New Approach for the Construction and Calibration of Gas-Tight Setups for Biohydrogen Production at the Small Laboratory Scale.

Biohydrogen production in small laboratory scale culture vessels is often difficult to perform and quantitate. One problem is that commonly used silicon tubing and improvised plastic connections used for constructing apparatus are cheap and easy to connect but are generally not robust for gases such as hydrogen. In addition, this type of apparatus presents significant safety concerns. Here, we demonstrate the construction of hydrogen-tight apparatus using a commercially available modular system, where plastic tubing and connections are made of explosion-proof dissipative plastic material. Using this system, we introduce a gas chromatograph calibration procedure, which can be easily performed without necessarily resorting to expensive commercial gas standards for the calibration of hydrogen gas concentrations. In this procedure, the amount of hydrogen produced by the reaction of sodium borohydride with water in a closed air-filled bottle is deduced from the observed decrease of the oxygen partial pressure, using the ideal gas law. Finally, the determined calibration coefficients and the gas-tight apparatus are used for the analysis of simultaneous oxygen consumption and hydrogen production of the purple photosynthetic bacterium, Rhodospirillum rubrum, during semi-aerobic growth in the dark.

The Methoxylated, Highly Conjugated C40 Carotenoids, Spirilloxanthin and Anhydrorhodovibrin, Can Be Separated Using High Performance Liquid Chromatography with Safe and Environmentally Friendly Solvents.

High performance liquid chromatography (HPLC) is a frequently used technique in carotenoid research. So far, however, little attention has been paid to the fact that many of the organic solvents used in HPLC separation of highly apolar C40 carotenoids impose a significant threat to both health (especially for women) and the general laboratory environment. Here, we developed a solvent combination capable of allowing high-resolution HPLC separation of the C40 carotenoid, spirilloxanthin, and all of its biosynthetic precursors beginning with phytoene, using relatively safe, environmentally friendly solvents. We show that separation of spirilloxanthin and its precursors anhydrorhodovibrin and lycopene using modern ultra-high performance chromatography (UHPLC) poses particular problems for apolar carotenoid separation, due to the long residence times in the sample delivery system, which facilitates carotenoid aggregation. We resolved these problems by developing the solvent delivery combination acetone/acetonitrile/isopropanol/methanol (65/30/5/2 (v/v/v/v)), which allows excellent column separation using the safe isocratic solvent system methanol/tetrahydrofuran (98/2 (v/v)). We also demonstrate that the development strategy for optimizing a solvent system for carotenoid separation can be well-described by the use of the average dielectric constant of the total sample delivery solvent, and present a formal method for analysis of the efficiency of separation.

The monofunctional cobalamin biosynthesis enzyme precorrin-3B synthase (CobZRR) is essential for anaerobic photosynthesis in Rhodospirillum rubrum but not for aerobic dark metabolism.

The in vivo physiological role of the gene cobZ, which encodes precorrin-3B synthase, which catalyzes the initial porphyrin ring contraction step of cobalamin biosynthesis via the cob pathway, has been demonstrated here for the first time. Cobalamin is known to be essential for an early step of bacteriochlorophyll biosynthesis in anoxygenic purple bacteria. The cobZ (cobZRR) gene of the purple bacterium Rhodospirillum rubrum was localized to a 23.5 kb insert of chromosomal DNA contained on the cosmid pSC4. pSC4 complemented several mutants of bacteriochlorophyll and carotenoid biosynthesis, due to the presence of the bchCX and crtCDEF genes at one end of the cosmid insert, flanking cobZRR. A second gene, citB/tcuB, immediately downstream of cobZRR, shows homologies to both a tricarballylate oxidoreductase (tcuB) and a gene (citB) involved in signal transduction during citrate uptake. CobZRR shows extensive homology to the N-terminal domain of the bifunctional CobZ from Rhodobacter capsulatus, and the R. rubrum citB/tcuB gene is homologous to the CobZ C-terminal domain. A mutant, SERGK25, containing a terminatorless kanamycin interposon inserted into cobZRR, could not grow by anaerobic photosynthesis, but grew normally under dark, aerobic and microaerophilic conditions with succinate and fructose as carbon sources. The anaerobic in vivo activity of CobZ indicates that it does not require oxygen as a substrate. The mutant excreted large amounts of protoporphyrin IX-monomethylester, a brown precursor of bacteriochlorophyll biosynthesis. The mutant was complemented either by the cobZRR gene in trans, or when exogenous cobalamin was added to the medium. A deletion mutant of tcuB/citB did not exhibit the cob phenotype. Thus, a role for tcuB/citB in cobalamin biosynthesis could not be confirmed.

Quantum Redirection of Antenna Absorption to Photosynthetic Reaction Centers.

The early steps of photosynthesis involve the photoexcitation of reaction centers (RCs) and light-harvesting (LH) units. Here, we show that the historically overlooked excitonic delocalization across RC and LH pigments results in a redistribution of absorption amplitudes that benefits the absorption cross section of the optical bands associated with the RC of several species. While we prove that this redistribution is robust to the microscopic details of the dephasing between these units in the purple bacterium Rhodospirillum rubrum, we are able to show that the redistribution witnesses a more fragile, but persistent, coherent population dynamics which directs excitations from the LH toward the RC units under incoherent illumination and physiological conditions. Even though the redirection does not seem to affect importantly the overall efficiency in photosynthesis, stochastic optimization allows us to delineate clear guidelines and develop simple analytic expressions in order to amplify the coherent redirection in artificial nanostructures.

A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies.

Mechanistic details of methanol oxidation catalyzed by the periplasmically-located pyrroloquinoline quinone-dependent methanol dehydrogenase of methylotrophs can be elucidated using site-directed mutants. Here, we present an in situ colony assay of methanol dehydrogenase, which allows robotic screening of large populations of intact small colonies, and regrowth of colonies for subsequent analysis.

Random mutagenesis and overexpression of rhodopin-3,4-desaturase allows the production of highly conjugated carotenoids in Rhodospirillum rubrum.

The crtD gene of the purple bacterium Rhodospirillum rubrum, encoding rhodopin desaturase, was cloned into a broad-host range expression plasmid (pRKCAG53) and transferred to the R. rubrum crtD(-) mutant, ST4, which restored the wild-type phenotype and produced the carotenoid spirilloxanthin. pRKCAG53 was randomly mutated in an Escherichia coli mutator strain and then transferred to ST4 for selection of non-wild-type phenotypes. Strains containing the mutated expression plasmid exhibited two coloured phenotypes: a "brown" phenotype, corresponding to 3,4,3',4'-tetrahydrospirilloxanthin, arising from plasmids containing an inactivated crtD gene, and secondly, a "dark pink" phenotype. Absorption and mass spectra and HPLC analysis obtained from hexane extracts of brown mutants, confirmed the carotenoid assignment above. DNA sequence analysis of the crtD genes from the brown transconjugants showed frameshifts at the extreme C-terminus, suggesting that this domain forms part of the active site. Spectral analysis of the dark pink strains showed an additional, non-natural double bond formed at the carotenoid end, yielding the asymmetric carotenoids, 3,4,3',4'-tetradehydrorhodopin - and 3',4'-didehydroanhydrorhodovibrin, each containing 14 conjugated double bonds. For only two dark pink strains, was a mutation in crtD detected, in both cases at the N-terminus of CrtD. Otherwise, the higher conjugation was ascribed to an elevated plasmid copy number.