Photosynthetic Bacterium Rhodobacter Sphaeroides Research Articles

Structure and function of a bacterial Fasciclin I Domain Protein elucidates function of related cell adhesion proteins such as TGFBIp and periostin

Fasciclin I (FAS1) domains have important roles in cell adhesion, which are not understood despite many structural and functional studies. Examples of FAS1 domain proteins include TGFBIp (βig-h3) and periostin, which function in angiogenesis and development of cornea and bone, and are also highly expressed in cancer tissues. Here we report the structure of a single-domain bacterial fasciclin I protein, Fdp, in the free-living photosynthetic bacterium Rhodobacter sphaeroides, and show that it confers cell adhesion properties in vivo. A binding site is identified which includes the most highly conserved region and is adjacent to the N-terminus. By mapping this onto eukaryotic homologues, which all contain tandem FAS1 domains, it is concluded that the interaction site is normally buried in the dimer interface. This explains why corneal dystrophy mutations are concentrated in the C-terminal domain of TGFBIp and suggests new therapeutic approaches.

Fluorescence Assays for Photosynthetic Capacity of Bacteria

The fast kinetics of induction and relaxation of bacteriochlorophyll prompt and delayed fluorescence together with absorption changes of the reaction center (RC) dimer (P) were measured by combination of flashes from laser diodes in intact cells of wild type, carotenoidless (R-26) and cytochrome c2 deficient (CYCA) mutants of photosynthetic bacteria Rhodobacter sphaeroides. The fluorescence induction under high intensity of continuous light splits into fast and slow rises both overlapped by the (carotenoid and/or bacteriochlorophyll) triplet quenching. The fast phase is purely photochemical as it depends strongly on the number of photons absorbed. The slow phase is the combination of thermal and photochemical reactions and reflects the multiple turnover of the system. Upon short flash, the fluorescence yield cannot reach the maximum due to partial reopening of the RCs by rapid donor and acceptor side reactions. Longer flashes are needed to close the RC completely. Contrary to higher plants, the kinetics of induction and relaxation of the fluorescence yield in bacteria are controlled principally by P+. The reactions on the quinone side play minor role. The quantitative determination of the cyclic electron transfer rate can be based on calibration to the quantity of P+.View Large Image | View Hi-Res Image | Download PowerPoint Slide

RpoH2 sigma factor controls the photooxidative stress response in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7

Bacteria belonging to the Alphaproteobacteria normally harbour multiple copies of the heat shock sigma factor (known as σ(32), σ(H) or RpoH). Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours five copies of rpoH genes, one of which is an rpoH2 homologue. The genes around the rpoH2 locus in A. brasilense show synteny with that found in rhizobia. The rpoH2 of A. brasilense was able to complement the temperature-sensitive phenotype of the Escherichia coli rpoH mutant. Inactivation of rpoH2 in A. brasilense results in increased sensitivity to methylene blue and to triphenyl tetrazolium chloride (TTC). Exposure of A. brasilense to TTC and the singlet oxygen-generating agent methylene blue induced several-fold higher expression of rpoH2. Comparison of the proteome of A. brasilense with its rpoH2 deletion mutant and with an A. brasilense strain overexpressing rpoH2 revealed chaperone GroEL, elongation factors (Ef-Tu and EF-G), peptidyl prolyl isomerase, and peptide methionine sulfoxide reductase as the major proteins whose expression was controlled by RpoH2. Here, we show that the RpoH2 sigma factor-controlled photooxidative stress response in A. brasilense is similar to that in the photosynthetic bacterium Rhodobacter sphaeroides, but that RpoH2 is not involved in the detoxification of methylglyoxal in A. brasilense.

Excited state dynamics in photosynthetic reaction center and light harvesting complex 1

Key to efficient harvesting of sunlight in photosynthesis is the first energy conversion process in which electronic excitation establishes a trans-membrane charge gradient. This conversion is accomplished by the photosynthetic reaction center (RC) that is, in case of the purple photosynthetic bacterium Rhodobacter sphaeroides studied here, surrounded by light harvesting complex 1 (LH1). The RC employs six pigment molecules to initiate the conversion: four bacteriochlorophylls and two bacteriopheophytins. The excited states of these pigments interact very strongly and are simultaneously influenced by the surrounding thermal protein environment. Likewise, LH1 employs 32 bacteriochlorophylls influenced in their excited state dynamics by strong interaction between the pigments and by interaction with the protein environment. Modeling the excited state dynamics in the RC as well as in LH1 requires theoretical methods, which account for both pigment-pigment interaction and pigment-environment interaction. In the present study we describe the excitation dynamics within a RC and excitation transfer between light harvesting complex 1 (LH1) and RC, employing the hierarchical equation of motion method. For this purpose a set of model parameters that reproduce RC as well as LH1 spectra and observed oscillatory excitation dynamics in the RC is suggested. We find that the environment has a significant effect on LH1-RC excitation transfer and that excitation transfers incoherently between LH1 and RC.

Culture conditions affecting H2 production by phototrophic bacterium Rhodobacter sphaeroides KD131

The effect of culture conditions on photo-H2 production was investigated using the photosynthetic bacterium Rhodobacter sphaeroides KD131. When the initial cell concentrations were either below or above a threshold of 0.56g-dcw/L, the H2 production decreased due to an imbalance between the biomass and the substrate. Malate- and succinate-fed cultures exhibited the highest substrate conversion to H2 production, whereas more than 85% of the substrate was utilized for cell growth in acetate- and butyrate-fed cultures. Compared with (NH4)2SO4, glutamate as a nitrogen source was more appropriate for the initial H2 production, but inhibited H2 evolution during extended cultivation due to released NH4+ ion. Even though the KD131 strain grew well under slightly acidic conditions, the pH value should be maintained in a neutral range in order to enhance H2 production. The highest H2 yield of 3.65mol-H2/mol-succinate was achieved when the KD131 strain grew in the succinate–glutamate medium with an initial cell concentration of 0.56g-dcw/L and the pH level controlled to 7.5.

Adaptation of intracytoplasmic membranes to altered light intensity in Rhodobacter sphaeroides

The model photosynthetic bacterium Rhodobacter sphaeroides uses a network of bacteriochlorophyll (BChl)–protein complexes embedded in spherical intracytoplasmic membranes (ICM) to collect and utilise solar energy. We studied the effects of high- and low-light growth conditions, where BChl levels increased approximately four-fold from 1.6×106 to 6.5×106 molecules per cell. Most of this extra pigment is accommodated in the proliferating ICM system, which increases from approximately 274 to 1468 vesicles per cell. Thus, 16×106nm2 of specialised membrane surface area is made available for harvesting and utilising solar energy compared to 3×106nm2 under high-light conditions. Membrane mapping using atomic force microscopy revealed closely packed dimeric and monomeric reaction centre-light harvesting 1-PufX (RC-LH1-PufX) complexes in high-light ICM with room only for small clusters of LH2, whereas extensive LH2-only domains form during adaptation to low light, with the LH2/RC ratio increasing three-fold. The number of upper pigmented band (UPB) sites where membrane invagination is initiated hardly varied; 704 (5.8×105 BChls/cell) and 829 (4.9×105 BChls/cell) UPB sites per cell were estimated under high- and low-light conditions, respectively. Thus, the lower ICM content in high-light cells is a consequence of fewer ICM invaginations reaching maturity. Taking into account the relatively poor LH2-to-LH1 energy transfer in UPB membranes it is likely that high-light cells are relatively inefficient at energy trapping, but can grow well enough without the need to fully develop their photosynthetic membranes from the relatively inefficient UPB to highly efficient mature ICM.

The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 μM) of mercury (Hg²⁺) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg²⁺ sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c₂ and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc₁ complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.

Effects of the Cryptochrome CryB from Rhodobacter sphaeroides on Global Gene Expression in the Dark or Blue Light or in the Presence of Singlet Oxygen

Several regulators are controlling the formation of the photosynthetic apparatus in the facultatively photosynthetic bacterium Rhodobacter sphaeroides. Among the proteins affecting photosynthesis gene expression is the blue light photoreceptor cryptochrome CryB. This study addresses the effect of CryB on global gene expression. The data reveal that CryB does not only influence photosynthesis gene expression but also genes for the non-photosynthetic energy metabolism like citric acid cycle and oxidative phosphorylation. In addition several genes involved in RNA processing and in transcriptional regulation are affected by a cryB deletion. Although CryB was shown to undergo a photocycle it does not only affect gene expression in response to blue light illumination but also in response to singlet oxygen stress conditions. While there is a large overlap in these responses, some CryB-dependent effects are specific for blue-light or photooxidative stress. In addition to protein-coding genes some genes for sRNAs show CryB-dependent expression. These findings give new insight into the function of bacterial cryptochromes and demonstrate for the first time a function in the oxidative stress response.

Effects of pH and Carbon Sources on Biohydrogen Production by Co-Culture of Clostridium butyricum and Rhodobacter sphaeroides

To improve the hydrogen yield from biological fermentation of organic wastewater, a co-culture system of dark- and photo-fermentation bacteria was investigated. In a pureculture system of the dark-fermentation bacterium Clostridium butyricum, a pH of 6.25 was found to be optimal, resulting in a hydrogen production rate of 18.7 ml-H₂/l/h. On the other hand, the photosynthetic bacterium Rhodobacter sphaeroides could produce the most hydrogen at 1.81 mol-H₂/mol-glucose at pH 7.0. The maximum specific growth rate of R. sphaeroides was determined to be 2.93 h⁻¹ when acetic acid was used as the carbon source, a result that was significantly higher than that obtained using either glucose or a mixture of volatile fatty acids (VFAs). Acetic acid best supported R. sphaeroides cell growth but not hydrogen production. In the co-culture system with glucose, hydrogen could be steadily produced without any lag phase. There were distinguishable inflection points in a plot of accumulated hydrogen over time, resulting from the dynamic production or consumption of VFAs by the interaction between the dark- and photofermentation bacteria. Lastly, the hydrogen production rate of a repeated fed-batch run was 15.9 ml-H₂/l/h, which was achievable in a sustainable manner.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Influence of environment induced correlated fluctuations in electronic coupling on coherent excitation energy transfer dynamics in model photosynthetic systems

Two-dimensional photon-echo experiments indicate that excitation energy transfer between chromophores near the reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides occurs coherently with decoherence times of hundreds of femtoseconds, comparable to the energy transfer time scale in these systems. The original explanation of this observation suggested that correlated fluctuations in chromophore excitation energies, driven by large scale protein motions could result in long lived coherent energy transfer dynamics. However, no significant site energy correlation has been found in recent molecular dynamics simulations of several model light harvesting systems. Instead, there is evidence of correlated fluctuations in site energy-electronic coupling and electronic coupling-electronic coupling. The roles of these different types of correlations in excitation energy transfer dynamics are not yet thoroughly understood, though the effects of site energy correlations have been well studied. In this paper, we introduce several general models that can realistically describe the effects of various types of correlated fluctuations in chromophore properties and systematically study the behavior of these models using general methods for treating dissipative quantum dynamics in complex multi-chromophore systems. The effects of correlation between site energy and inter-site electronic couplings are explored in a two state model of excitation energy transfer between the accessory bacteriochlorophyll and bacteriopheophytin in a reaction center system and we find that these types of correlated fluctuations can enhance or suppress coherence and transfer rate simultaneously. In contrast, models for correlated fluctuations in chromophore excitation energies show enhanced coherent dynamics but necessarily show decrease in excitation energy transfer rate accompanying such coherence enhancement. Finally, for a three state model of the Fenna-Matthews-Olsen light harvesting complex, we explore the influence of including correlations in inter-chromophore couplings between different chromophore dimers that share a common chromophore. We find that the relative sign of the different correlations can have profound influence on decoherence time and energy transfer rate and can provide sensitive control of relaxation in these complex quantum dynamical open systems.

The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of PA and BB bacteriochlorophylls

To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl PA or BChl BB. Contrary to expectations, the double mutation I(L177)H+H(L173)L does not bring about a heterodimer RC but causes a 46nm blue shift of the long-wavelength P absorbance band. The histidine L177 or a water molecule were suggested as putative ligands for PA in the RC I(L177)H+H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9Å shows changes at the interface region between the BChl PA and the monomeric BChl BB. Spectral and pigment analysis provided evidence for β-coordination of the BChl BB in the double mutant RC I(L177)H+H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the β-coordination of the BChl BB. Possible structural consequences of the L177 mutation affecting the coordination of the two BChls PA and BB are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Incorporation of the bacterial reaction centre into dendrimersomes

For the first time the ability of the first generation dendrimer belonging to the family of polyester-benzylether, (3,5)12G1-PE-BMPA-(OH)4, to form dendrimersomes is presented together with their capability to reconstitute the integral membrane protein complex called Reaction Centre (RC) purified from the photosynthetic bacterium Rhodobacter sphaeroides. Size, polydispersity and time stability of the empty and protein containing dendrimersomes are presented together with the photochemical activity of the guest protein. The RC presence appears to strongly enhance the self-assembly properties of the Janus dendrimer, leading to the formation of proteo-dendrimersomes showing a photochemical activity similar to that found for RC in solution. The interaction of the embedded RC with reduced cyt-c has also been investigated, indicating that the incorporation of the protein is vectorial (almost 90% of the guest protein faces the dendrimerosomes exterior with its cyt-c docking site) at variance with lecithin liposomes where the reconstitution is essentially statistical.

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%.

A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes

Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment–protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption–fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption–fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Role of a short light, oxygen, voltage (LOV) domain protein in blue light- and singlet oxygen-dependent gene regulation in Rhodobacter sphaeroides

The facultatively photosynthetic bacterium Rhodobacter sphaeroides harbours an unusual light, oxygen, voltage (LOV) domain protein, RsLOV. While showing a characteristic photocycle, the protein lacks a C-terminal output domain, similar to PpSB2 in Pseudomonas putida. Oxygen tension and light quantity are the two factors mainly responsible for controlling the expression of photosynthesis genes in R. sphaeroides. Two photoreceptor proteins are known to be involved in this regulation: the intensively studied AppA protein and the more recently identified cryptochrome-like protein CryB. Here we show by transcriptome and physiological studies that RsLOV is also involved in the regulation of photosynthetic gene expression. Our data further hint at a connection between RsLOV, carbohydrate metabolism and chemotaxis, as well as with the cellular response to photooxidative stress. RsLOV affects not only blue light-dependent gene expression but also redox-dependent regulation.

The RSP_2889 gene product of Rhodobacter sphaeroides is a CueR homologue controlling copper-responsive genes

Metal homeostasis is important in all living cells in order to provide sufficient amounts of metal ions for biological processes but to prevent toxic effects by excess amounts. Here we show that the gene product of RSP_2889 of the facultatively photosynthetic bacterium Rhodobacter sphaeroides is homologous to CueR, a regulator of copper metabolism in Escherichia coli and other bacteria. CueR binds to the promoter regions of genes for a copper-translocating ATPase and for a copper chaperone and is responsible for their high expression when cells are exposed to elevated levels of copper ions. While deletion of RSP_2889 has no significant effect on copper resistance, expression from a low-copy-number plasmid mediates increased sensitivity to copper.

Pathways Involved in Reductant Distribution during Photobiological H 2 Production by Rhodobacter sphaeroides

We used global transcript analyses and mutant studies to investigate the pathways that impact H(2) production in the photosynthetic bacterium Rhodobacter sphaeroides. We found that H(2) production capacity is related to the levels of expression of the nitrogenase and hydrogenase enzymes and the enzymes of the Calvin-Benson-Bassham pathway.

Monomeric RC–LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre–light-harvesting 1–PufX (RC–LH1–PufX) ‘core’ complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX −). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX − mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC–LH1–PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX − membranes, resulting in locally ordered clusters of monomeric RC–LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.

Enhancing photo-fermentative hydrogen production by Rhodobacter sphaeroides KD131 and its PHB synthase deleted-mutant from acetate and butyrate

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H 2) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H 2 yield (HY) of 0.65 mol H 2/mol acetate- and 2.50 mol H 2/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H 2/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H 2 production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H 2 production. When pH was controlled to 7.0, strain KD131 wt evolved more H 2 by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H 2 production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH 4) 2SO 4. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H 2 production in expending reducing power.