Reverse transcription (RT) PCR analysis of transcript levels in mutants attenuated in hydrogen photoproduction. ZUZANNA CZERNIK (Scripps College Claremont, CA 91711) MATT WECKER (National Renewable Energy Laboratory, Golden, CO, 89401)

The green alga, Chlamydomonas reinhardtii is capable of sustained H2 photoproduction under anaerobic conditions. Algae could be an excellent renewable, cheap and clean source of hydrogen, if the levels of production could be increased. One hope for increasing hydrogen levels is the creation of algae mutants which are more efficient producers of the gas. In order to do this the genetic pathway has to be properly understood. NREL has many mutants ready for study to. However the current method for studying these mutants is long. In order to streamline the process a new protocol using RT-PCR was developed. RT-PCR allows for the presence of RNA to be detected, and the amount present to be quantified. Various parameters involved in the procedure were tested, adjusted and changed to create a working protocol. Once the protocol was working a dark adaptation experiment was repeated for mutant strain 103-9 and wild-type 425. RNA was isolated, and PCR on control genes and genes involved in the hydrogen pathway was performed. The results confirm the previous results obtained: mRNA for HydEF, which assembles a working hydrogenase enzyme, is not present in the mutant. Since the new method also allows quantification a new discovery was made. The relative abundance of HydA, which codes for the hydrogenase enzyme, is higher in the mutant than in the wild-type. Since the mutant is unable to produce hydrogen, perhaps it reacts by up-regulating hydrogenase production under conditions that would normally force it to create hydrogen. These results will need to be verified still. The protocol can now be used to study other mutants as well.

The Use of Spectroscopic Analysis Tools To Evaluate Changes in the Composition of Pretreated Biomass. KILEY MACK (University of Colorado Boulder, CO 80309) STEVE KELLEY (National Renewable Energy Laboratory, Golden, CO, 89401)

The biomass that is used for production of ethanol must generally be pretreated before it is converted. These pretreatments change the chemical and structural properties of biomass and make it more easily converted to ethanol. The changes can be evaluated using spectroscopic techniques like Near Infrared (NIR) spectroscopy and Fourier Transform Infrared (FT-IR) spectroscopy. These methods offer comprehensive information on changes in the chemical composition of pretreated biomass. They are also faster and less expensive than traditional wet chemical methods used to analyze biomass. The purpose of this study was to use these spectroscopic tools to understand changes in the composition and structure of spruce wood chips that had been subjected to different pretreatment processes. A secondary goal was to compare the rapid analysis methods in terms of their reproducibility and chemical insight. Multivariate statistical tools were used to relate changes in the composition of the samples to the pretreatment conditions (temperature, time, acid concentration, etc.) and to compare the rapid analysis methods. The chemical analysis indicated a strong correlation between the concentration of the acid used in the pretreatment and the loss of acetate from the hemicellulose. Both of the spectroscopic tools, in combination with multivariate analysis (MVA) techniques, were valuable for differentiating between the samples; however, the FT-IR was more useful for discerning the chemical changes that took place during the pretreatment process.