Comparative physiological parameters for the analysis of H2 production by the microalga Chlamydomonas reinhardtii and the photosynthetic purple bacterium Rhodospirillum rubrum

Abstract

The main focus of this thesis in Part I is the investigation and establishment of growth benchmarks to enable better comparison of H2 evolution in the microalga Chlamydomonas reinhardtii with other non-related bioH2 producers, especially the purple bacterium Rhodospirillum rubrum. For R. rubrum, the physiological benchmarks have been worked out by the Ghosh group (Grammel, H., Gilles, E.D. and Ghosh, R. (2003). Appl. Environ. Microbiol. 69, 6577-6586.) The major objectives adopted towards the fulfilment of our goal have been as follows: (1) the establishment of standard growth and aerobic culture conditions for C. reinhardtii strain SAG 18.79 (CC-1418) for establishment of biochemical benchmarks and (2) evaluating the H2 production capacity of C. reinhardtii on the basis of biochemical benchmarks and culture volume considerations in cultures grown under sulphur depletion conditions. Extensive method development has been carried out to meet these objectives. First, commercially available gas-tight (conducting) tubing and connections were used to construct professional small-scale gas sampling vessels. Secondly, the calibration of the apparatus was performed by measuring the H2 evolved from the well-known reaction of NaBH4 with water. With the H2 evolution data, I demonstrated that, the application of gas laws enables determination of an unknown gas (here H2) concentration by gas chromatography, in the absence of standard gas calibration mixtures. Thirdly, enzymatic and colorimetric assays were developed for the measurement of key substrates: (1) starch in C. reinhardtii; and (2) fructose in R. rubrum. A comprehensive protocol for the extraction and hydrolysis of starch from cells, and the assay of glucose by the glucose oxidase-horseradish peroxidase enzyme reaction along with chromogenic substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) has been described. For the determination of fructose, the well-known Kulka method was substantially modified and adapted to a small scale assay, presented in Part III of this thesis. The modified assay contains a smaller volume and a safer formulation (5.4 M) of HCl, than the original method, and is shown to be suitable for use in high-throughput systems biology or enzymatic applications. The assay was used for monitoring the consumption of fructose during growth experiments as well as during the H2 production phase of R. rubrum. Additionally, the use of this assay has been extended for the rapid measurement of the phosphorylated sugar 1-deoxy-D-xylulose-5-phosphate, obviating the necessity of lengthy HPLC analysis. This work has now been published. With the use of methodologies established above, we have determined a physiological dataset for C. reinhardtii which can be objectively compared to the reference data from R. rubrum. For C. reinhardtii, we present a systematic comparison of “benchmark” parameters such as biomass units, cell number, chlorophyll, starch and protein content of cultures measured at two different light intensities. I show that, a simple measurable parameter, the culture turbidity, can be interconverted via linear curve fitting functions within the exponential growth phase to estimate any of the benchmark parameters at both light intensities. To our knowledge, no systematic correlation of growth parameters for C. reinhardtii has ever been published. A comparison of the amounts and rates of H2 produced by the C. reinhardtii strain SAG 18.79 has been made under both “intermediate” light intensity (70 µmol photons/m2/s) and “very low” light intensity (5 µmol photons/m2/s). The values obtained, have been compared to those of other bioH2 producers in terms of culture volume and biomass wherever available. The kinetics of H2 accumulation in the bioreactor headspace for C. reinhardtii strain SAG 18.79 during sulphur depletion, showed a H2 production phase lasting 40 - 75 h depending on the light intensity, followed by an equivalent H2 uptake phase. For most of the widely studied strains, H2 uptake has been observed only under special conditions. For the strain used here, H2 uptake is considerable and may be a good model for studying this phenomenon. A discussion of the present state of bioH2 technology and the degree of improvement required to achieve conventional production levels have been calculated at the end of Part I. In purple bacteria, H2 production occurs primarily via the nitrogenase, which is only expressed at limiting [NH4+]. In Part II of the thesis, the effect of varied [NH4+] on R. rubrum growth and nif gene expression was examined under photosynthetic, anaerobic and dark, semi-aerobic conditions by the use of a newly developed, modified M2SF medium- here designated as M2NF medium. I show that nif gene expression in R. rubrum is regulated by [NH4+] within the concentration range of 0-10 mM. At the highest end of this concentration range, nif gene expression is totally repressed. At intermediate levels of [NH4+], nif gene expression occurs almost linearly at the expense of lower attainable cell densities, due to nitrogen limitation. This work forms the basis for further examination of nif gene regulation and consequently H2 production by use of other N- sources such as amino acids.