Towards establishment of axenic cyanobacteria cultures

K. Häggqvist, Jaana Rikkinen, Liisa Nohynek, Heiko Rischer, T. Lindholm, J. Meriluoto

Research output: Contribution to conferenceConference PosterScientific

Abstract

In the EU FP7 project MAREX (www.marex.fi), bioactive properties of various marine organisms are studied. In the framework of the project, rock pool cyanobacteria were collected from the Åland Islands, Finland, identified and monoalgal cultures established. Axenic algae cultures are crucial when exploring bioactivity, as they provide certainty that the relevant compounds are actually produced by the algae and not associated bacteria (Enss & Rischer 2012). A protocol modified from Su et al. (2007) was tested for production of axenic cultures of cyanobacteria. The cyanobacteria were cultured for two weeks (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1) in Z8+N growth medium (Spoof & Meriluoto 2005). In order to fragment the filamentous cyanobacteria, steel marbles (ø 4 mm) were added to 25 ml cultures, followed by vortexing in periods of 10 s (max. three periods). Cells were harvested by sequential filtration (5, 20, 80 µm pore size), washed three times in 50 ml Z8+N (2000 * g, 5 min), and incubated in 50 ml wash medium (Z8+N, 0,1 M EDTA, 0,0005 % Tween-20) for 1 h at 24 °C. Lysozyme (1.2 g/l, or 0.8 g/l) was added, the cells incubated for 10 min, washed two times (2000 * g, 5 min) and finally resuspended in Z8+N. Antibiotics (timentin 150 µg/ml, gentamycin 100 µg/ml, cefotaxime 250 µg/ml, ampicillin 250 µg/ml, meropenem 10 µg/ml) were added to the algal suspensions. The cultures were incubated 24 h in dark conditions and subsequently in light (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1). Antibiotics were removed from aliquots of the cultures after one and three weeks. The axenity of the cultures was tested by plating of bacteria on Difco Marine Agar 2216. After three week incubation, no bacterial growth was observed. A method for confirmation of the axenity of viable cyanobacteria cultures will be developed. So far the cyanobacterial cells appear intact, but growth was not noticed in the aliquots. The viability of the cells will be tested by fluorescence staining using methods comparable to those used by Sato et al. (2004) and de los Ríos et al. (2004). According to our results, the employed method is not directly suitable for establishment of axenic cyanobacteria cultures. However, our work showed that the tested cyanobacteria can resist short incubation in quite high lysozyme concentration, while a short incubation in SDS, even in low concentration (final concentration 0.005 %), bleached the cells instantly.
Original languageEnglish
Publication statusPublished - 2012
MoE publication typeNot Eligible
EventNordic Environmental Chemistry Conference, NECC 2012 - Turku, Finland
Duration: 4 Jun 20127 Jun 2012

Conference

ConferenceNordic Environmental Chemistry Conference, NECC 2012
Abbreviated titleNECC 2012
CountryFinland
CityTurku
Period4/06/127/06/12

Fingerprint

Cyanobacteria
lysozyme
algae
antibiotics
cells
cefotaxime
axenic culture
bacteria
gentamicin
ampicillin
steel
cell viability
Finland
microbial growth
bioactive properties
culture media
agar
rocks
fluorescence
organisms

Cite this

Häggqvist, K., Rikkinen, J., Nohynek, L., Rischer, H., Lindholm, T., & Meriluoto, J. (2012). Towards establishment of axenic cyanobacteria cultures. Poster session presented at Nordic Environmental Chemistry Conference, NECC 2012, Turku, Finland.
Häggqvist, K. ; Rikkinen, Jaana ; Nohynek, Liisa ; Rischer, Heiko ; Lindholm, T. ; Meriluoto, J. / Towards establishment of axenic cyanobacteria cultures. Poster session presented at Nordic Environmental Chemistry Conference, NECC 2012, Turku, Finland.
@conference{9edad351011747809d9c0824ba9eebc7,
title = "Towards establishment of axenic cyanobacteria cultures",
abstract = "In the EU FP7 project MAREX (www.marex.fi), bioactive properties of various marine organisms are studied. In the framework of the project, rock pool cyanobacteria were collected from the {\AA}land Islands, Finland, identified and monoalgal cultures established. Axenic algae cultures are crucial when exploring bioactivity, as they provide certainty that the relevant compounds are actually produced by the algae and not associated bacteria (Enss & Rischer 2012). A protocol modified from Su et al. (2007) was tested for production of axenic cultures of cyanobacteria. The cyanobacteria were cultured for two weeks (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1) in Z8+N growth medium (Spoof & Meriluoto 2005). In order to fragment the filamentous cyanobacteria, steel marbles ({\o} 4 mm) were added to 25 ml cultures, followed by vortexing in periods of 10 s (max. three periods). Cells were harvested by sequential filtration (5, 20, 80 µm pore size), washed three times in 50 ml Z8+N (2000 * g, 5 min), and incubated in 50 ml wash medium (Z8+N, 0,1 M EDTA, 0,0005 {\%} Tween-20) for 1 h at 24 °C. Lysozyme (1.2 g/l, or 0.8 g/l) was added, the cells incubated for 10 min, washed two times (2000 * g, 5 min) and finally resuspended in Z8+N. Antibiotics (timentin 150 µg/ml, gentamycin 100 µg/ml, cefotaxime 250 µg/ml, ampicillin 250 µg/ml, meropenem 10 µg/ml) were added to the algal suspensions. The cultures were incubated 24 h in dark conditions and subsequently in light (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1). Antibiotics were removed from aliquots of the cultures after one and three weeks. The axenity of the cultures was tested by plating of bacteria on Difco Marine Agar 2216. After three week incubation, no bacterial growth was observed. A method for confirmation of the axenity of viable cyanobacteria cultures will be developed. So far the cyanobacterial cells appear intact, but growth was not noticed in the aliquots. The viability of the cells will be tested by fluorescence staining using methods comparable to those used by Sato et al. (2004) and de los R{\'i}os et al. (2004). According to our results, the employed method is not directly suitable for establishment of axenic cyanobacteria cultures. However, our work showed that the tested cyanobacteria can resist short incubation in quite high lysozyme concentration, while a short incubation in SDS, even in low concentration (final concentration 0.005 {\%}), bleached the cells instantly.",
author = "K. H{\"a}ggqvist and Jaana Rikkinen and Liisa Nohynek and Heiko Rischer and T. Lindholm and J. Meriluoto",
year = "2012",
language = "English",
note = "Nordic Environmental Chemistry Conference, NECC 2012, NECC 2012 ; Conference date: 04-06-2012 Through 07-06-2012",

}

Häggqvist, K, Rikkinen, J, Nohynek, L, Rischer, H, Lindholm, T & Meriluoto, J 2012, 'Towards establishment of axenic cyanobacteria cultures' Nordic Environmental Chemistry Conference, NECC 2012, Turku, Finland, 4/06/12 - 7/06/12, .

Towards establishment of axenic cyanobacteria cultures. / Häggqvist, K.; Rikkinen, Jaana; Nohynek, Liisa; Rischer, Heiko; Lindholm, T.; Meriluoto, J.

2012. Poster session presented at Nordic Environmental Chemistry Conference, NECC 2012, Turku, Finland.

Research output: Contribution to conferenceConference PosterScientific

TY - CONF

T1 - Towards establishment of axenic cyanobacteria cultures

AU - Häggqvist, K.

AU - Rikkinen, Jaana

AU - Nohynek, Liisa

AU - Rischer, Heiko

AU - Lindholm, T.

AU - Meriluoto, J.

PY - 2012

Y1 - 2012

N2 - In the EU FP7 project MAREX (www.marex.fi), bioactive properties of various marine organisms are studied. In the framework of the project, rock pool cyanobacteria were collected from the Åland Islands, Finland, identified and monoalgal cultures established. Axenic algae cultures are crucial when exploring bioactivity, as they provide certainty that the relevant compounds are actually produced by the algae and not associated bacteria (Enss & Rischer 2012). A protocol modified from Su et al. (2007) was tested for production of axenic cultures of cyanobacteria. The cyanobacteria were cultured for two weeks (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1) in Z8+N growth medium (Spoof & Meriluoto 2005). In order to fragment the filamentous cyanobacteria, steel marbles (ø 4 mm) were added to 25 ml cultures, followed by vortexing in periods of 10 s (max. three periods). Cells were harvested by sequential filtration (5, 20, 80 µm pore size), washed three times in 50 ml Z8+N (2000 * g, 5 min), and incubated in 50 ml wash medium (Z8+N, 0,1 M EDTA, 0,0005 % Tween-20) for 1 h at 24 °C. Lysozyme (1.2 g/l, or 0.8 g/l) was added, the cells incubated for 10 min, washed two times (2000 * g, 5 min) and finally resuspended in Z8+N. Antibiotics (timentin 150 µg/ml, gentamycin 100 µg/ml, cefotaxime 250 µg/ml, ampicillin 250 µg/ml, meropenem 10 µg/ml) were added to the algal suspensions. The cultures were incubated 24 h in dark conditions and subsequently in light (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1). Antibiotics were removed from aliquots of the cultures after one and three weeks. The axenity of the cultures was tested by plating of bacteria on Difco Marine Agar 2216. After three week incubation, no bacterial growth was observed. A method for confirmation of the axenity of viable cyanobacteria cultures will be developed. So far the cyanobacterial cells appear intact, but growth was not noticed in the aliquots. The viability of the cells will be tested by fluorescence staining using methods comparable to those used by Sato et al. (2004) and de los Ríos et al. (2004). According to our results, the employed method is not directly suitable for establishment of axenic cyanobacteria cultures. However, our work showed that the tested cyanobacteria can resist short incubation in quite high lysozyme concentration, while a short incubation in SDS, even in low concentration (final concentration 0.005 %), bleached the cells instantly.

AB - In the EU FP7 project MAREX (www.marex.fi), bioactive properties of various marine organisms are studied. In the framework of the project, rock pool cyanobacteria were collected from the Åland Islands, Finland, identified and monoalgal cultures established. Axenic algae cultures are crucial when exploring bioactivity, as they provide certainty that the relevant compounds are actually produced by the algae and not associated bacteria (Enss & Rischer 2012). A protocol modified from Su et al. (2007) was tested for production of axenic cultures of cyanobacteria. The cyanobacteria were cultured for two weeks (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1) in Z8+N growth medium (Spoof & Meriluoto 2005). In order to fragment the filamentous cyanobacteria, steel marbles (ø 4 mm) were added to 25 ml cultures, followed by vortexing in periods of 10 s (max. three periods). Cells were harvested by sequential filtration (5, 20, 80 µm pore size), washed three times in 50 ml Z8+N (2000 * g, 5 min), and incubated in 50 ml wash medium (Z8+N, 0,1 M EDTA, 0,0005 % Tween-20) for 1 h at 24 °C. Lysozyme (1.2 g/l, or 0.8 g/l) was added, the cells incubated for 10 min, washed two times (2000 * g, 5 min) and finally resuspended in Z8+N. Antibiotics (timentin 150 µg/ml, gentamycin 100 µg/ml, cefotaxime 250 µg/ml, ampicillin 250 µg/ml, meropenem 10 µg/ml) were added to the algal suspensions. The cultures were incubated 24 h in dark conditions and subsequently in light (24 °C, 16:8 light:dark, intensity 38 µmol m-2 s-1). Antibiotics were removed from aliquots of the cultures after one and three weeks. The axenity of the cultures was tested by plating of bacteria on Difco Marine Agar 2216. After three week incubation, no bacterial growth was observed. A method for confirmation of the axenity of viable cyanobacteria cultures will be developed. So far the cyanobacterial cells appear intact, but growth was not noticed in the aliquots. The viability of the cells will be tested by fluorescence staining using methods comparable to those used by Sato et al. (2004) and de los Ríos et al. (2004). According to our results, the employed method is not directly suitable for establishment of axenic cyanobacteria cultures. However, our work showed that the tested cyanobacteria can resist short incubation in quite high lysozyme concentration, while a short incubation in SDS, even in low concentration (final concentration 0.005 %), bleached the cells instantly.

M3 - Conference Poster

ER -

Häggqvist K, Rikkinen J, Nohynek L, Rischer H, Lindholm T, Meriluoto J. Towards establishment of axenic cyanobacteria cultures. 2012. Poster session presented at Nordic Environmental Chemistry Conference, NECC 2012, Turku, Finland.