Amino acids as possible alternative nitrogen source for growth of Euglena gracilis Z in life support systems.

In recent times Euglena gracilis Z was employed as primary producer in closed environmental life-support system (CELSS), e.g. in space research. The photosynthetic unicellular flagellate is not capable of utilizing nitrate, nitrite, and urea as nitrogen source. Therefore, ammonium is supplied as an N-source in the lab (provided as diammonium-dihydrogenphosphate, (NH4)2HPO4) to E. gracilis cultures. While nitrate exerts low toxicity to organisms, ammonium is harmful for many aquatic organisms especially, at high pH-values, which causes the ionic NH4+ (low toxicity) to be partially transformed into the highly toxic ammonia, NH3. In earlier reports, Euglena gracilis was described to grow with various amino acids as sole N-source. Our aim was to investigate alternatives for (NH4)2HPO4 as N-source with lower toxicity for organisms co-cultivated with Euglena in a CELSS. The growth kinetics of Euglena gracilis cultures was determined in the presence of different amino acids (glycine, glutamine, glutamic acid, leucine, and threonine). In addition, uptake of those amino acids by the cells was measured. Cell growth in the presence of glycine and glutamine was quite comparable to the growth in (NH4)2HPO4 containing cultures while a delay in growth was observed in the presence of leucine and threonine. Unlike, aforementioned amino acids glutamate consumption was very poor. Cell density and glutamate concentration were almost unaltered throughout the experiment and the culture reached the stationary phase within 8 days. The data are compared with earlier studies in which utilization of amino acids in Euglena gracilis was investigated. All tested amino acids (glutamate with limitations) were found to have the potential of being an alternative N-source for Euglena gracilis. Hence, these amino acids can be used as a non-toxic surrogate for (NH4)2HPO4.

The influence of microgravity on Euglena gracilis as studied on Shenzhou 8.

The German Aerospace Center (DLR) enabled German participation in the joint space campaign on the unmanned Shenzhou 8 spacecraft in November 2011. In this report, the effect of microgravity on Euglena gracilis cells is described. Custom-made dual compartment cell fixation units (containing cells in one chamber and fixative - RNA lysis buffer - in another one) were enclosed in a small container and placed in the Simbox incubator, which is an experiment support system. Cells were fixed by injecting them with fixative at different time intervals. In addition to stationary experiment slots, Simbox provides a 1 g reference centrifuge. Cell fixation units were mounted in microgravity and 1 g reference positions of Simbox. Two Simbox incubators were used, one for space flight and the other as ground reference. Cells were fixed soon after launch and shortly before return of the spaceship. Due to technical problems, only early in-flight samples (about 40 min after launch microgravity and corresponding 1 g reference) were fully mixed with fixative, therefore only data from those samples are presented. Transcription of several genes involved in signal transduction, oxidative stress defence, cell cycle regulation and heat shock responses was investigated with quantitative PCR. The data indicate that Euglena cells suffer stress upon short-term exposure to microgravity; various stress-induced genes were up-regulated. Of 32 tested genes, 18 were up-regulated, one down-regulated and the rest remained unaltered. These findings are in a good agreement with results from other research groups using other organisms.

Gravitaxis and graviperception in flagellates.

There is strong evidence that gravitactic orientation in flagellates and ciliates is mediated by an active physiological gravireceptor rather than by passive alignment of the cells in the water column. In flagellates the threshold for graviorientation was found to be at 0.12 x g on a slow rotating centrifuge during the IML-2 mission on the Shuttle Columbia and a subsequent parabolic rocket flight (TEXUS). During the IML-2 mission no adaptation to microgravity was observed over the duration of the space flight, while gravitaxis was lost in a terrestrial closed environmental system over the period of almost two years. Sedimenting statoliths are not likely to be involved in graviperception because of the small size of the cells and their rotation around the longitudinal axis during forward locomotion. Instead the whole cytoplasmic content of the cell, being heavier than the surrounding aqueous medium (1.05 g/ml), exerts a pressure on the lower membrane. This force activates stretch-sensitive calcium specific ion channels which can be inhibited by the addition of gadolinium which therefore abolishes gravitaxis. The channels seem to mainly allow calcium ions to pass since gravitaxis is blocked by the addition of the calcium ionophore A23187 and by vanadate which blocks the Ca-ATPase in the cytoplasmic membrane. Recently, a gene for a mechanosensitive channel, originally sequenced for Saccharomyces, was identified in Euglena by PCR. The increase in intracellular free calcium during reorientation can be visualized by the fluorophore Calcium Crimson using laser excitation and image intensification. This result was confirmed during recent parabolic flights. The gated calcium changes the membrane potential across the membrane which may be the trigger for the reorientation of the flagellum. cAMP plays a role as a secondary messenger. Photosynthetic flagellates are suitable candidates for life support systems since they absorb CO2 and produce oxygen. Preliminary experiments are discussed.