1. What, in your opinion, makes Vibrio natriegens so special and potentially interesting for the biotech industry?

V. natriegens is the fastest growing non-pathogenic organism on our planet and therefore has the potential to speed-up industrial fermentation processes, cell free protein synthesis and routines in molecular biology.

2. Can you think of potential disadvantages that V. natriegens holds?

Adapted fermentation methods have to be developed to meet the requirements due to the high metabolic rates.

3. Compared to Escherichia coli there is still a lot of questions that need to be answered to fully understand V. natriegens. What do you think are the most important problems that need to be understood or improved before using the strain for biotechnological production processes?

In order to fully exploit the potential of V. natriegens in the future, further work must contribute significantly to the understanding of the metabolism and its regulation.

4. Rare codons and respective tRNAs are a bottle neck and thus a regulation mechanism in protein expression. Our goal is to enhance protein expression by introducing rare tRNAs into V.o natriegens. Do you think this might be a good way to make the strain more interesting for the biotech industry?

Your results will reveal the answer to this question. :-)

Being an expert in the field of algae biomass production, he informed us about the different cultivation methods his company uses, like tubular and flat-panel photobioreactors, open ponds, cascade raceways and fermenters. He recommended to use a photobioreactor which fits our algae, scale and aim best.
Our non-axenic culture and the scale we chose for cultivation were the main arguments to build an air lift photobioreactor instead of a fermenter or open ponds.
We also gained insight in harvesting and processing technologies. Therefore, we tried different downstream processes to optimize the extraction with acid and ball mills. Additionally we asked how he estimated the feasibility of using microalgae as basis of cultivation media for bacterial cultivation.

He really liked the idea of a CO2-friendly bioprocess and already did some research on this field on his own. We thank Mr Vitor Verdelho for the interesting input which helped us choosing the right cultivation method as well as the optimal extraction method.

Prof. Dr. Heyer told us that you work with algae.

We have Chlorella vlugaris as an endosymbiont in Euplotes and Hydra. Otherwise, we use Chlorella as feed for mussel and cancer larvae. We do not co-cultivate with bacteria, but in general,  Chlorella cultures are in a non-axenic medium, meaning that there is always with a mixture of different bacteria in the medium.

How would you get the culture axenic?

By applying an antibiotic to which the algae do not respond. Then, centrifuge accordingly and transfer into a new medium and possibly place the culture in sunlight to remove the last residues of antibiotic. There is also a method in which the culture is more and more diluted, but this is probably out of the question. You could contact SAG Göttingen for further information.

Do you have any idea how we can effectively decompose the algae (Chlorella vulgaris)?

There is a gentle method where you use detergent like SDS. Sonification with a rod sonifier might also be a method. You could also ask Prof. Dr. Ingrid Weiss (University of Stuttgart) if you can use her ball mills. We have 3 of them which can each hold 10 x 50 ml Falcons. You should also try out acid-hydrolysis with different concentrations of various acids. Afterwards, examine the algae under the microscope to analyze the disruption

Prof. Dr. Heyer told us something about endospores that are supposed to form in Chlorella vulgaris, which makes digestion more difficult

Chlorella forms endospores when the cell is stressed. You should see if you can find anything in the literature on this.

How could one measure growth of Chlorella vulgaris to get a growth curve?

It would be possible to carry out cell counts with algae in a Neubauer chamber.

What is the best way to cultivate algae like Chlorella vulgaris?

There are different approaches for the cultivation of biomass. For example, companies have meter-long tube systems that they have installed on the roofs where the algae are pumped through. Mr. Heyer also has a reactor with which he has cultivated algae on a small scale. In a non-axenic algae culture, bacteria usually are also cultivated because they provide the algae with certain supplements. In axenic cultivation, these would have to be added. You could ask a company like SAG Göttingen what to look out for in cultivation. They are usually very interested in helping.

Our first challenge in this year's iGEM competition was to further strengthen Vibrio natriegens. After the iGEM team from Marburg 2018 contributed their part to provide new strains and a toolbox to accelerate cloning with V. natriegens, we want to contribute further with our project. Therefore, we planned to develop a plasmid-based system to increase the cellular availability of tRNAs with the aim to increase the expression of heterologous genes with V. natriegens. In the first part of our project we determined which codons are less used within V. natriegens and how we can express them within V. natriegens. After identifying the rare codons with the HIVE-CUT tool available online, we had to find out how to increase the availability of the corresponding tRNAs. A plasmid-based approach was used to demonstrate the principle and to fine tune the expression.

With this raw idea and information from the literature we went to Dr. rer. nat. Josef Altenbuchner (Institute for Industrial Genetics at the University of Stuttgart), an expert in the field of industrial genetics who has great expertise in the design of expression vectors for a variety of hosts. In our first meeting with Dr. rer. nat. Josef Altenbuchner, he gave us excellent indications for the molecular handling of V. natriegens as host. Thanks to his input on tolerances for certain antibiotics, which V. natriegens keeps, we were able to concentrate on two antibiotics from the beginning - chloramphenicol and tetracycline.

His first question about using V. natriegens was why we wanted to use another Gram-negative host. After an interesting conversation about the benefits we see in V. natriegens, he told us about the different ways of transforming V. natriegens. His advice was to use electrocompetent cells or, for an even better transformation rate, to switch to an OriT plasmid for conjugation with E. coli.

After the useful and important advice for the first molecular methods, we talked about our next expression goal - heterologous genes. He suggested using a two plasmid-based approach from the beginning. On the one hand, he experienced that V. natriegens did not prefer large plasmids, on the other hand, in order to be able to control the expression of each individual, the tRNA and the heterologous protein, in order to be more flexible. When he came to plasmid design, he suggested using a p15A Ori and, since he is familiar with the Biobrick plasmids used in iGEM, using an iGEM vector as the backbone. With this information and interesting lectures on the different areas of genetics, we have started to develop our expression vectors. We gladly accepted his advice and used a p15A Ori and pSB3T5-I52001 as a template for the combined rrnA P1 promoter (from V. natriegens) and the rrnA terminator (derived from E. coli K12) in our new ptRNA.

Being the expert in plant biotechnology at our university, we contacted Prof. Heyer in april at the beginning of our project to ask whether the project was feasible.
His opinion was that the idea of growing bacteria on media based on algae was possible itself. However, he also had concerns disrupting the cell walls of the microalgae as they are very solid.
He reinforced our choice of microalgae, as chlorella is a fast biomass producer. To better analyze the contents of chlorella Prof. Heyer kindly offered us his support in analyzing sugars using HPLC in his lab. For further information and for starting a microalgae culture, he gave us various contacts.

We would like to thank Prof Heyer for the very helpful and informative talk and especially for his great support during our project concerning HPLC analysis of the disrupted cells.

We were very grateful, that Dr. Tröndle was impressed of our project „PhyCoVi“. He was particularly interested in the idea to quantify tRNA species specifically. In his opinion, tRNA quantification provides great potential for industrial applications as well as for fundamental research. Nevertheless, he identified two potential problems in our method: After telling him, that we may have problems regarding the reverse transcription, he noticed that the optimum working temperature of the reverse transcriptase is quite low. While performing reverse transcription at 42 °C to generate cDNA from mRNA is sufficient, in our case this temperature might be too low. tRNA is characterized by heavy secondary structures that may impair reverse transcription. Therefore, he recommended to use “HotScriptase RT”. This modified polymerase was developed by Genaxxon bioscience GmbH and is able to carry out the reverse transcription and the amplification in one step using RNA as a template. Because “HotScriptase RT” requires extension temperatures above 68 °C, tRNA secondary structures should be broken up, leading to better results. In addition, he suggested that the binding sites of all primers should be located at the same position in the four-leaf clover shaped structure of the tRNA (e.g. TΨC-loop). This should lead to better results, as this loop is broken down relatively early, compared to the stems, allowing perfect annealing. The results of different tRNAs can also be better compared, since the difference in the melting temperatures required for reverse transcription between different tRNA primer pairs is minimized.

Problem

With almost no foreknowledge about microalgae cultivation and growth, we talked to several experts on this topic (Prof. Dr. Arnd G. Heyer, Vitor Verdelho, Luft and PD Dr. Michael Schweikert) helping us to implement the cultivation of Chlorella vulgaris and Chlorella sorokiniana in our lab. Although cultivation of the microalgae was with a constant setup (16:8 hours of illumination, constant gassing and mixing) the growth curve (measurement of optical density (OD)) of the microalgae exhibited several discrepancies compared to our expected growth curve. As an example, the growth of the algae was different for each day, sometimes exhibiting an exponential growth, sometimes a linear or stagnating growth. With our fear that constantly varying growth behavior might cause different compositions of our algae extract we were keen on fixing the growth behavior of the microalgae in order to obtain a constant, non-changing growth rate.

The role of MicroBiolytics

We reached out to MicroBiolytics, a local company offering quality control services for the food and pharmaceutical industry. The CEO of the company (Andreas Wolf) was very interested in our project, especially the idea to develop an eco-friendlier process. We set up a meeting with Andreas in which we told him about our iGEM project, whereas he explained us what MicroBiolytics offers. In short, MicroBiolytics analyzes fluids using infrared-spectroscopy, being able to analyze the changes of compounds in the fluid. With the inconsistent algae growth in mind we came to the conclusion that we could detect and measure the changes in the growth medium helping us understand the algae growth. Therefore, we collected daily samples of the airlift bioreactor in which we cultivated our algae and noted the corresponding OD at 750 nm. With the captured OD we can deduce on which day the algae exhibit stagnating growth and link that to the liquid in the reactor by analyzing the collected sample. Furthermore, we were offered to analyze our algae extraction batches for purity and differences in compounds.

After the meeting, we collected the airlift samples over the next 2 weeks and brought them to MicroBiolytics. We were then explained the analyzation method and were able to use their device for measurements. Afterwards, we were explained how they deduce the relevant data from the huge data sets which accumulate for infrared spectroscopy and how to interpret the results.

Results

Samples were taken according to the following scheme.

On each date four samples were taken from the reactor. Two for the measurement and two for pH determination. Two additional samples C1 DSNA1 and C2 DSNA2 were taken as control for the analysis of composition. We also calculated the concentrations of each supplement of the DSN media which was used for the Chlorella sorokiniana cultivation as additional information for Micro Biolytic. The concentrations are shown in the following table.

In a presentation results were presented and explained. Each sample was measured 3 times. They did a quantitative analysis of known spectra from a database on oure samples.

In the following figure the concentrations of sulfate are shown in each sample.

Figure 1: Concentrations of sulfate in samples taken on a certain date. The dotted indicates the calculated concentration of sulfate in the DSN media. C1 and C2 represent the real concentrations of sulfate in the DSN media.

It is notable that the sulfate concentration is steady increasing over the time in the reactor except for sample A01 and A02. I may have something to do with the evaporation of the media during the cultivation. Sulfate may accumulate.

The next figure shows the nitrate concentration in each sample.

Figure 2: Concentrations of nitrate in samples taken on a certain date. The dotted indicates the calculated concentration of nitrate in the DSN media. C1 and C2 represent the real concentrations of nitrate in the DSN media.

In figure 2 the real nitrate concentration (C1 and C2) seam to perfectly match the calculated concentrations of nitrate. The nitrate concentration in A01 and A02 seem to be divergent to the other from samples A03 to A08. Concentrations in A03 to A08 seem not to change much. From sample A09 on there is a significant jump in concertation from near 0.30 g/L to almost 58 g/l. This can be explained by the fact, that we inoculated 3-4 L of DSN media on this date (10.07.2019) because the corresponding volume evaporated.

The concentration of sodium chlorin in our samples are shown in the next figure.

Figure 3: Concentrations of sodium chloride in samples taken on a certain date. The dotted indicates the calculated concentration of sodium chloride in the DSN media. C1 and C2 represent the real concentrations of sodium chloride in the DSN media.

It is noteworthy that sodium chloride concentrations in all samples are much lower than they should be according to the recipe for the DSN media. The values are slightly increasing from sample A09. This can be again an effect of the addition of DSN media.

Phosphate was not detected in any samples.

Draw-back

With the measurement of the composition of the media in our reactor, we were able to draw back at least one important part for future algae cultivation. As can be seen, after addition of new media in the reactor (A09). After this timepoint we were able to detect growth of the microalgae. Since the only significant effect was able to be detected for nitrate and the other components not exhibiting a significant effect we wanted to test if weekly addition of supplemental nitrate would increase the algae growth and remove the shaky growth of the algae.
With this in mind, we exemplary tested the nitrate supplementation for the next two weeks. The additional nitrate supplementation was able to stabilize algae growth with the daily OD measurement not exhibiting any stagnating growth (Figure 4).

Figure 4: Chlorella vulgaris growth after nitrate supplementation measured at an optical density of 750 nm.

Thanks to the weekly nitrate supplementation we were able to stabilize the growth of the microalgae, thereby increasing our biomass yield. Furthermore, due to the detected evaporation effect we decided to weekly refill with new media. This was only possible with the help of MicroBiolytics which kindly supported us along our project.