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A modified medium and method for the cultivation of Chloroflexus
ELSEVIER
Journal
of Microbiological
Methods
27 (1996)
Journal ofMicrobiological Methods
147-150
A modified medium and method for the cultivation Khursheed
of ChZoroJexus
A. Malik*
DSMZ-Deutsche Sammlung van Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, W-38124 Braunschweig, Germany Received
1 August 1996; accepted 18 August 1996
Abstract A modified
anaerobic
medium
and cultivation
for Chloroflexaceae are described which assured almost All tested strains of Chlorojkxus ouruntiacus and Chlorojlexus
conditions
complete anaerobiosis for growth and maintenance.
aggregans were succer,sfully grown within relatively short times with full pigmentation, indicating that the new medium and reduced cultivation conditions were most suited for photoautotrophic growth. The anaerobic conditions and the presence of sulfide also favored prolonged viability during maintenance as liquid cultures. Keywords:
Chloroj?exus;
Cultivation; Anaerobic media and conditions
2. Material
1. Introduction
Photosynthetic flexibacteria Chloroflexaceae are generally grown under aerobic or semianaerobic conditions. However, to achieve good growth, cultures are usually mixed with known strains which during growth consume oxygen to ensure anaerobiosis [ 1,2]. Previously, it was observed that mostly phototrophically grown cultures showed their sensitivity to oxygen in the presence of light [3,4]. In view of this, the influence of anaerobic conditions and the presence of sulfide in the media was investigated for the cultivation of Chlouo~exus. The present study presents data on a modified medium and a successful cultivation method for Chloroflexus aurantiacus and Chloroflexus aggregans.
*Fax: +49 531 2616418. 0167-7012/96/$15.00 Copyright PII SO167-7012(96)00941-4
0
and methods
2.1. Microorganisms Four strains of Chlorojlexus aurantiacus (DSM 635, 636, 637 and 638) and two strains of Chloroflexus aggregans (DSM 9485 and 9486) were used throughout this work. All bacteria were from the German Collection of Microorganisms and Cell Cultures (DSMZ).
2.2. Media and cultivation procedure For comparison, the cultures were grown under semiaerobic conditions (in lOO-ml flat glass bottles with screw-caps) in the conventional medium (described by Schmidt [S], originally from Pierson and Castenholz [6]) and under anaerobic conditions (in lOO-ml flat glass bottles with screw caps with holes for the injection of the samples and butyl rubber septa) in modified medium (Table 1). For Chloroflexus aggregans, 1.0 ml/l, sterile vitamin solution
1996 Elsevier Science B.V. All rights reserved
K.A. Malik I Jow~ud of Mic~rohiologicul Mrthods 27 ( 1996) 147- 150
148
Table I Modified growth medium for Chloroflexaccae 1.0 g 1.0 g 0.1 g 0.1 g 0.1 g KNO, 0.5 g NaNO, 0.1 g NaCl 0.05 g CaClZX2H,0 5.0 ml Ferric citrate solution (0.1 g in 100 ml H1O) 1.0 ml Trace element solution SL-6 1050.0 ml Distilled water The pH of this medium was adjusted to 8.2. The medium was boiled under a stream of NL for a few minutes and 90 ml medium was distributed into 100.ml screw-capped bottles. Each bottle was bubbled with NZ and closed immediately with a rubber septum and screw tightened. The bottles were autoclaved at 121°C for 15 min. After autoclaving, 1.0 ml of neutralized sulfide solution (0.015% end concentration, see details below) was injected into each bottle. For Chk~&.rus aggregans, 1.O ml/l. sterile vitamin solution CA [7] was added. This medium can be stored for several months under anaerobic conditions.
Yeast extract Glycyl-glycine Na,HPO,XZH,O MgS0,X7HZ0
Trace element solution SL-6: ZnS0,X7H10 MnCllX4Hz0
0.1 g 0.03 g 0.3 g 0.2 g 0.01 g 0.02 g 0.03 g 1000 ml
HW, COCllX6HL0 CuCI,X2HI0 NiCllX6H.0 NalMo0,X2HZ0 Distilled water
Neutralized sulfide solution: 100 ml Distilled water 1.5 g Na,SX9H20 The solution was prepared in a screw-capped bottle with a butyl rubber septum and a magnetic stirrer. It was bubbled with nitrogen gas. closed and autoclaved for I5 min at 121°C. After cooling to room temperature the pH was adjusted to about 7.3 by injecting sterile 2 M H,SO, drop-wise with a syringe. It was stirred continuously to avoid precipitation of elemental sulfur. The final solution should be clear and is yellow in colour.
CA [7] was added to each medium. The cultures were incubated at 50°C at a light intensity of 300500 lx. 2.3. Comparison
of photoautotrophic
growth
The cultures were grown in 15-ml screw-cap tubes (modified medium was filled in Hungate tubes with septa, Bellco Glass Inc., 2047-16125) to the logarithmic growth phase in two different media under
semiaerobic conditions and modified anaerobic conditions. Three parallel tubes were prepared for each strain from each media. Equal volumes of conventional and modified medium were inoculated with an equal volume of inoculum from freshly grown cultures. The increase in absorbance A were measured every day at 650 nm using a Bausch and Lomb (Belgium) Spectronic 88 spectrophotometer.
3. Results and discussion Although photosynthetic energy conversion and photoautotrophic growth require anaerobic conditions, generally semianaerobic or aerobic conditions have been used to cultivate phototrophic bacteria [4,8,9]. Thus, it is often difficult to grow and conventional maintain such cultures under semianaerobic conditions as it has been observed that phototrophically grown cultures mainly show their sensitivity to oxygen in the presence of light [4]. The genus Chlorojlexus resembles Chlorobium in cell structure and chlorophyll pigments, but is similar to the purple non-sulfur bacteria in being both photoheterotrophic and facultatively chemoheterotrophic, in addition to the ability to grow photoautotrophically [9- 1 I]. Thus anaerobic conditions and the presence of sulfide in the modified medium favoured growth of Chloroj?exus aurantiacus and ChloroJlexus aggregans. Although the medium described (Table 1) is not totally new, slight modification in its composition has resulted in improved growth for all the tested strains. All the cultures grown under modified conditions showed full pigmentation indicating that the cultivation conditions were optimal for photoautotrophic growth. During parallel growth experiments, after equal periods of incubation, a marked increase in optical density was observed in modified medium as compared to the conventional medium (Table 2). The results in Table 2 show that the period of incubation was shortened by several days to obtain equal cell density. Sodium sulfide, which was injected to lower the redox potential, was favored during growth and maintenance as Chlorojlexus apparently also grows photoautotrophically with sulfide as electron donor [10,12].
K.A. Malik I Journal
Table 2 Photoautotrophic
growth
Strain and DSM No.
of Chloroflexaceae
of Microbiological
Methods
under modified and conventional
Conditions
Increase
27 (1996)
149
147-150
conditions
in A (650 nmj”
Growth in modified and conventional
medium after (days)
1
2
3
4
7
8
9
10
A B
0.04 0.03
0.08 0.04
0.25 0.05
0.30 0.06
0.65 0.04
0.85 0.03
1.15 0.02
1.35 0.02
A B
0.03 0.02
0.15 0.03
0.30 0.04
0.45 0.05
0.60 0.04
0.65 0.03
0.75 0.02
0.95 0.02
A B
0.04 0.02
0.07 0.03
0.25 0.03
0.55 0.04
0.65 0.04
0.75 0.03
0.85 0.03
0.95 0.02
A B
0.03 0.02
0.08 0.03
0.25 0.04
0.40 0.05
0.69 0.04
0.85 0.03
0.95 0.02
1.15 0.02
A B
0.05 0.05
0.08 0.07
0.35 0.15
0.65 0.30
0.85 0.45
0.95 0.55
1.35 0.60
1.55 0.50
Chlorojle.xus aurantiacus
DSM 635 Chlorojlexus
aurantiacus
DSM 636 ChloroJlexus
aurantiacus
DSM 637 Chlorojfexus
aurantiacus
DSM 638 ChlorojGxus
aggregansh
DSM 9485
A, cultures grown in moduied medium under modified conditions; B, cultures grown in conventional medium under conventional conditions. For more details, see Section 2. aEqual volumes of conventional and modified medium were inoculated with an equal volume of inoculum from freshly grown cultures. The tube cultures were grown under conventional and modified conditions. Increase in A values (rounded to two decimal) were measured at 650 nm every day until 10 days of incubation. The A values at zero time were about 0.01 in all cases. The results given are from one experiment that was typical of three. h Cultures formed aggregates and the exact measurement of optical density was a problem.
Under such anaerobic conditions, the liquid cultures could be mainrained much longer compared to the cultures grown and maintained under conventional semiaerobic conditions. Detailed studies on the maintenance of such liquid cultures for long time periods have been reported elsewhere [ 131.
Acknowledgments I am thankful assistance.
to Andrea
Schutze
for technical
References [1] Castenholz, R.W. and Pierson, B.K. (1981) Isolation of members of the family Chloroflexaceae. In: The Prokaryotes (Eds. M.P. Starr, H. Stolp, H.G. Trtlper, A. Balows and H.G. Schlegel). Springer, Berlin, pp. 290-298.
[2] Pierson. B.K and Castenholz. R.W. (1992) The family Chloroflexaceae. In: The Prokaryotes (Eds. A. Balows, H.G. Triiper. M. Dworkin, W. Harder and K.H. Schleifer), Second Edition. Springer, Berlin, pp. 3754-3774. [31 Malik. K.A. ( 1983) A modified method for the cultivation of phototrophic bacteria. J. Microbial. Methods 1, 343-352. [41 Malik. K.A. (1991) Maintenance of phototrophic bacteria. In: Maintenance of Microorganisms and Cultured Cells (Eds. B.E. Kirsop and A. Doyle). Academic Press, London, pp. 82-100. [51 Schmidt. K. (1980) A comparative study on the composition of chlorosomes (Chlorobium vesicles) and cytoplasmic membranesfrom Chlorojems aurantiacus strain OK-70-fl and Chlorobium limicola f. thiosulfatophilum strain 6230. Arch. Microbial. 124, 2 l-3 1. [61 Pierson, B.K and Castenholz, R.W. (1974) A phototrophic gliding filamentous bacterium of hot springs, Chlorojlexus trurarztiacus, gen. and sp. nov. Arch. Microbial. 100. 5-24. 171 Hanada, S., Hiraishi, A., Shimada. K. and Matsura, K., ( 1995) Chlorojexus aggregam sp. nav., a phototrophic filamentous bacterium which forms dense aggregates by active gliding movement. Int. J. Syst. Bacterial. 45, 676681. for the enrichment, WI Van Niel, C.B. (1971) Techniques isolation and maintenance of the photosynthetic bacteria. In: Methods in Enzymology,Vol. 23, Part A (Ed. A. San Pi&o). Academic Press, New York, pp. 2-3.
150
K.A. Malik I Journal of Microbiological
[9] Pfennig, N. and Triiper, H.G. (1989) Anoxygenic phototrophic bacteria. In: Bergey’s Manual of Systematic Bacteriology, Vol. 3 (Eds. J.T. Staley, M.P. Bryant, N. Pfennig and J.G. Halt). Williams and Wilkins, Baltimore, pp. 16351709. [lo] Madigan. M.T., Petersen, S.R. and Brock, T.D. (1974) Nutritional studies on Ctrloroje..wus. a filamentous photosynthetic, gliding bacterium. Arch. Microbial. 100, 97-103. [l l] Castenholz, R.W. (1989) Genus ChlorojJexus. In: Bergey’s Manual of Systematic Bacteriology. Vol. 3 (Eds. J.T. Staley.
Methods 27 (1996) 147-150 M.P. Bryant, N. Pfennig and J.G. Holt). Williams and Wilkins, Baltimore, pp. 1698-1709. [12] Madigan, M.T. and Brock, T.D. (1975) Photosynthetic sulfide oxidation by Clrlorojlexus aurantiacus. a filamentous photosynthetic. gliding bacterium. J. Bacterial. 122, 782784. [13] Malik, K.A. (1996) A convenient method of maintaining Chlorojkmu for long time periods as slow growing liquid cultures. J. Microbial. Methods 27, 151-155.
Journal
of Microbiological
Methods
27 (1996)
Journal ofMicrobiological Methods
147-150
A modified medium and method for the cultivation Khursheed
of ChZoroJexus
A. Malik*
DSMZ-Deutsche Sammlung van Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, W-38124 Braunschweig, Germany Received
1 August 1996; accepted 18 August 1996
Abstract A modified
anaerobic
medium
and cultivation
for Chloroflexaceae are described which assured almost All tested strains of Chlorojkxus ouruntiacus and Chlorojlexus
conditions
complete anaerobiosis for growth and maintenance.
aggregans were succer,sfully grown within relatively short times with full pigmentation, indicating that the new medium and reduced cultivation conditions were most suited for photoautotrophic growth. The anaerobic conditions and the presence of sulfide also favored prolonged viability during maintenance as liquid cultures. Keywords:
Chloroj?exus;
Cultivation; Anaerobic media and conditions
2. Material
1. Introduction
Photosynthetic flexibacteria Chloroflexaceae are generally grown under aerobic or semianaerobic conditions. However, to achieve good growth, cultures are usually mixed with known strains which during growth consume oxygen to ensure anaerobiosis [ 1,2]. Previously, it was observed that mostly phototrophically grown cultures showed their sensitivity to oxygen in the presence of light [3,4]. In view of this, the influence of anaerobic conditions and the presence of sulfide in the media was investigated for the cultivation of Chlouo~exus. The present study presents data on a modified medium and a successful cultivation method for Chloroflexus aurantiacus and Chloroflexus aggregans.
*Fax: +49 531 2616418. 0167-7012/96/$15.00 Copyright PII SO167-7012(96)00941-4
0
and methods
2.1. Microorganisms Four strains of Chlorojlexus aurantiacus (DSM 635, 636, 637 and 638) and two strains of Chloroflexus aggregans (DSM 9485 and 9486) were used throughout this work. All bacteria were from the German Collection of Microorganisms and Cell Cultures (DSMZ).
2.2. Media and cultivation procedure For comparison, the cultures were grown under semiaerobic conditions (in lOO-ml flat glass bottles with screw-caps) in the conventional medium (described by Schmidt [S], originally from Pierson and Castenholz [6]) and under anaerobic conditions (in lOO-ml flat glass bottles with screw caps with holes for the injection of the samples and butyl rubber septa) in modified medium (Table 1). For Chloroflexus aggregans, 1.0 ml/l, sterile vitamin solution
1996 Elsevier Science B.V. All rights reserved
K.A. Malik I Jow~ud of Mic~rohiologicul Mrthods 27 ( 1996) 147- 150
148
Table I Modified growth medium for Chloroflexaccae 1.0 g 1.0 g 0.1 g 0.1 g 0.1 g KNO, 0.5 g NaNO, 0.1 g NaCl 0.05 g CaClZX2H,0 5.0 ml Ferric citrate solution (0.1 g in 100 ml H1O) 1.0 ml Trace element solution SL-6 1050.0 ml Distilled water The pH of this medium was adjusted to 8.2. The medium was boiled under a stream of NL for a few minutes and 90 ml medium was distributed into 100.ml screw-capped bottles. Each bottle was bubbled with NZ and closed immediately with a rubber septum and screw tightened. The bottles were autoclaved at 121°C for 15 min. After autoclaving, 1.0 ml of neutralized sulfide solution (0.015% end concentration, see details below) was injected into each bottle. For Chk~&.rus aggregans, 1.O ml/l. sterile vitamin solution CA [7] was added. This medium can be stored for several months under anaerobic conditions.
Yeast extract Glycyl-glycine Na,HPO,XZH,O MgS0,X7HZ0
Trace element solution SL-6: ZnS0,X7H10 MnCllX4Hz0
0.1 g 0.03 g 0.3 g 0.2 g 0.01 g 0.02 g 0.03 g 1000 ml
HW, COCllX6HL0 CuCI,X2HI0 NiCllX6H.0 NalMo0,X2HZ0 Distilled water
Neutralized sulfide solution: 100 ml Distilled water 1.5 g Na,SX9H20 The solution was prepared in a screw-capped bottle with a butyl rubber septum and a magnetic stirrer. It was bubbled with nitrogen gas. closed and autoclaved for I5 min at 121°C. After cooling to room temperature the pH was adjusted to about 7.3 by injecting sterile 2 M H,SO, drop-wise with a syringe. It was stirred continuously to avoid precipitation of elemental sulfur. The final solution should be clear and is yellow in colour.
CA [7] was added to each medium. The cultures were incubated at 50°C at a light intensity of 300500 lx. 2.3. Comparison
of photoautotrophic
growth
The cultures were grown in 15-ml screw-cap tubes (modified medium was filled in Hungate tubes with septa, Bellco Glass Inc., 2047-16125) to the logarithmic growth phase in two different media under
semiaerobic conditions and modified anaerobic conditions. Three parallel tubes were prepared for each strain from each media. Equal volumes of conventional and modified medium were inoculated with an equal volume of inoculum from freshly grown cultures. The increase in absorbance A were measured every day at 650 nm using a Bausch and Lomb (Belgium) Spectronic 88 spectrophotometer.
3. Results and discussion Although photosynthetic energy conversion and photoautotrophic growth require anaerobic conditions, generally semianaerobic or aerobic conditions have been used to cultivate phototrophic bacteria [4,8,9]. Thus, it is often difficult to grow and conventional maintain such cultures under semianaerobic conditions as it has been observed that phototrophically grown cultures mainly show their sensitivity to oxygen in the presence of light [4]. The genus Chlorojlexus resembles Chlorobium in cell structure and chlorophyll pigments, but is similar to the purple non-sulfur bacteria in being both photoheterotrophic and facultatively chemoheterotrophic, in addition to the ability to grow photoautotrophically [9- 1 I]. Thus anaerobic conditions and the presence of sulfide in the modified medium favoured growth of Chloroj?exus aurantiacus and ChloroJlexus aggregans. Although the medium described (Table 1) is not totally new, slight modification in its composition has resulted in improved growth for all the tested strains. All the cultures grown under modified conditions showed full pigmentation indicating that the cultivation conditions were optimal for photoautotrophic growth. During parallel growth experiments, after equal periods of incubation, a marked increase in optical density was observed in modified medium as compared to the conventional medium (Table 2). The results in Table 2 show that the period of incubation was shortened by several days to obtain equal cell density. Sodium sulfide, which was injected to lower the redox potential, was favored during growth and maintenance as Chlorojlexus apparently also grows photoautotrophically with sulfide as electron donor [10,12].
K.A. Malik I Journal
Table 2 Photoautotrophic
growth
Strain and DSM No.
of Chloroflexaceae
of Microbiological
Methods
under modified and conventional
Conditions
Increase
27 (1996)
149
147-150
conditions
in A (650 nmj”
Growth in modified and conventional
medium after (days)
1
2
3
4
7
8
9
10
A B
0.04 0.03
0.08 0.04
0.25 0.05
0.30 0.06
0.65 0.04
0.85 0.03
1.15 0.02
1.35 0.02
A B
0.03 0.02
0.15 0.03
0.30 0.04
0.45 0.05
0.60 0.04
0.65 0.03
0.75 0.02
0.95 0.02
A B
0.04 0.02
0.07 0.03
0.25 0.03
0.55 0.04
0.65 0.04
0.75 0.03
0.85 0.03
0.95 0.02
A B
0.03 0.02
0.08 0.03
0.25 0.04
0.40 0.05
0.69 0.04
0.85 0.03
0.95 0.02
1.15 0.02
A B
0.05 0.05
0.08 0.07
0.35 0.15
0.65 0.30
0.85 0.45
0.95 0.55
1.35 0.60
1.55 0.50
Chlorojle.xus aurantiacus
DSM 635 Chlorojlexus
aurantiacus
DSM 636 ChloroJlexus
aurantiacus
DSM 637 Chlorojfexus
aurantiacus
DSM 638 ChlorojGxus
aggregansh
DSM 9485
A, cultures grown in moduied medium under modified conditions; B, cultures grown in conventional medium under conventional conditions. For more details, see Section 2. aEqual volumes of conventional and modified medium were inoculated with an equal volume of inoculum from freshly grown cultures. The tube cultures were grown under conventional and modified conditions. Increase in A values (rounded to two decimal) were measured at 650 nm every day until 10 days of incubation. The A values at zero time were about 0.01 in all cases. The results given are from one experiment that was typical of three. h Cultures formed aggregates and the exact measurement of optical density was a problem.
Under such anaerobic conditions, the liquid cultures could be mainrained much longer compared to the cultures grown and maintained under conventional semiaerobic conditions. Detailed studies on the maintenance of such liquid cultures for long time periods have been reported elsewhere [ 131.
Acknowledgments I am thankful assistance.
to Andrea
Schutze
for technical
References [1] Castenholz, R.W. and Pierson, B.K. (1981) Isolation of members of the family Chloroflexaceae. In: The Prokaryotes (Eds. M.P. Starr, H. Stolp, H.G. Trtlper, A. Balows and H.G. Schlegel). Springer, Berlin, pp. 290-298.
[2] Pierson. B.K and Castenholz. R.W. (1992) The family Chloroflexaceae. In: The Prokaryotes (Eds. A. Balows, H.G. Triiper. M. Dworkin, W. Harder and K.H. Schleifer), Second Edition. Springer, Berlin, pp. 3754-3774. [31 Malik. K.A. ( 1983) A modified method for the cultivation of phototrophic bacteria. J. Microbial. Methods 1, 343-352. [41 Malik. K.A. (1991) Maintenance of phototrophic bacteria. In: Maintenance of Microorganisms and Cultured Cells (Eds. B.E. Kirsop and A. Doyle). Academic Press, London, pp. 82-100. [51 Schmidt. K. (1980) A comparative study on the composition of chlorosomes (Chlorobium vesicles) and cytoplasmic membranesfrom Chlorojems aurantiacus strain OK-70-fl and Chlorobium limicola f. thiosulfatophilum strain 6230. Arch. Microbial. 124, 2 l-3 1. [61 Pierson, B.K and Castenholz, R.W. (1974) A phototrophic gliding filamentous bacterium of hot springs, Chlorojlexus trurarztiacus, gen. and sp. nov. Arch. Microbial. 100. 5-24. 171 Hanada, S., Hiraishi, A., Shimada. K. and Matsura, K., ( 1995) Chlorojexus aggregam sp. nav., a phototrophic filamentous bacterium which forms dense aggregates by active gliding movement. Int. J. Syst. Bacterial. 45, 676681. for the enrichment, WI Van Niel, C.B. (1971) Techniques isolation and maintenance of the photosynthetic bacteria. In: Methods in Enzymology,Vol. 23, Part A (Ed. A. San Pi&o). Academic Press, New York, pp. 2-3.
150
K.A. Malik I Journal of Microbiological
[9] Pfennig, N. and Triiper, H.G. (1989) Anoxygenic phototrophic bacteria. In: Bergey’s Manual of Systematic Bacteriology, Vol. 3 (Eds. J.T. Staley, M.P. Bryant, N. Pfennig and J.G. Halt). Williams and Wilkins, Baltimore, pp. 16351709. [lo] Madigan. M.T., Petersen, S.R. and Brock, T.D. (1974) Nutritional studies on Ctrloroje..wus. a filamentous photosynthetic, gliding bacterium. Arch. Microbial. 100, 97-103. [l l] Castenholz, R.W. (1989) Genus ChlorojJexus. In: Bergey’s Manual of Systematic Bacteriology. Vol. 3 (Eds. J.T. Staley.
Methods 27 (1996) 147-150 M.P. Bryant, N. Pfennig and J.G. Holt). Williams and Wilkins, Baltimore, pp. 1698-1709. [12] Madigan, M.T. and Brock, T.D. (1975) Photosynthetic sulfide oxidation by Clrlorojlexus aurantiacus. a filamentous photosynthetic. gliding bacterium. J. Bacterial. 122, 782784. [13] Malik, K.A. (1996) A convenient method of maintaining Chlorojkmu for long time periods as slow growing liquid cultures. J. Microbial. Methods 27, 151-155.