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 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.

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.