A variety of unicellular green algae have the ability to photo-produce molecular hydrogen (H2). Using sunlight to power the production of H2 from water is attractive due to the abundant supply of both resources and the potential for the technology to address global warming and energy supply concerns. Increasing levels of H2 production from those currently achievable with algal systems is a necessity for the technology to become economically feasible. Green unicellular algae are rare amongst organisms in that some have an ability to switch to an H2-producing metabolism when environmental conditions become anaerobic. The process of H2 production is greatly accentuated in the light due to the role of the photosynthetic apparatus directing electron flow to hydrogenase enzymes located in the chloroplast. Difficulties in maintaining continuous systems of H2 production largely result from the O2 sensitivity of hydrogenase enzymes. As O2 is generally produced through photosynthesis, the process of H2 production has always been short-lived. Recently, a process of inducing H2 production for several days was accomplished by depriving the growth medium of sulphur (Melis et al., 2000). Lacking sulphur, photosystem II activity diminishes to a point where any O2 evolved is consumed by respiration; this leads to the culture becoming anaerobic and to the onset of H2 production. The method of sulphur depletion has proven to be very useful for studies of H2 production due to enhanced rates over longer time periods being possible. This work was performed to search for new H2-producing Australian algal species and to shed light upon the molecular and biochemical interactions occurring when algal species move from aerobic photosynthetic growth to an anaerobic H2-producing status. An assay to test new species for an H2-producing ability was developed and implemented; leading to the isolation of new H2-producing species from Australian waters. The assay involved purging algal cultures in the dark with N2, sealing them in bioreactors and then exposing them to light. Metabolic profiling performed during this assay revealed cells to rapidly enter a fermentative metabolism upon the onset of anoxia. Acetate, formate and ethanol were key metabolites produced alongside H2 during this period. Metabolomics was used as a tool to understand the biochemical interactions occurring during 120 h of sulphur depleted H2 production. Extraction protocols were developed that allowed the detection and identification of over 100 metabolites using gas chromatography coupled to mass spectrometry, nuclear magnetic resonance spectroscopy and thin layer chromatography. Shifts in primary energy metabolism when cells switch from O2 production to H2 production were revealed. Indications are that both starch and triacylglyceride accumulate during the first 24 h of sulphur depletion prior to anoxia. Following the onset of anoxia, fermentative metabolism begins, H2 is produced and amino acids generally increase. A build-up of toxic fermentative end products and a lack of sulphur are believed to cause the termination of H2 production, rather than a lack of energy reserves. Key achievements of this work have been: • The establishment of an assay that can be used for future bio-prospecting work aimed at finding H2-producing algal species. • The isolation of new H2-producing green algal species from Australian waters. • The establishment of protocols for the extraction of metabolites from small volumes (1 ml) of Chlamydomonas reinhardtii cultures for analysis on a variety of analytical platforms. • The mapping of changes in metabolism of C. reinhardtii during the switch from an aerobic environment to an anaerobic H2-producing environment. • A range of recommendations for future research that may lead to higher H2 production.
| Identifer | oai:union.ndltd.org:ADTP/279178 |
| Creators | Matthew Timmins |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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