The Production Workflow
After harvesting the algae biomass, the algae pellet is very viscous. For optimal handling, a suspension of the algae in a watery solution is required. Therefore we used eight times the volume of the algae to dissolve in 1M HCl. Less solution would not only mean worse handling, but also a higher buffering effect of algae lysis products. Therefore, hydrolysis of proteins would have been less efficient . Additionally, high amounts of HCl would mean a higher salt proportion in the end product. The solution was heated to 100 °C for more than one hour to increase the stress on the algae cells integrity. To maximize the protein hydrolysis, the algae’s cell wall and lipid layers were disrupted with a bead mill, using glass beads with a size of 200 µm (see Figure 1 A). After that, the solution had to be neutralized. Since standard saline is required in all growth media, we used NaOH for neutralizing, until a pH of 7.0 was reached. Amino acids, sugars and other biodegradable components are now in solution, while lipids and cell wall fragments sediment quickly. Since these components often cannot be metabolized by bacteria, we removed them via centrifugation. Additionally, some components coagulate during the sterilization process in an autoclave. To prevent this, the whole product was autoclaved and centrifuged, before the extract was dried. The end product was a white yellow powder similar to other cell extracts like soy peptone stock or BHIN stock. In solution, a bright yellow color appeared similar to the classic LB medium (see Figure 1 B).
Figure 1: A) shows glass beads used during cell extraction by a bead mill. B) shows the PhyCoVi medium with its typical yellowish color.
Validation of bacterial growth on PhyCoVi medium
To validate the applicability of PhyCoVi medium, for cultivation of all kinds of bacteria, we measured the growth of Vibrio natriegens, Escherichia coli (strain MG1655), Bacillus subtillis and Corynebacterium glutamicum photometrically at 600 nm over the curse of 315 to 360 minutes. We compared two different additional NaCl concentrations 1 % and 2 % (w/v) in five medium variants: first our PhyCoVi (PCV) medium containing algae extract, secondly, PCV with additional 1 % soy peptone. For control LB medium consisting of 0.5 % yeast extract and 1 % soy peptone, Brain-Heart-Infusion medium (BHIN) anda medium solemnly consisting of soy peptone and NaCl were used. For the exact compositions, see Table 1.
Table 1 - Quantity specification for all used mediums during the algae based PhyCoVi medium validation.
|Ingredients||PCV [g/L]||PCV + Soy Peptone [g/L]||LB medium [g/L]||BHIN [g/L]||Soy Peptone Control [g/L]|
|NaCl||10 or 20|
While V. natriegens, E. coli (strain MG1655) and Bacillus subtillis have their optimal growing temperature at 37 °C1, 2, 3, Corynebacterium glutamicum grows best at 30 °C4. For the experiment design, we used 3 individual culture tubes for each condition to have three independent replicas. From each tube we transfered a 100 µL sample to a 96-well plate and measured the optical density at 600 nm every 45 minutes. The plots of the absorbance over time shows growth on all used medium variants.
Growth curve of Vibrio natriegens
Since V. natriegens lives in salt marshes mud, it requires a high salt amount in its growth medium. The used concentrations of 1 and 2 % NaCl are recommended by further studies1 and indicated by the numbers 10 for 10 mg/ml and 20 for 20 mg/ml. Furthermore, the PCV medium variants already contain salt due to the extraction method, which is not taken in account for the value of the end concentration of NaCl. As seen in Fig. 2, the PCV medium with additional soy peptone showed the highest absorbance in the last three samples, followed by the two BHIN variants. Interestingly, the LB medium showed very low absorbance values similar to only soy peptone and the PCV medium on its own. The BHIN variants showed the shortest lag phases, probably due to its high content of free amino acids and additional sugar in form of glucose or dextrose5, 6.
Figure 2 - Growth curve of V. natriegens on PhyCoVi medium (PCV) and PCV + Soy pepton. LB-medium, Brain-Heart infusion medium (BHIN) and soy pepton served as control. The medium contained different amounts of NaCl (10 or 20 g/L). Samples for measuring absorbance were taken every 45 minutes up to 315 minutes.
Growth curve of Escherichia coli MG1655
Looking at the growth of the E. coli strain MG1655, we saw a much narrower distribution of absorbance. Still our PCV + Soy Peptone medium showed the highest values atop of BHIN, but with a longer lag phase compared with the growth of V. natriegens. The worst conditions seem to be in the PCV samples with 20 mg/mL NaCl, where no growth is detected until minute 225. Other strains of E. coli where tested on our medium by our collaboration partners, who received our two PCV medium variants and an LB control medium.
Figure 3: Growth curve of E. coli on PhyCoVi medium (PCV) and PCV + Soy pepton. LB-medium, Brain-Heart infusion medium (BHIN) and soy pepton served as control. The medium contained different amounts of NaCl (10 or 20 g/L). Samples for measuring absorbance were taken every 45 minutes up to 315 minutes.
Growth curve of Bacillus subtilis and Corynebacterium glutamicum
The last two organisms tested on our medium were much more sensitive concerning high salt concentrations. Also we already proved the usability of our medium. Therefore, we only compared our two PCV variants with and without soy peptone to LB and BHIN, all with additional 10 mg/mL NaCl. It can be seen, that both bacteria struggle much longer in these conditions, but still manage to grow. This time though, both Bacillus subtilis and Corynebacterium glutamicum prefer LB and BHIN over our PCV variants.
Figure 4 - Growth curve of Bacillus subtillis on PhyCoVi medium (PCV) and PCV + Soy pepton. LB-medium, Brain-Heart infusion medium (BHIN) and soy pepton served as control. The medium contained 10 g/L NaCl. Samples for measuring absorbance were taken every 45 minutes up to 360 minutes.
Figure 5: Growth curve of Corynebacterium glutamicum on PhyCoVi medium (PCV) and PCV + Soy pepton. LB-medium, Brain-Heart infusion medium (BHIN) and soy pepton served as control. The medium contained 10 g/L NaCl. Samples for measuring absorbance were taken every 45 minutes up to 360 minutes.
Since we only worked in a rather small scope with our RAPtor, consistent medium production is not guaranteed, but could be held on a consistent level. Another problem would be the scale up of boiling algaes in 1 M HCl and the neutralization of this solution. Yet for this scale, we could maintain every safety standard. Furthermore there are still possibilities of a more effective algae cell disruption and more efficient hydrolysis. Looking at the results, it was already efficient enough, to maintain stable bacterial cultures with a relatively small amount of algae extract. Lastly, we could not figure out, how to utilize the cell wall fragments and lipids of our algaes, since they were discarded after being centrifuged to a pellet. Harnessing their metabolic value would further increase the efficacy of our medium 7. Other possibilities would lie in the fields of bio fuels, where already many projects are ongoing8, 9.
As a conclusion the developed PhyCoVi medium is applicable for cultivation of V. natriegens and E. coli. In both cases the PhyCoVi medium with additional soy peptone outcompetes the common growth medium for these bacteria. PhyCoVi medium has the advantage that it only contains source derived from plants or algae and therefore, no animal is harmed. Thus, the new medium is not only more efficient but also ethically harmless.
- Henry H. Lee, Nili Ostrov, Brandon G. Wong, Michaela A. Gold, Ahmad S. Khalil, George M. Church (2016), Vibrio natriegens, a new genomic powerhouse, bioRxiv, doi: https://doi.org/10.1101/058487
- Bronikowski, A. M., Bennett, A. F. and Lenski, R. E. (2001), EVOLUTIONARY ADAPTATION TO TEMPERATURE. VIII. EFFECTS OF TEMPERATURE ON GROWTH RATE IN NATURAL ISOLATES OF ESCHERICHIA COLI AND SALMONELLA ENTERICA FROM DIFFERENT THERMAL ENVIRONMENTS. Evolution, 55: 33-40. doi:10.1111/j.0014-3820.2001.tb01270.x
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- Ohnishi, J., Hayashi, M., Mitsuhashi, S. et al. (2003), Appl Microbiol Biotechnol 62: 69. https://doi.org/10.1007/s00253-003-1254-2
- Brain Heart Infusion (BHI) Broth: Composition, Preparation and Uses, June 20, 2019, Nisha Rijal, last access October 20, 2019, https://microbeonline.com/brain-heart-infusion-bhi-broth-composition-preparation-and-uses/
- Brain Heart Infusion Broth for microbiology, 2019, Merck KGaA, Darmstadt, last access October 20, 2019, https://www.sigmaaldrich.com/catalog/product/sial/53286?lang=de®ion=DE
- Bateman D.F., Basham H.G. (1976) Degradation of Plant Cell Walls and Membranes by Microbial Enzymes. In: Heitefuss R., Williams P.H. (eds) Physiological Plant Pathology. Encyclopedia of Plant Physiology (New Series), vol 4. Springer, Berlin, Heidelberg
- Yanqun Li, Mark Horsman, Nan Wu, Christopher Q. Lan, Nathalie Dubois‐Calero (2008), Biofuels from Microalgae, Biotechnology ProgressVolume 24, Issue 4, https://doi.org/10.1021/bp070371k
- René H. Wijffels, Maria J. Barbosa (2010) An Outlook on Microalgal Biofuels, Science, 13 Aug 2010 : Vol. 329, Issue 5993, pp. 796-799, DOI: 10.1126/science.1189003
Cloning of ptRNA_backbone
The self-designed ptRNA_backbone was synthesized by Integrated DNA Technologies as a gBlock with 5´ phosphorylation to ensure blunt end ligation. For verification we transformed the ligated plasmid into E.coli DH5α resulting in colonies on tetracycline LB agar plates only for DH5α containing ptRNA_backbone (see Figure 1). We were able to demonstrate that the ptRNA_backbone itself is functional and mediates tetracycline resistance.
After plasmid preparation the expected length of the construct was validated by agarose gel electrophoresis. Looking at Figure 2 isolated plasmids run at the desired length which corresponds to the length of the ptRNA_backbone 2159 bp.
Transformation of pRARE into Vibrio natriegens
As an alternative to cloning our self-designed ptRNA_backbone containing tRNAs of rare codons into Vibrio natriegens we used the pRARE plasmid. It carries genes for several rare tRNAs in Escherichia coli, which allows their concentration to be increased. The pRARE plasmid was prepared from E. coli Rosetta and transformed into Vibrio natriegens. Growth curves of the wild type Vibrio natriegens and Vibrio natriegens containing pRARE were performed. The growth curves represented in Figure 1 show no differences in the growth velocity between both Vibrio natriegens strain variants. Thus, we could demonstrate that enhanced expression of tRNAs has no obvious negative effect on the metabolic activity of Vibrio natriegens cells.