Team:TU-Munich/Results/Moss

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Growing Physcomitrella patens on solid materials

For implementation of the PhyscoFilter it is elementary to know about the mosses growth behavior on different surfaces. We therefore tried 5 different materials and came to the conclusion that the moss grows very well on all of them, but especially felt material would suit our plans for implementing the filter system in form of a remediation raft since the moss can easily cling to the fibers, which are also dense enough to prevent the moss from being washed away. Also the spongy properties of felt make it an ideal surface for the plant as it ensures a constant supply of water. The second best material is probably agar or metal grid on agar, but this has the disadvantage of being washed away gradually.

Determination of growth rates for different liquid culture forms

In parallel to the determination of growth conditions for solid materials we examined different growth conditions in liquid culture, too. In general the handling of liquid cultures of moss is more easy than the one of those growing on solid media as the moss can be disrupted mechanically with an Ultra-Turrax. That way homogenized cultures can be achieved very comfortably. Moreover the growth of moss in liquid suspension cultures provides an improved and constant nourishment of cells without any nutrition gradients and continuously pH adjusted. Even if the up scaling process up to volumes of around 20 L in standard stirred tank bioreactors is more convenient at a first glance, the further up scaling process is often limited by an insufficient light input resulting in suboptimal growth rates. This is physically determined as the volume increases in the third potency whereas the surface only increases in the second potency. Therefore larger suspension volumes require different and often technically more challenging bioreactor forms as tube reactors, plate or wave reactors.

In our approaches we tested mainly the influence of mixing and coupled to this of aeration on the growth of moss. For this purpose 500 mL flasks containing 250 mL Knob media were inoculated with 50 mL moss suspension which had been disrupted 24 h ago (corresponding a moss concentration of 80 mg dry mass per liter). In triplicates the growth conditions in standing, shaken and aerated flasks were determined for 9 days at room temperature and a normal dark/light rhythm (8/16h).

Figure 1: Different experimental setups for the growth of wild type moss in liquid culture. A: Aerated flasks, B: Standing flask, C: Shaken flask
Figure 2: Dryweight determined for the different cultivation methods after nine days of incubation at normal growth conditions.

All flasks were inoculated with 80 mg dry weight per litre moss. After nine days of incubation the biomass in the standing flask stayed approximately constant (82 mg per litre) compared to the beginning of the experiment. The biomasses in the shaken and in the aerated flasks increased in the same time to 118 and 168 mg per litre respectively. As all flaks were incubated under the same temperature and illumination conditions the internal mixing and especially linked to this the aeration seems to be of importance for biomass generation. Normally at the Reski laboratory the bioreactors are aerated with 0,3 vvm at a light intensity of 55 μmol m-2 s-1 . So far unpublished results indicate that aeration with up to 6 volume percent carbon dioxide improves the growth rate if the light intensity is increased as well. Nevertheless an increased light intensity automatically requires a stronger cooling capacity of the bioreactor due to photons which are not absorbed by the photo systems. Therefore the development of illumination which only serves the wavelengths required by the photosystems in plants could be an interesting alternative. Moreover the addition of further carbon sources than carbon dioxide could boost the growth of the moss as well. So far the addition of glucose leads to a change of colour to brown of the moss plants if applied for longer than 14 days. An optimization of the media composition as well as the testing of different feeding strategies could help to solve this problem.

Tolerance to relevant environmental pollutants and toxins

To test whether and how the moss reacts to toxins and pollutants, which can occur in waste and surface water and which our PhyscoFilter should remove, wild type plants were incubated in serial dilutions of the toxic substances. As a negative control distilled water was used. After 4, 7, 10 and 19 days the plants were screened with a light microscope, where one could easily differentiate between alive and dead plants. The latter occurred in two different phenotypes, one appearing transparent (dead moss 1) because it lost its chlorophyll, the other black (dead moss 2).

Figure 3: Toxicity assay for wild type moss
Table 1: Typical concentrations of toxic substances
Substance Application Concentration
Ampicillin Antibiotic agar plate 0.1 g/L[1]
Chloramphenicol Antibiotic agar plate 0.025 g/L[1]
Kanamycin Antibiotic agar plate 0.05 g/L[1]
Tetracycline Antibiotic agar plate 0.01 g/L[1]
Geneticin (G418) Antibiotic agar plate (for moss selection) 12.5 mg/L[2]
Diclofenac 1 tablet (25-50 mg) dissolved in 6 L (blood circuit) 4.2-8.3 mg/L
NaCl Sea water 3.5 g/L
Catechol Death of Arabidopsis 55 mg/L[3]
Erythromycin 1 tablet (500 mg) dissolved in 6 L (blood circuit) 0.08 g/L

The result of this toxicity assay is, that wild type plants are not negatively affected by waste water treatment plant (WWTP) effluents, which were sampled from the local WWTPs Großlappen (waste water 1) and Garching (waste water 2). So the filter system could work effectively placed in the effluent stream of WWTPs or on surface water. However, sea water seems to influence the vitality of the moss, so the implementation of the PhyscoFilter in salt water is not recommended. Furthermore we can conclude that substances the genetically modified moss should degrade (Erythromycin, Catechol) or accumulate (Diclofenac) only affect the plant - if they do at all - at concentrations much higher than they occur naturally (see table 1). Also the assay indicates that it is possible to grow the plant on agar plates with often used antibiotics (Tetracycline, Ampicillin, Chloramphenicol, Kanamycin), since the working concentration has no influence on the moss. This can be very useful to prevent bacterial contamination of plates. As expected G418 shows toxic influence on wild type moss and can therefore be used as selection substance for transformed plants, though it takes a few days to take effect.

References:

[University of Toledo, Department of Environmental Sciences]
[plant-biotech.net ]
[Physcomitrella cell culture conditions]
[Liao,Y. et al, 2006] Liao,Y. et al, (2006). The Key Role of Chlorocatechol 1,2-Dioxygenase in Phytoremoval and Degradation of Catechol by Transgenic Arabidopsis. Plant Physiology, 142(2): 620–628.

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