Team:Linkoping Sweden/Results

Cultivation

Growth

Predators

Symbiosis

Growth Model

Halorhodopsin

References

Results

Cultivation of Synechocystis sp. PCC 6803

Successful and efficient growth of Synechocystis sp. PCC 6803 is vital to enable testing of the parts involved in the CyaSalt desalination system and ultimately, to enable implementation of the desalination system in a seawater environment. Therefore, our project contained excessive wet-lab and dry-lab studies of the cultivation of Synechocystis sp. PCC 6803 under different conditions.

Growth Conditions

As an effect of the slow growth rate of Synechocystis sp. PCC 6803 (between 4-8 hours doubling time), optimizing its growth conditions was a crucial part of our project [1-3]. Therefore, analysis of the growth conditions for Synechocystis sp. PCC 6803 constituted a large part of our wet-lab work. The growth media used for cultivation was BG-11. The composition of BG-11 can be found here. Synechocystis sp. PCC 6803 is photosynthetic and hence it requires light to grow. Accordingly, the light source was one of the external factors evaluated. Supported by cultivation protocols obtained from two separate research groups working with Synechocystis sp. PCC 6803, the lights eventually used were of 340 Lumens and approximately 5500 Lux. Cultures were grown at a distance of 10 cm from the light source. Additional details about lamps used can be found here. A further aspect that was studied was the access to CO₂ atmosphere provided to the culture flasks. After expert consultation, we realized that an open atmosphere would most likely increase the growth rate due to it providing the cultures with additional CO₂. To obtain this, the culture flasks were sealed with cotton plugs which resulted in a substantial increase in culture density, as observed in Figure 1. Figure 1. gives a visual comparison of the growth in culture flasks sealed with cotton plugs and aluminium foil. The final growth setup used is illustrated in Figure 2.

Figure 1. Comparison of Synechocystis sp. PCC 6803 cultures. The Erlenmeyer flask furthest to the left contains BG-11 media and the bacteria has been grown with open atmosphere access. The middle flask also contains BG-11 but it has been cultivated while sealed with foil. A substantially higher culture density is observed for the one sealed with a cotton plug. The flask furthest to the right contains Synechocystis sp. PCC 6803 grown in autoclaved water retrieved on Gotland (salinity 1%). Compared to the other two cultures, it has a yellow color, possibly due to a lack of nutrients during growth.

Figure 2. Illustration of the cultivation set-up used for the growth of Synechocystis sp. PCC 6803. The incubator used for plates and flasks is visualized. The Erlenmeyer flasks used are sealed with cotton plugs in order to enable open atmosphere access. The lamps were controlled by timers set to be turned on during 12 hours of the day and turned off during the remaining 12. The light intensities that were measured at different positions in the incubator are written in Lux.

Succeeding the growth optimization in BG-11, the growth of Synechocystis sp. PCC 6803 was studied under various other conditions. Since our desalination system ultimately relies on the phototrophs growing in seawater, we collected several water samples to test the growth. Water samples with a salt percentage of approximately 1% were retrieved from the Baltic Sea. Furthermore, samples were collected from the North Sea with a salinity of approximately 3%. The graph in Figure 3. illustrates the results obtained from growing Synechocystis sp. PCC 6803 in these samples, both autoclaved and non-autoclaved, in addition to in BG11.

Figure 3. Growth curve for Synechocystis sp. PCC 6803. The X-axis shows the time in days from seeding and the y-axis shows the measured optical density at 544 nm. The media which Synechocystis sp. PCC 6803 used is displayed in the legend to the right in the figure. Seawater samples with ~3% salinity, retrieved from the North Sea in Gothenburg, Sweden are abbreviated GBG and brackish seawater samples retrieved from the Baltic Sea on Gotland, Sweden have a salinity of ~1%. The smaller graph included in the figure shows the optical density in the BG-11, GBG not autoclaved and Gotland not autoclaved cultures during the first 6 days. This was done to highlight that Synechocystis sp. PCC 6803 started dying in the not autoclaved water during that time.

The graph displays substantial growth in the BG-11 media. The growth in the autoclaved seawater also exhibits a rather steady growth curve, although at a considerably slower rate than in BG-11. The cultures grown in autoclaved seawater had a much lower optical density of Synechocystis sp. PCC 6803 compared to the ones grown in BG-11. The cells also exhibited a yellow color as observed in Figure 1., most likely due to deficiency of a nutrient. The results that correlate best with real-world implementation are from the non-autoclaved seawater cultures. In these samples, the graph shows a slight growth initially. However, after only 4 days, the Synechocystis cultures in non-autoclaved seawater began to die. This was further visually observed by the green color of the cultures fading.
Moreover, samples from the dying cultures were looked at under an epifluorescence microscope. Here, observations further suggested that the Synechocystis sp. PCC 6803 were dying. Figures 4A and 4B show images of healthy Synechocystis sp. PCC 6803 cultures taken with an epifluorescence microscope whereas Figure 4C. shows a dying culture. Figure 4C. shows that no fluorescence was obtained from the dying Synechocystis sp. PCC 6803 cultures.

Figure 4. Images showing A) A microscopic image (40x) of a healthy Synechocystis sp. PCC 6803 culture excited at 535 nm. B) A spectral microscopic image (40x) of a healthy Synechocystis sp. PCC 6803 culture excited at 560 nm. C) A spectral microscopic image (40x) of a dead Synechocystis sp. PCC 6803 culture excited at 560 nm.

Predators Native to Seawater

Additional microscopic observations of the non-autoclaved seawater cultures presented a possible reason for Synechocystis sp. PCC 6803 dying. Organisms, shown in Figure 5., possibly preying on Synechocystis sp. PCC 6803 were observed and possibly identified. These observations lead us to speculate that predators living in the seawater, possibly amoebae, have caused the death of Synechocystis sp. PCC 6803. Previous studies have suggested that a potential solution to amoebas preying on cyanobacteria is to impair the O-antigen production in Synechocystis sp. PCC 6803. This would result in them not being recognized by the amoebae. However, a potential risk to this solution is that it motivates evolution in the amoeba that enables them to identify the modified cyanobacteria [4].

Figure 5. Bright-field images (40x) of the potential eukaryotic predators found in non-autoclaved seawater samples retrieved from Gotland (salinity ~1%) with Synechocystis sp. PCC 6803. The organisms in the image did not exhibit any fluorescence.

Symbiotic Phototrophic Organisms Native to Seawater

Another suggestion is to use another phototrophic organism that is not targeted by the predator. Evidence encouraging this possibility is that various phototrophic organisms were still thriving in symbiosis in the non-autoclaved seawater after Synechocystis sp. PCC 6803 had died (appearing after 25 days). These phototrophic organisms were noticed through microscopic studies and are shown in Figure 6.

Figure 6. Native phototrophs that grew in non-autoclaved seawater from Gotland (A-D) and Gothenburg (E, F). The phototrophs were identified as (1) Unknown, (2) Aphanizomenon, (3) Nitzschia, (4) Unknown, (5) Unknown, (6) Unknown, (7) Unknown, (8) Unknown, (9-10) Nostoc sp or Nodularia, (11) Unknown, (12) Unknown, (13) Gloeocapsa, (14) Woronichinia sp. All the phototrophs were identified by comparing morphology [5]. The images were generated with an epifluorescence microscope (Leica Spectraview). Excitation wavelength for the corresponding picture (A) 480 nm, (B) 405 nm, (C-D) 436 nm, (E) 480 nm, (F) 350 nm.

Some of the observed organisms were identified by comparing morphology. The identified species could be Aphanizomenon, Nitzschia, Nostoc sp. or Nodularia, Gloeocapsa and Woronichinia sp. [5]. Since these phototrophic organisms exhibit superior growth in seawater compared to Synechocystis sp. PCC 6803, they could be better suited to implement the CyaSalt desalination system in. Especially Aphanizomenon and Nodularia could work for desalination of Baltic seawater, as they normally bloom in large quantities in the Baltic Sea [6]. For future work, one option is to modify multiple of the observed phototrophic organisms to obtain a symbiotic desalinating system. Another option is to continue with one of the organisms and attempt to implement our desalination system in it. Conclusively, the best approach needs to be determined by further studies.

Growth Prediction by the Model

A model was created to enable growth predictions of phototrophic organisms. Experimental data obtained from Synechocystis sp. PCC 6803 cultivation under various conditions was applied to the model and predicted growth curves were generated. The model is adaptable so that the growth of any phototrophic organism, wild-type or modified, can be predicted. Read more about modelling results and how it has affected our project here.

Expression of Halorhodopsin

The part BBa_K3892001, coding for the ion pump Halorhodopsin, were ordered from Integrated DNA Technologies (IDT) cloned into the plasmid pUCIDT-Amp. However, they experienced problems with producing it and were ultimately only able to produce a low amount of product. After receiving this product, it was transformed into E. coli (strain DH5 alpha) as well as V. natriegens (strain Vmax). However, the resulting colonies were small and they also grew slower than anticipated. When sending a number of plasmid preps for sequencing, the results showed that the sequence encoding Halorhodopsin was not conserved in our plasmids. The last part of the gene had been mutated at various positions in all products, causing a significantly shorter Halorhodopsin to be expressed. Based on these results, it is plausible that Halorhodopsin is toxic to its host organism and that the sequence mutates in order to preserve the survival of the host organism. The possibly toxic effect of Halorhodopsin is something that requires further research to be confirmed, but also something that should be taken into consideration in future research done with this ion pump.

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[1] Cordara A, Re A, Pagliano C, Van Alphen P, Pirone R, Saracco G, Branco Dos Santos F, Hellingwerf K, Vasile N. Analysis of the light intensity dependence of the growth of Synechocystis and of the light distribution in a photobioreactor energized by 635 nm light. PeerJ. 2018 Jul 27;6:e5256. doi: 10.7717/peerj.5256. PMID: 30065870; PMCID: PMC6065478.

[2] van Alphen P, Abedini Najafabadi H, Branco Dos Santos F, Hellingwerf KJ. Increasing the Photoautotrophic Growth Rate of Synechocystis sp. PCC 6803 by Identifying the Limitations of Its Cultivation. Biotechnol J. 2018 Aug;13(8):e1700764. doi: 10.1002/biot.201700764. Epub 2018 May 2. PMID: 29577667.

[3] Zavřel T, Sinetova MA, Búzová D, Literáková P, Červený J. Characterization of a model cyanobacterium Synechocystis sp: PCC 6803 autotrophic growth in a flat-panel photobioreactor. Eng Life Sci. 2015;15(1):122–32. doi: 10.1002/elsc.201300165.

[4] Simkovsky R, Daniels EF, Tang K, Huynh SC, Golden SS, Brahamsha B. Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proc Natl Acad Sci U S A. 2012;109(41):16678–83. doi: 10.1073/pnas.1214904109.

[5] Wiśniewska K, Lewandowska AU, Śliwińska-Wilczewska S. The importance of cyanobacteria and microalgae present in aerosols to human health and the environment - Review study. Environ Int. 2019 Oct;131:104964. doi: 10.1016/j.envint.2019.104964. Epub 2019 Jul 24. PMID: 31351382.

[6] Walve J, Larsson U. Blooms of Baltic Sea Aphanizomenon sp. (Cyanobacteria) collapse after internal phosphorus depletion. Aquat Microb Ecol. 2007;49(1):57–69.