Intermittent illumination increases biophotolytic hydrogen yield by Anabaena cylindrica.

Intermittent illumination increased H2 and C2H4 yields per unit of light from growing cells and from nitrogren-starved cells by 1.7- and 1.35-fold, respectively, as compared with continuous illumination.     

Analysis of ligand binding curves in terms of species fractions

The ligand binding curve for a macromolecular system presents the average number or ligand molecules bound per macromolecule as a function of the chemical potential or the logarithm of the ligand concentration. We show that various observable properties of this curve, for example its asymptotes and derivatives, are expressible in terms of linear combinations of the mole fractions α_i of macromolecules binding i molecules of ligand. Whenever enough such properties of the binding curve are known, the linear equations in α_i can be solved to give the mole fractions of each of the various macromolecular species. An application of these results is that a Hill plot for hemoglobin-ligand equilibrium where the asymptotes approach unit slope can be made to yield the four Adair constants by a simple algebraic method. A second use is that a knowledge of the first and second derivatives of the binding curve at points along the curve can yield the species fractions as functions of the degree of saturation without direct knowledge of the ligand binding constants. These methods are illustrated by some numerical examples.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain

We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for GND2 and HXK2 with the genes for transaldolase (TAL1), transketolase (TKL1), and pyruvate kinase (PYK1) in the Saccharomyces cerevisiae strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for TAL1, TKL1, and PYK1 were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be GND2-TAL1-HXK2-TKL1-HXK2-PYK1. The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.     

The relationship between fungal preservation method and secondary metabolite production in Metarhizium anisopliae and Fusarium oxysporum

The effects of preservation regime on secondary metabolite production in two genera of economically important fungi, Metarhizium anisopliae and Fusarium oxysporum, was assessed using thin layer chromatography and high performance liquid chromatography over a 2-year testing period. Five preservation regimes, commonly used in microbial culture collections throughout the world were investigated: continual sub-culture, lyophilization, storage of mycelial plugs in water, storage at –20 °C and cryopreservation with liquid nitrogen. Preservation regime influenced secondary metabolite production in the test fungi. Changes in secondary metabolite profiles occurred after relatively short storage periods in most strains, irrespective of the preservation treatment used. Although no preservation treatment can be guaranteed to provide total stability of secondary metabolite production, cryopreservation was the best of the methods tested. Response to preservation and storage also differed between strains of the same species. Therefore, there is a need to develop new and existing preservation criteria with an emphasis on strain-specific criteria in order to reduce the prospects of instability in secondary metabolite production.     

Antagonistic Properties of Two Bacterial Biocontrol Agents of Grey Mould Disease

Bacillus pumilus NCIMB 13374 and Pseudomonas fluorescens NCIMB 13373 inhibit the growth of Botrytis cinerea , the cause of grey mould of strawberries. Both antagonists increased the pH of the growth medium from pH 6 to pH 8-8.5 and both produced antifungal agents. The compound(s) produced by B. pumilus had a fungicidal effect during conidial germination, whereas the compound(s) produced by P. fluorescens had a fungistatic effect. There was no evidence for the production of inhibitory volatile compounds. Both isolates also showed the ability to inhibit other strawberry fungal pathogens, and have potential for a wider range of biocontrol of plant diseases.     

Quantification of mycelial development of arbuscular mycorrhizal fungi using image analysis

Quantitative and reproducible information concerning the development of the extraradical mycelium of arbuscular mycorrhizal fungi (AMF) is lacking due to the difficulties in extracting, identifying and estimating hyphal lengths. In this study, using a rhizobox growth system, the lengths of hyphae of AMF estimated using an image analysis system were not significantly different from data obtained by a trained observer using a modified grid-line intersect method. The assessment of lengths of hyphae on membrane filters or slides was, however, much quicker using image analysis, and allowed the complete sample to be quantified, unlike the grid-line method where a limited number of fields of view are assessed. The image analysis procedure is objective, observer-independent and less laborious than the manual method of assessment. Of the four different methods of sample preparation compared, membrane filter methods were found to be the most appropriate for quantitative sampling from three non-soil substrates. Glomus monosporum (UKC M3) produced twice as much extraradical mycelium and hyphal length per centimetre of colonised root than G. geosporum (BEG 11) on both leek and linseed in a durite sand at final harvest (63 days). Both AMF also produced more hyphal length per centimetre of colonised root on linseed than on leek. The spatial distribution of both AMF, however, was similar in durite sand and no correlation with levels of NaHCO₃-extractable phosphorus was noted. In a third experiment, with G. manihotis (UKC INDO-1) colonising a tropical forage legume, Pueraria phaseoloides, in two other growth substrates, a different pattern of development of the extra-radical mycelium was observed. Because of a higher content of particulate matter, which collected on the membrane filters, the extraction technique had to be modified to give optimal performance of the image analysis system.