hupSL genes and hydrogenase activity. Two fast-growing strains without hupSL genes showed high nitrogenase activity and hydrogen accumulation during growth on Ormerod medium. These strains were capable of H₂ photoproduction using non-treated dark culture (75% in water) after dark fermentation of starch at 30 g L⁻¹, unlike control strains, Rhodobacter capsulatus B10 and Rb. sphaeroides GL. New N7 and 13 strains identified as Rb. sphaeroides can be recommended for application in a two-stage H₂ production system.     

Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides

Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize. For large-scale hydrogen production, an integrated view of the overall metabolism is necessary in order to interpret results properly and facilitate experimental design. In this study, a summary of the hydrogen production metabolism of the photosynthetic purple non-sulfur (PNS) bacteria will be presented.Practically all hydrogen production by PNS bacteria occurs under a photoheterotrophic mode of metabolism. Yet results show that under certain conditions, alternative modes of metabolism—e.g. fermentation under light deficiency—are also possible and should be considered in experimental design.Two enzymes are especially critical for hydrogen production. Nitrogenase promotes hydrogen production and uptake hydrogenase consumes hydrogen.Though a wide variety of substrates can be used for growth, only a portion of these is suitable for hydrogen production. The efficiency of a certain substrate depends on factors such as the activity of the TCA cycle, the carbon-to-nitrogen ratio, the reduction-state of that material and the conversion potential of the substrate into alternative metabolites such as PHB.All these individual components of the hydrogen production interact and are subject to strict regulatory controls. An overall scheme for the hydrogen production metabolism is presented.     

Photofermentation of malate for biohydrogen production— A modeling approach

A kinetic model for photofermentative biohydrogen production is developed in this study to predict the dynamics of the process. The proposed model contains 17 parameters to describe cell growth, substrate consumption, and hydrogen evolution as well as inhibition of the process by biomass, light intensity, and substrate. Batch experimental results from the literature were used to calibrate and validate the model with malic acid as a model substrate, using Rhodobacter sphaeroides as a model biomass. Temporal hydrogen evolution and cell growth predicted by the proposed model agreed well with the experimentally measured data obtained from four literature reports, with statistically significant correlation coefficients exceeding 0.9. Based on sensitivity analysis performed with the validated model, only six of the 17 parameters were found to be significant. Model simulations indicated that the range of optimal light intensity for maximum hydrogen yield from malate by R. sphaeroides was 150–250 W/m².     

Recent advanced biotechnological strategies to enhance photo-fermentative biohydrogen production by purple non-sulphur bacteria: An overview

Photo-fermentation seems to be an attractive hydrogen production pathway. However, the light conversion efficiency and photo-hydrogen production of purple non-sulphur bacteria (PNSB) are very low, and hence, various biotechnological approaches are investigated to improve biohydrogen production. This article presents an overview of the advanced biotechnological approaches to enhance the photo-fermentative biohydrogen production. The advancements reviewed include optimisation of the medium, abiotic factors, the lighting regime, immobilisation techniques, application of photoluminating nanomaterials, genetic engineering, and other strategies. These approaches show positive results in the enhancement of photo-hydrogen production by PNSB. Some recommendations are suggested for further studies in the enhancement of photo-hydrogen production, such as green nanomaterials application, integrated dark- and photo-fermentation, genetic manipulation, and the application of the non-technological analysis approaches.     

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides was studied in batch experiments. Results showed that in all batches hydrogen was produced after a lag phase of about 10 h; pure culture of R. sphaeroides produced hydrogen at rates substantially lower than C. butyricum. In co-culture systems, R. sphaeroides even with cell populations 5.9 times higher still could not compete with C. butyricum for glucose. In co-culture systems, R. sphaeroides syntrophically interacted with C. butyricum, using the acetate and butyrate produced by the latter as substrate for hydrogen production. Hydrogen production was ceased in all batches when the pH was lowered to the level of pH 6.5, resulting from the accumulation of fatty acids. It was also demonstrated in this study that fluorescence in situ hybridization (FISH) was an effective means for the quantification of the relative abundance of individual bacteria in a co-culture system.     

A pneumatically agitated flat-panel photobioreactor with gas re-circulation: anaerobic photoheterotrophic cultivation of a purple non-sulfur bacterium

The application of hydrogen as a clean and efficient energy carrier in the near future becomes more and more evident. Within the process of photobiological hydrogen production, purple non-sulfur bacteria are an interesting subject of study because of their high hydrogen producing capacity. In a previous study, the used Rhodopseudomonas sp. had proven to efficiently produce hydrogen from acetic acid and light energy. We constructed a pneumatically agitated flat-panel photobioreactor as a model system for optimization of photoheterotrophic hydrogen production. Batch experiments and a chemostat experiment were performed to investigate the proper functioning of the new photobioreactor. During the first experiments, argon gas was sparged through the system for mixing and inhibition of growth was observed. Experimental results indicate that the stripping of carbon dioxide from the culture liquid caused this inhibition of growth. Possibly, the Rhodopseudomonas sp. used requires carbon dioxide during growth on a highly reduced substrate like acetate. Recirculating the gas prevented the carbon dioxide from being stripped from the system. In this mode of operation, growth was supported.     

Flux balance analysis of mixed anaerobic microbial communities: Effects of linoleic acid (LA) and pH on biohydrogen production

The internal fluxes of mixed anaerobic cultures fed 2000 mg l⁻¹ linoleic acid (LA) plus glucose at 6 initial pH conditions and maintained at 37 °C were estimated using a flux balanced analysis (FBA). In cultures fed LA at pH 7, less than 8% of the flux was diverted to CH₄. At an initial pH ≥ 5.5, the quantity of glucose removed was greater than 95%; however, at pH 4.5 and 5.0 the quantity consumed were 38% and 75%, respectively. The FBA output showed that the acetogenic H₂-consumers were responsible for more than 20% of the H₂ consumed. Adding LA and decreasing the pH was ineffective in reducing the activity of acetogenic H₂-consumers. As the initial pH decreased, the acetogenic H₂-consuming flux decreased in the presence of 2000 mg l⁻¹ LA. A maximum H₂ yield of 1.55 mol mol⁻¹ glucose consumed (peak hydrogenase flux (R12)) was attained when the acetogenic H₂-consuming flux reached 0.42 mol at a pH of 5.5.     

Isolation of anoxygenic photosynthetic bacteria from Songkhla Lake for use in a two-staged biohydrogen production process from palm oil mill effluent

We are developing a process to produce biohydrogen from palm oil mill effluent. Part of this process will involve photohydrogen production from volatile fatty acids under low light conditions. We sought to isolate suitable bacteria for this purpose from Songkhla Lake in Southern Thailand. Enrichment for phototrophic bacteria from 34 samples was conducted providing acetate as a major carbon source and applying culturing conditions of anaerobic-low light (3000 lux) at 30 °C. Among the independent isolates from these enrichments 19 evolved hydrogen with productivities between 4 and 326 ml l⁻¹ d⁻¹. Isolate TN1 was the most efficient producer at a rate of 1.85 mol H₂ mol acetate⁻¹ with a light conversion efficiency of 1.07%. The maximum hydrogen production rate for TN1 was determined to be 43 ml l⁻¹ h⁻¹. Environmentally desirable features of photohydrogen production by TN1 included the absence of pH change in the cultures and no detectable residual CO₂.     

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%.     

Mathematical model of biohydrogen production in microbial electrolysis cell: A review

Microbial electrolysis cell (MEC) is a promising reactor. However, currently, the reactor cannot be adapted for industrial-scale biohydrogen production. Nevertheless, this drawback can be overcome by modeling studies based on mathematical equations. The limitation of analytical instrumentation to record the non-linearity of the dynamic behavior for biohydrogen processes in an MEC has led to the introduction of computational approach that has the potential to reduce time constraints and optimize experimental costs. Reviews of comparative studies on bioelectrochemical models are widely reported, but there is less emphasis on the MEC model. Therefore, in this paper, a comprehensive review of the MEC mathematical model will be further discussed. The classification of the model with respect to the assumptions, model improvement, and extensive studies based on the model application will be critically analyzed to establish a methodology algorithm flow chart as a guideline for future implementation.     

Kinetic analysis of hydrogen production using anaerobic bacteria in reverse micelles

The micellar formation and entrapment of bacteria cell in reverse micelles were investigated by ultraviolet spectrum (UV), fluorescence spectrum, and scanning electron microscope (SEM). The hydrogen production in reverse micelles was confirmed. The Gompertz equation was employed to evaluate the hydrogen-producing behavior in reverse micellar systems. Different systems including dioctyl sulfosuccinate sodium salt (AOT)–isooctane, sodium dodecyl sulfate (SDS)–benzene and SDS–carbon tetrachloride (CCl₄) reverse micelles were analysized. The results revealed that the maximum rate of hydrogen production (R_m) was also suitable to formulate the relationship between hydrogen-producing rate and hydrogen productivity in reverse micelles.     

Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria

Beer lees are the main by-product of the brewing industry. Biohydrogen production from beer lees using anaerobic mixed bacteria was investigated in this study, and the effects of acidic pretreatment, initial pH value and ferrous iron concentration on hydrogen production were studied at 35 °C in batch experiments. The hydrogen yield was significantly enhanced by optimizing environmental factors such as hydrochloric acid (HCl) pretreatment of substrate, initial pH value and ferrous iron concentration. The optimal environmental factors of substrate pretreated with 2% HCl, pH = 7.0 and 113.67 mg/l Fe²⁺ were observed. A maximum cumulative hydrogen yield of 53.03 ml/g-dry beer lees was achieved, which was approximately 17-fold greater than that in raw beer lees. In addition, the degradation efficiency of the total reducing sugar, and the contents of hemicellulose, cellulose, lignin and metabolites are presented, which showed a strong dependence on the environmental factors.     

Modeling microbial growth of dynamic membrane in a biohydrogen production bioreactor

Biohydrogen is renewable and has a huge potential to replace fossil fuels. Understanding mechanisms of controlling microbial processes of the dynamic membrane is critical for effective dark fermentative biohydrogen production in a dynamic membrane bioreactor (DMBR). This paper aims to develop a sophisticated model of biofilm growth, dynamic membrane formation, and dark fermentative hydrogen production within a platform of coupled lattice Boltzmann and cellular automata. The model was validated against the experimental data available and then was applied for the investigation of biohydrogen production in bioreactors under different membrane structures and inlet velocities. The results showed that porous twisted channels in the dynamic membrane could significantly affect biohydrogen extraction and biofilm patterns. In all cases, the dynamic membrane formation has three phases: the initial bacteria deposit, stable biofilm growth, and stable maximum biofilm biomass. The biohydrogen production could increase by 16.4% by optimizing the porous structure and increase 30%–40% of the hydrogen extraction. Inlet velocity also affects biohydrogen extraction in a range of ?28.3%–71.2%. Both porous structure and inlet velocity would be critical operational parameters for continuous biohydrogen production. The present model demonstrated its capability to investigate dark fermentative hydrogen production and its potential applications to porous bioreactors.     

Enhancing biohydrogen production of the alkalithermophile Thermobrachium celere

In this study the effect of different buffering agents, pH control and N₂ sparging on biohydrogen production in Thermobrachium celere was investigated in batch cultivations. Among the tested buffers, none was able to prevent the medium acidification resulting in a premature interruption of the hydrogen production. Controlling the pH helped to sustain the growth, the complete substrate consumption and the H₂ production. However, in these conditions the increase of H₂ partial pressure induced a partial metabolic shift towards ethanol production resulting in a decreased H₂ yield. Analysis of formate accumulation during growth suggests that this compound might play a relevant role in the anabolic routes in T. celere. When frequent N₂ sparging was applied for H₂ removal, together with pH control, the H₂ yield was remarkably enhanced from 2.26 to 3.53 mol H₂/mol glucose, and the maximum H₂ production rate and specific H₂ production rate reached 41.5 mmol H₂/l/h and 142.3 mmol H₂/h/g, respectively. This result suggests that under proper conditions T. celere is able to produce hydrogen at high yield and production rate.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

S-systems sensitivity analysis of the factors that may influence hydrogen production by sulfur-deprived Chlamydomonas reinhardtii

We built a metabolic map of the hydrogen production process by the microalga Chlamydomonas reinhardtii, mathematically modeled this map in the S-systems formalism, then analyzed the effect of variations in the value of different model parameters on the overall response of the system. The mathematical model exhibited behavior similar to that described in literature for photosynthetic algal hydrogen production by sulfur-deprived algal cultures. This behavior consists of an initial phase during which oxygen is transiently generated and then consumed, followed by an anaerobic phase that is characterized by generation of hydrogen. Our analysis of the effect of independent variables on the hydrogen production process mostly agrees with previous work [Horner J, Wolinsky M. A power-law sensitivity analysis of the hydrogen-producing metabolic pathway in Chlamydomonas reinhardtii. Int J Hydrogen Energy 2002;27: 1251–1255]. Moreover, a more detailed study of the effects of parameter modification (rate constants and kinetic order) indicated that genetic engineering of the hydrogenase expression, activity and stability may lead to increased performance of the process.