Optimization of activation conditions of Rhodobacter sphaeroides in hydrogen generation process

AIMS: To examine the effects of the culture age, illuminance intensity and changes in these parameters during activation on hydrogen generation process carried out by purple nonsulfur Rhodobacter sphaeroides bacteria. METHODS AND RESULTS: The following parameters were determined in all experiments: the amount of hydrogen evolved (measured using gas chromatography), biomass increase as dry mass, pH values and consumption of organic substance as chemical oxygen demand (COD). The medium used in the process of activation and hydrogen generation contained malic acid (15 mmol) and sodium glutamate (2 mmol). The optimum age of bacteria was 12-24 h and the best intensity of illuminance was found to be 5 cd sr m-2 on activation and 9 cd sr m-2 on hydrogen generation. These conditions provided hydrogen evolution of 1.39 l l-1 of the medium with the highest specific hydrogen production of 0.146 l H2 l-1 medium h-1 g-1 inoculum. An increase in the illuminance intensity resulted in a slight inhibition of the process. CONCLUSIONS: The activation stage of bacteria has a significant effect on the parameters of hydrogen photogeneration. The optimization of the activation stages allowed a shortening of the time of hydrogen generation and of the period after which hydrogen evolution starts. SIGNIFICANCE AND IMPACT OF THE STUDY: An innovative method of bacteria activation before the initiation of the hydrogen generation process has been used to optimize this process. The shortening of the process duration as well as the twice higher hydrogen yield can help in the designing of other systems (including also those operating under solar irradiation) in which R. sphaeroides bacteria are to be applied.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host.

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.     

Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H(2)-producing conditions

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H(2)) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on (13)C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H(2)-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H(2) yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-β-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD(+). Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H(2) production.     

Effect of pH on Bacillus thermoamylovorans Growth and Glucose Fermentation

The effect of pH on the growth and physiology of Bacillus thermoamylovorans, a new moderately thermophilic and non-spore-forming bacterium isolated from palm wine, was studied. Growth occurred from pH 5.4 to 8.5, with optimum growth at 7.0. During the exponential growth phase at optimum pH, glucose was consumed at the maximum rate (qs), 17.87 mmol g(sup-1) h(sup-1), and was mainly fermented into acetate, ethanol, and formate (76.5% of metabolites produced). In acidic or alkaline conditions, glucose specific consumption rates were considerably reduced (qs = 8.06 mmol g(sup-1) h(sup-1) at pH 5.6 and 2.85 mmol g(sup-1) h(sup-1) at pH 8.4), and a switch in glucose metabolism toward lactate production (62.6% of metabolites produced at pH 5.6 and 41.2% of those produced at pH 8.4) was observed. Moreover, optimum cellular yield (Y(infx/ATP)), 14.8 g mol(sup-1), and optimum energy yield (Y(infATP/s)), 2.65 mol mol(sup-1), were observed at neutrality. The results of this study were compared with published data about lactic acid bacteria; this comparison allowed us to complement our previous taxonomic study of B. thermoamylovorans and to identify additional phenotypic differences between B. thermoamylovorans and lactobacilli.     

Hydrogen production with R. faecalis RLD-53 isolated from freshwater pond sludge

The Rhodopseudomonas faecalis strain RLD-53 was isolated from freshwater pond sludge and was demonstrated it could produce hydrogen. This study to investigate their ability of hydrogen production under some conditions in batch culture experiments. At pH 7.0, temperature 35 degrees C and light intensity of 4000 lux, the H(2) yield was 2.64 mol-H(2)/mol-acetate, 2.34 mol-H(2)/mol-propionate, 1.75 mol-H(2)/mol-lactate and 3.55 mol-H(2)/mol-malate, respectively. The maximal H(2) production rate of 32.62 ml-H(2)/l/h and H(2) yield of 2.84 mol-H(2)/mol-acetate were achieved when beef extract was used as nitrogen source. Light intensity is the most important factor for H(2) production, H(2) production yield and rate decreased with increasing light intensity and reached highest under light intensity of 3000-5000 lux. Result indicated the strain RLD-53 was a high efficient bacteria for hydrogen production.     

Isolation of propionate degrading bacterium in co-culture with a methanogen from a cattle dung biogas plant

Various anaerobic hydrolytic and methanogenic bacteria active in cattle dung biogas plants are reported in the literature. Anaerobic bacteria with ability to use volatile fatty acids constitute a vital bridge between hydrolytic bacteria and methanogenic bacteria. The present paper describes the isolation ofSyntrophobacter wolinii a propionate degrading bacterium in co-culture with a hydrogen utilizing methanogenviz.,Methanobacterium formicicum from the fermenting slurry of cattle dung biogas plant. Earlier studies on propionate and butyrate degradation indicatedMethanospirillum hungatei as the hydrogen utilizing partner of the co-culture whereas in the present studies this was not the case. Temperature 35° C, pH 7.5 and 20 mM of propionate were found optimal for growth and activity of co-culture.     

Siderophore utilization and iron uptake by Rhodopseudomonas sphaeroides

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.     

Microbial Consortia for Hydrogen Production Enhancement

Ten efficient hydrogen-producing strains affiliated to the Clostridium genus were used to develop consortia for hydrogen production. In order to determine their saccharolytic and proteolytic activities, glucose and meat extract were tested as fermentation substrates, and the best hydrogen-producing strains were selected. The C. roseum H5 (glucose-consuming) and C. butyricum R4 (protein-degrading) co-culture was the best hydrogen-producing co-culture. The end-fermentation products for the axenic cultures and co-cultures were analyzed. In all cases, organic acids, mainly butyrate and acetate, were produced lowering the pH and thus inhibiting further hydrogen production. In order to replace the need for reducing agents for the anaerobic growth of clostridia, a microbial consortium including Clostridium spp. and an oxygen-consuming microorganism able to form dense granules (Streptomyces sp.) was created. Increased yields of hydrogen were achieved. The effect of adding a butyrate-degrading bacteria and an acetate-consuming archaea to the consortia was also studied.     

光合细菌与产气肠杆菌协同产氢特性分析

对光合细菌(Rhodopseudomonas sp. DT)与产气肠杆菌(Enterobacter aerogenes)进行了发酵产氢试验, 考察了不同起始接种比例、培养温度及碳源条件下混合菌协同产氢特性。结果表明: 光合细菌与产气肠杆菌初始接种比例对协同产氢影响较大, 初始接种比例为1:1最有利于协同产氢, 产氢效率和产氢周期达到了3.1 mol H2/mol葡萄糖及81 h。进一步培养液pH动力学变化研究发现初始接种比例为1:1的混合菌培养液pH变化较小, 为pH 6~7, 利于混合菌协同产氢。28°     

Assessing the impact of denitrifier-produced nitric oxide on other bacteria

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low muM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.     

Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process

A fermentative hydrogen-producing bacterium, Clostridium tyrobutyricum JM1, was isolated from a food waste treating process using 16S rRNA gene sequencing and amplified ribosomal DNA restriction analysis (ARDRA). A fixed-bed bioreactor packed with polyurethane foam as support matrix for the growth of the isolate was operated at different hydraulic retention time (HRT) to evaluate its performance for hydrogen production. The reactor achieved the maximal hydrogen production rate of 7.2 l H(2)l(-1)d(-1) at 2h HRT, where hydrogen content in biogas was 50.0%, and substrate conversion efficiency was 97.4%. The maximum hydrogen yield was 223 ml (g-hexose)(-1) with an influent glucose concentration of 5 g l(-1). Therefore, the immobilized reactor using C. tyrobutyricum JM1 was an effective and stable system for continuous hydrogen production.     

Effects of initial lactic acid concentration, HRTs, and OLRs on bio-hydrogen production from lactate-type fermentation

A batch test and continuous operation were performed to identify the effect of lactate on hydrogen production at pH 4.5. When the initial lactic acid concentration was increased from 0 to 8 g/L in the batch test, the hydrogen yield also increased from 1.41 to 1.72 mol-H₂/mol-glucose. The system exhibited a long lag time and an insignificant hydrogen yield with 16 g-lactic acid/L. A continuous stirred tank reactor (CSTR) was operated at different organic loading rates (OLRs: 10, 15, 20 and 40 g/L/day) and hydraulic retention times (HRTs: 6, 12 and 24 h). At an OLR of 20 g-glucose/L/day and 12 h of HRT, the hydrogen yield was 1.2 mol-H₂/mol-glucose. The yield decreased with a 24 h HRT. Even though lactate was one of the major constituents of volatile fatty acids (VFAs), hydrogen production was feasible throughout the operation. Clostridium sp. was the dominant hydrogen-producing bacteria in the system.     

Relationship of proton motive force and the F0F1-ATPase with bio-hydrogen production activity of Rhodobacter sphaeroides: effects of diphenylene iodonium, hydrogenase inhibitor, and its solvent dimethylsulphoxide

Rhodobacter sphaeroides MDC 6521 was able to produce bio-hydrogen (H(2)) in anaerobic conditions under illumination. In this study the effects of the hydrogenase inhibitor-diphenylene iodonium (Ph(2)I) and its solvent dimethylsulphoxide (DMSO) on growth characteristics and H(2) production by R. sphaeroides were investigated. The results point out the concentration dependent DMSO effect: in the presence of 10?mM DMSO H(2) yield was ~6 fold lower than that of the control. The bacterium was unable to produce H(2) in the presence of Ph(2)I. In order to examine the mediatory role of proton motive force (?p) or the F(0)F(1)-ATPase in H(2) production by R. sphaeroides, the effects of Ph(2)I and DMSO on ?p and its components (membrane potential (?ψ) and transmembrane pH gradient), and ATPase activity were determined. In these conditions ?ψ was of -98?mV and the reversed ?pH was +30?mV, resulting in ?p of -68?mV. Ph(2)I decreased ?ψ in concentrations of 20?μM and higher; lower concentrations of Ph(2)I as DMSO had no valuable effect on ?ψ. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide. The 10-20?μM Ph(2)I did not affect the ATPase activity, whereas 40?μM Ph(2)I caused a marked inhibition (~2 fold) in ATPase activity. The obtained results provide novel evidence on the involvement of hydrogenase and the F(0)F(1)-ATPase in H(2) production by R. sphaeroides. Moreover, these data indicate the role of hydrogenase and the F(0)F(1)-ATPase in ?p generation. In addition, DMSO might increase an interaction of nitrogenase with CO(2), decreasing nitrogenase activity and affecting H(2) production.     

A complete set of flagellar genes acquired by horizontal transfer coexists with the endogenous flagellar system in Rhodobacter sphaeroides

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a gamma-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other alpha-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Kinetics and modelling of kojic acid production by Aspergillus flavus Link in batch fermentation and resuspended mycelial system

An unstructured model based on logistic and Luedeking-Piret equations was proposed to describe growth, substrate consumption and kojic acid production by Aspergillus flavus Link strain 44-1 in batch fermentation and also in a resuspended cell system. The model showed that kojic acid production was non-growth associated. The maximum kojic acid and cell concentrations obtained in batch fermentations using the fermenter with optimized dissolved oxygen control (32.5 g/l and 11.8 g/l, respectively) and using a shake-flask (36.5 and 12.3 g/l, respectively) were not significantly different. However, the maximum specific growth rate and a non-growth-associated rate constant for kojic acid formation (n) for batch fermentation using the fermenter (0.085/h and 0.0125 g kojic acid/g cell.h, respectively) were approximately three and two times higher than the values obtained for fermentation using a shake-flask, respectively. Efficient conversion of glucose to kojic acid was achieved in a resuspended pellet or mycelial system, in a solution containing only glucose with citrate buffer at pH 3.5 and at a temperature of 30 °C. The resuspended cell material in the glucose solution was still active in synthesizing kojic acid after prolonged incubation (up to about 600 h). The rate constant of kojic acid production (n) in a resuspended cell system using 100 g glucose/l was almost constant at an average value of 0.011 g kojic acid/g cell.h up to a cell concentration of 19.2 g/l, above which it decreased. A drastic reduction of n was observed at a cell concentration of 26.1 g/l. However, the yield based on glucose consumed (0.45 g/g) was similar for all cell concentrations investigated.     

Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in hook length control.

Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyhook phenotype. Morphological characterization of the mutant was done by electron microscopy. Polyhooks were obtained by shearing and were used to purify the hook protein monomer (FlgE). The apparent molecular mass of the hook protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the hook proteins of other flagellated bacteria indicated that the Rhodobacter hook protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyhook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed.     

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).     

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.