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A novel method for isolation of magnetic bacteria without magnetic collection using magnetotaxis
ELSEVIER
Journal
of Microbiological
Methods
26 (1996)
Journal ofMicrobiological Methods
139-145
A novel me,thod for isolation of magnetic bacteria without magnetic collection using magnetotaxis Toshifumi
Sakaguchi,
qf Biotechnology,
Departtnertt
Received
Noriyuki
Tokyo University
16 August
Tsujimura,
of Agriculture
Tadashi Matsunaga”
and Technology,
Koganei.
Tobo
184, Japan
1995: revised 29 February 1996; accepted 2 March 1996
Abstract A novel method was developed for isolating magnetic bacteria without magnetic collection using magnetotaxis. The method consists of incubation of sediments, enrichment of bacteria in the medium, isolation of enriched bacteria by colony formation, and optimization of conditions for growth and synthesis of magnetic particles. The water column above natural sediment, incubated at 25°C under dim light, and containing many species of bacteria, was employed as the inoculum. Collection of magnetc bacteria using magnets was not carried out. Ferric quinate was used as the main iron source in the liquid isolation medium. Due to iron sulfide precipitation, formation of black crystals was observed in the enriched culture of magnetic bacteria. Magnetic bacteria were purified by colony formation from enriched cultures which formed the black crystals. Culturing condition was optimized by addition of appropriate nutrients which behave as electron acceptors or donors. This method allows isolation of non-motile and non- or weakly-magnetotactic bacteria, which would not accumulate in the presence of an applied magnetic field. A sulphate-reducing magnetic anaerobe. RS-1, which is weakly magnetotactic, was isolated by this method. In addition, the successful isolation of RS-1 by this method suggests the presence of magnetic bacteria which exist in a non-magnetic state in sediments. Keywords:
Enrichment;
Isolation;
Sediment;
Magnetic
bacteria; Sulfate-reducing
1. Introduction Magnetic intracellular
bacteria
have
nano-sized
the
ability
fine magnetic
cells are able to respond terrestrial or artificial
to synthesize particles.
The
and orient along the lines of magnetic fields. Therefore,
since the discovery of magnetic bacteria in 1975 [4], all isolation methods of magnetic bacteria have relied on harvesting with a magnet or artificial magnetic fields [ 1,7,18,20,25,28,35] and many types of magnetic bacteria of various morphologies, such as *Corresponding 857713
author.
0167-7012/96/$15.00
Tel.:
0
+81
423 887020:
fax:
+81
423
1996 Elsevier Science B.V. All rights reserved
PII SO167-7012(96)00905-O
bacteria
spirilla, vibrioids, cocci, rods, and multicellular bacteria [ 12,161 have been observed in various ecological niches and in enriched samples [1,5,6,11,13,15,17,25,26,29,30,33,34,36]. However, it is difficult to maintain magnetic bacteria in an artificial environment. The number of axenically cultured magnetic bacteria is extremely limited in comparison with the diversity in natural sediments. Axenic cultures of magnetic bacteria are typically obtained by magnetic collection using their magnetotactic properties [2,3,7,10,20-23,281. On the other hand, the presence of forms of magnetic bacteria which are non-magnetic or nonmotile as a result of environmental factors such as
140
T. Sakaguchi et al. I Journal of Microbiological
oxygen concentration or the carbon source, has been demonstrated in axenic culture [7,8,14,19,20,28]. It is impossible to distinguish magnetic bacteria in a non-magnetic state from other microorganisms without using molecular techniques [9,31,32]. Therefore, in natural environments also, we expect the existence of non-motile, non-magnetic, and weakly magnetotactic bacteria which can not be detected as magnetic bacteria. Thus, we report here a novel enrichment and isolation method for magnetic bacteria without magnetic collection using their magnetotaxis, which has been applied to the enrichment and isolation of a weakly magnetotactic sulfate-reducing bacterium, strain RS-1 [27].
2. Materials and methods 2.1. Collection
of sediment
Water and sulfide-rich sediment samples were taken from a waterway near Kameno River in Wakayama Prefecture, western Japan. Sediment was divided into lOOO-ml glass bottles with plastic caps, and water from that location or distilled water was poured into the bottle so that each contained sediment and water in a ratio of 1:2. These samples were incubated with the caps loose to allow gaseous exchange at room temperature (approximately 25°C). Changes in the samples were observed during incubation.
2.2. Observations of microorganisms in the water column above incubated sediment and crude enrichment culture Microorganisms in the water column (not the sediment) of sample vessels after incubation and in enrichment media added to this water, were observed using a phase-contrast microscope (Model BH-2PC, Olympus, Tokyo, Japan). To provide a magnetic field for microscope slide preparations, a rectangular samarium-cobalt (Sm-Co) magnet of size 17 X 15 X 15 mm (TDK, Tokyo, Japan) was placed on the stage of a microscope. The magnetic sensitivity of
Methods 26 (1996) 139-145
the microorganisms which appeared in water and enrichments was determined by observing rotation of cells when the Sm-Co magnet on the microscope was rotated by hand. To measure cell weight and growth in the water and enrichments, dry weights were determined, and optical densities of solutions were measured using a spectrophotometer (UV-2200, Shimadzu, Kyoto, Japan) at 660 nm. Cell concentrations were determined using a hemacytometer. Redox potential and dissolved oxygen concentration were measured using Ag-AgC1 and oxygen electrodes (BO-219, Able, Tokyo, Japan).
2.3. Preparation of medium for enrichment of magnetic bacteria and iron source optimization The composition of the medium for enrichment of magnetic bacteria was as follows (per liter of distilled water): 0.1 g of potassium dihydrogen phosphate, 0.06 g of ammonium nitrate, 0.05 g of yeast extract, 0.02 g of succinate, 2.0 ml of Wolfe’s mineral solution (ferrous sulfate was omitted), 0.05 g of sodium thioglycolate, and 2.0 ml of each different iron source solution. To determine the optimum iron source for synthesis of intracellular magnetic particles of magnetic bacteria in the medium, five 17 mM iron sources, ferric chloride, ferrous sulfate, ferric quinate, ferric gallate, and ferric citrate, were prepared. These solutions were made and used as principal iron sources in the medium [20]. Media containing different iron sources (34 PM) were adjusted to pH 6.9 with NaOH solution before autoclaving (120°C 2 atm, 10 min). After sterilization and cooling, 35 ml of each medium were poured into a 50-ml conical flask. The medium was sparged with 100% argon gas to establish anaerobic conditions, and the gas phase was replaced with 100% argon. The flasks were sealed with butyl gum stoppers, and inocula were injected using disposable syringes. Water (0.2 ml) above the sediment incubated at room temperature, containing non-magnetic microorganisms, was inoculated into 50-ml conical flasks containing 35 ml of the prepared medium and iron source to be tested. To enrich for magnetic bacteria, the flasks were incubated at room temperature (approximately 25°C) under dim light for 7 days after inoculation.
T. Sakaguchi et al. I Joumal
2.4. Investigation oj’ the effects of sulfur and nitrogen compounds on the growth of magnetic bacteria Sulfur compounds (sodium sulfide, sodium thioglycolate, cysteine and sodium sulfate) and nitrogen compounds (sodium nitrate, sodium nitrite) were used to investigate which optimum electron acceptor was required for griowth of enriched magnetic bacteria. Each compound was added separately at concentrations of 0.3-l.5 mM to the crude culture in which the rod-shaped magnetic bacteria occurred. Ferric quinate (32 I was used as the Fe source. After 5 days, cell concentration was measured using a hemacytometer.
2.5. Purification of enriched magnetic colony formation
bacteria by
Magnetic bacteria which were enriched in the crude culture by ad’dition of the appropriate nutrient (as an electron acceptor) into the enrichment medium were purified by colony formation on agar plates (0.7% w/v agar) in an anaerobic jar (Oxoid, Basingstoke, UK) [20]. Succinate was removed from the enrichment medium. Ferric quinate and sodium sulfate were used as Fe and S sources, respectively.
2.6. IdentiJcation of optimum growth of magnetic bacteria
141
of Microbiological Methods 26 (1996) 139-145
electron donor for
To maintain large numbers of axenic magnetic bacteria, the optimum electron donor (carbon source) for growth was determined. Solutions of each carbon source (100 n-M) and yeast extract (Difco) were sterilized by filtration through nitrate cellulose membranes (pore size 0.2 pm, Advantec Toyo, Tokyo, Japan). These sterilized solutions were added to the enrichment medium lacking a carbon source at concentrations of 0.75-4.5 mM. Ferric quinate (32 ,uM) and sodium sulfate (0.93 mM) were used as the Fe and S sources, respectively. After 5 days, cell growth in the presance of each test compound was measured using a hemacytometer. The effect of the addition was estimated by comparison with a control to which no test compound was added.
3. Results 3.1. Observations of microorganisms sediment ana’ water column
in natural
Grey sludge with an odor of hydrogen sulfide was collected from the bottom of a waterway (depth; approximately 30 cm). Morphologically diverse magnetic bacteria such as cocci, spirilla and rods were observed in natural sediment before incubation at room temperature. The number of magnetic bacteria was approximately lo’-10’ cells per cm3 of the sediment. Approximately 90% of these cells were similar to rapidly swimming motile cocci Bilophococcus magnetotacticus [24] and their diameter was 2-3 pm. After 7 days, many microorganisms appeared in the water column above sediment incubated at room temperature, and the solution became turbid. The turbidity (optical density at 660 nm> increased from 0.058 to 0.080. The solution contained lo*-lo9 cells and, 12 mg of dry cells per liter. The redox potential (Eh) was 241-250 mV at pH 8.05. The dissolved oxygen concentration in the water column was almost 0 ppm. Cells in the water column did not respond to the magnetic field of a Sm-Co magnet (did not show magnetotaxis). However, some of the non-magnetic microorganisms were morphologically similar to magnetic bacteria which were collected magnetically at the sampling site. Increase in bacterial turbidity could be repeatedly observed in several samples of sediments. Bacterial growth in the aquatic zone of the sample vessels was therefore reproducible. 3.2. Growth of magnetic bacteria from samples of non-magnetic microorganisms in enrichment media When ferric quinate was used as an iron source in the enrichment medium, the color of the culture changed from colorless to blackish 7 days after incubation, and rod-shaped magnetic bacteria were present (Fig. 1). The redox potentials of the culture decreased to about -20 mV (pH 7.18-7.23). The cultures smelled of hydrogen sulfide and contained non-magnetic bacteria, such as spirilla, rods and vibrioids. The optical density at 660 nm of the cultures was 0.095-0.125 (107-lo8 cells/ml). How-
142
T. Sakaguchi et al. I Journal of Microbiological Method.7 26 (1996) 139- 14.5
quinate enriched medium, growth of magnetic bacteria was observed. These microorganisms had similar morphology to the non-magnetic bacteria isolated from the aquatic part of the sediment samples. These magnetic bacteria grew at densities of 5 X lo5 to 1 X lo6 cells/ml. They were motile, with a swimming speed of approximately 20 pm/s. In addition, it was possible to maintain these bacteria in complex crude culture by continued subculturing in the laboratory. The rod shaped magnetic bacteria appeared 5-10 days after 10% inoculation of this crude culture into fresh enrichment medium. 3.3. Effect of sulfur and nitrogen compounds on growth of magnetic bacteria in the enrichment medium
Fig. 1. Blackish culture containing rod shaped magnetic bacteria (A: (1) inoculated sample, (2) before the inoculation) and a phase contrast photomicrograph of enriched rod-shaped magnetic cells in the blackish culture (B: bar = 100 pm).
ever, when ferric citrate was used as the iron source, no color change occurred and the culture contained fewer types of bacteria than the blackish cultures. When ferric chloride, ferric gallate and ferrous sulfate were used as iron sources, growth of black crystals was observed in the cultures but no growth of magnetic bacteria was observed. However, when the same microorganisms were grown in ferric Table 1 Effects of added sulfur compounds
on numbers
When sodium sulfate was added to the enrichment medium, the population of magnetic bacteria increased more than lo-fold (lo7 cells/ml) compared to other sulfur compounds (Table 1). The concentration of magnetic bacteria gradually increased with increasing sulfate concentration of the medium (Fig. 2). The color of the culture changed to blackish, and hydrogen sulfide was generated. On the other hand, other sulfur compounds (sodium thioglycolate, cysteine and sodium sulfide) did not noticeably enhance the concentration of magnetic bacteria. When nitrate or nitrite was added to the medium, the concentration of magnetic bacteria did not increase. 3.4. Isolation of magnetic formation
bacteria by colony
Magnetic bacteria which increased in the enrichment medium were spread on agar plates. A rodshaped magnetic bacterium (RS-1) formed white irregular round colonies on agar plates under an-
of rod shaped magnetic
bacterial
in complex
crude culture
Source
Concentration
No addition Sodium thioglycolate (0.88 mM) Cysteine (0.57 n&l) Sodium sulfide (0.31 mM) Sodium sulfate (0.42 mM)
Less than lo5 cells/ml 8.1 X 105-1.0 X lo6 cells/ml 1.3-5.2 X lo5 cells/ml 1.3-3.2 X lo6 cells/ml More than 10’ cells/ml
“After 5 day, each growth was measured,
and the initial cell concentration
of magnetic
of rod” shaned magnetic bacteria
cells was approximately
lo4 cells/ml.
143
T. Sakaguchi et al. I Journal of Microbiological Methods 26 (1996) 139-145
u
0.0
0.4
0.8
1.2
Sulfate concentration
1.6 Carbon source concentration
(mM)
Fig. 2. Effect of sulfate concentration in the isolation medium on growth of rod-shaped magnetic bacteria. The original Fe salt in these media was ferric quinate (32 PM). Initial cell concentration: 5 X lo4 cells/ml.
aerobic conditions (Fig. 3), and was isolated as a pure strain. The micro-colonies appeared 14-20 days after the inoculation. Their diameter was less than 1.5 mm. Appears that choice is random. Cells in the colony were not magneto-sensitive, due to iron deficiency or a metabolic reason, but were the same size and morphology as the rod-shaped magnetic bacteria observed in the crude culture. Although the cells were not magneto-sensitive in colonial form, they recovered their ability to synthesize intracellular
(mM)
Fig. 4. Effects of carbon sources on growth of rod-shaped magnetic bacteria. Yeast extract 0.3 g/l, 0.93 mM of sodium sulfate and 32 /.LM of ferric quinate were added to each medium. Initial cell concentration: 2.5 X lo5 cells/ml, temperature 25°C. 5 days growth after the cell inoculation.
magnetic particles when cultured in the liquid enrichment medium to which 0.31 mM sodium sulfate and 0.3 g/l yeast extract (Difco) were added. They were therefore identified as magnetic bacteria. 3.5. Effect of carbon sources on growth of magnetic bacteria in the enrichment medium Addition of 0.3-0.5 g/l of yeast extract (Difco) increased the number of magnetic cells in enrichment media lacking carbon sources. Over 1.0 g/l of yeast extract inhibited growth. Lactate, malate, pyruvate, ethanol, and fumarate enhanced growth of the isolated rod-shaped magnetic bacteria (RS-l), and served as electron donors for growth under sulfatereducing conditions. These carbon sources were effective in increasing cell numbers of magnetic bacteria. As shown in Fig. 4, pyruvate was a particularly good carbon source, and the optimum concentration was over 1.5 mM. A final cell concentration of 1 X lo* cells/ml was reached in liquid culture when an initial cell concentration of 2.5 X lo5 cells/ml was employed. However, succinate and acetate did not enhance growth.
4. Discussion Fig. 3. Colony formaticn of enriched magnetic bacteria (RS-1) on enrichment medium containing agar (0.7% w/v). Bar indicates I .O cm.
This novel isolation method mainly consists of the following steps: (1) incubation of sediment, (2)
144
T. Sakaguchi et al. I Journal of Microbiological Methods 26 (1996) 139-145
enrichment of bacteria in the medium, (3) isolation of enriched bacteria by colony formation, (4) optimization of nutrients and conditions for growth and for synthesis of bacterial magnetic particles. In particular, artificial magnetic fields were not used for the collection of magnetic bacteria. Bar magnets were used only to identify cells rotated by the magnetic force as magnetic bacteria. The characteristic feature of this method was the use of a water column above sediment incubated at 25°C (room temperature) under dim light as the inoculum. This water was turbid and contained various non-magnetic microorganisms, which appeared to be from the sediment, and may have included a non-magnetic form of magnetic bacteria. When the culture was enriched for these bacteria, their color changed to blackish, due to precipitation of metal sulfide compounds by sulfate reduction. Therefore, it was possible to utilize this color change as an indicator of the presence of magnetic bacteria like RS-1. In fact when we succeeded in enriching rod-shaped magnetic bacteria from other sampling points (data not shown), each of the enrichments turned the color to blackish. For further enrichment of magnetic bacteria, growth optimization tests were carried out by addition or modification of an appropriate nutrient which was capable of acting as an electron acceptor or donor for growth. This is a valuable procedure for establishing a pure culture, determining the conditions under which magnetic inclusions are synthesized, investing the properties of isolated bacteria. In previous pure cultures of magnetic bacteria [3,7,20,23,28], magnetotaxis (swimming along magnetic field lines) was important for collection of the bacteria. Magnetic isolation is limited to microorganisms with strong and stable magnetotaxis. Magnetic cells of RS-1 showed different aerotaxis to magnetic microaerophiles in slide preparations. They accumulated at a single point in the center of the cover slip under artificial magnetic fields (Fig. 5). Although artificial magnetic fields elicited a magnetic response from RS-1 cells, these cells could not be collected, due to their strong anaerotaxis and weak magnetotaxis. Furthermore, the ratio of magnetic north to south seeking cells was approximately 1:l in a population of the cells, and they reverse their direction frequently. These observations indicate that RS1 is difficult to isolate and purify magnetically. This suggests that the range of magnetic bacteria which
___.~ -
-... lOOplIt
Fig. 5. Anaerotaxis of rod shaped magnetic bacteria (RS-I) towards a single point at the center of a microscope slide. Arrow represents direction of magnetic axis (bar = 100 pm).
can be collected using artificial magnetic fields is limited, and our method allows isolation of nonmotile and non- or weakly magnetotactic bacteria. This successful isolation of RS-1 shows that it is possible to isolate magnetic bacteria without use of magnetophoresis due to magnetotaxis, and implies the presence of magnetic bacteria which can not be detected as magnetic bacteria because of existing in a non-magnetic form in natural sediments.
References 111Adamkiewicz,
VW., Authier, A., Dumont, S., Garzon, S., Leduc, S., Morency, D., Nakhostin, N. and Strykowski, H. (1991) A simple procedure for enriching and cultivating magnetic bacteria in low agar-mud medium. J. Microbial. Methods 13, 2.55-258. 121Bazylinski, D.A. (1990) Anaerobic production of singledomain magnetite by the marine, magnetotactic bacterium, strain MV-1. In: Iron Biominerals (Eds. R.B. Frankel and R.P. Blakemore). Plenum Press, New York, pp. 69-77. 131 Bazylinski, D.A., Frankel, R.B. and Jannasch. H.W. (1988) Anaerobic magnetite production by a marine, magnetotactic bacterium. Nature 334. 518-519. [41 Blakemore, RX (1975) Magnetotactic bacteria. Science 190, 377-379. PI Blakemore, RI? (1982) Magnetotactic bacteria. Annu. Rev. Microbial. 36, 217-238. [61 Blakemore, R.P., Blakemore, N.A., Bazylinski, D.A. and Moench, T.T. (1989) Magnetotactic bacteria. In: Bergey’s Manual of Systematic Bacteriology, Vo1.3. (Eds. J.T. Staley, M.P. Bryant, N. Pfenning and J.G. Holt). Williams & Wilkins, Baltimore, pp. 1882-1889.
T. Sakaguchi
et al. I Journal of Microbiological
[7] Blakemore, RX, Maratea, D. and Wolfe., R.S. (1979) Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium. J. Bacterial. 140, 720-729. [8] Blakemore, R.P., Short, K.A., Bazylinski, D.A., Rosenblatt, C. and Frankel, R.B. (1985) Microaerobic conditions are required for magnetire formation within Aquaspirillum magnetotacticum. Geomicrobiology 4, 53-71. [9] Burgess, J.G., Kawaguchi, R., Sakaguchi, T., Thomhill, R.H. and Matsunaga, T. (1993) Evolutionary relationship among Magnetospirillum strains inferred from phylogenetic analysis of 16s rDNA sequences. J. Bacterial. 175, 6689-6694. [lo] Delong, E.F., Frankel, R.B. and Bazylinski, D.A. (1993) Multiple evolutionary origins of magnetotaxis in bacteria. Science 259, 803-806. [ll] Esquivel, D.M.S. and Lins de Barros, H.G.P. (1986) Motion of magnetotactic microorganisms. J. Exp. Biol. 121, 153163. [12] Farina, F., EsquiveI, D.S.M. and Lins de Barros, H.G.P. (1990) Magnetic iron-sulphur crystals from a magnetotactic microorganism. Nature 343, 256-258. [ 131 Fassbinder, J.W.E., Stajek, H. and Vali, H. (1990) Occurrence of magnetic bacteria in soil. Nature 343, 161-163. [14] Fmnkel, R.B., Blakemore, R.P. and Wolfe, R.S. (1979) Magnetite in freshwater magnetotactic bacteria. Science 203, 1355-1356. [15] Mann, S., Sparks, N .H.C. and Board, R.G. (1990) Magnetotactic bacteria: microbiology, biomineralization, palaeomagnetism and biotechnology. Adv. Microb. Physiol. 31, 125181. [16] Mann, S., Sparks, 1N.H.C.. Frankel, R.B., Bazylinski, D.A. and Jannasch, H.W (1990) Biomineralization of ferrimagnetic greigite (Fe,S,) and iron pyrite (Fe&) in a magnetotactic bacterium. Nature 343, 258-261. [17] Matitashvili, E.A., Matojan, D.A., Gendlar, T.S., Kurzchalia, T.V. and Adamia, R.S. (1992) Magnetotactic bacteria from freshwater lakes in Georgia. J. Basic Microbial. 32, 185192. [18] Matsunaga, T. and Kamiya, S. (1987) Use of magnetic particles isolated from magnetotactic bacteria for enzyme immobilization. Appl. Microbial. Biotechnol. 26, 328-322. [19] Matsunaga, T. and Sakaguchi, T. (1992) Production of biogenic magnetite by aerobic magnetic spirilla strains AMB-1 and MGT.-1. In: Fe&es, Proceeding of the Sixth International Conference on Ferrites (ICF 6). Japan Society of Powder and Powder Metallurgy, Tokyo and Kyoto, Japan, pp. 262-267. [20] Matsunaga, T., Sakaguchi, T. and Tadokoro, F. (1991) Magnetite formation by magnetic bacteria capable of growing aerobically. Appl. Microbial. Biotechnol. 35, 651-655. (211 Matsunaga, T, Tad’akoro, F. and Nakamura, N. (1990) Mass culture of magnetic bacteria and their application to flow type immunoassay:;. IEEE Trans. Magnet. 26, 1557-1559. [22] Meldrum, F.C., Mann, S., Heywood, B.R., Frankel, R.B. and Bazylinski, D.A. (1993) Electron microscopy study of
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[26]
[27]
[28]
[29]
[30]
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[34]
[35]
[36]
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magnetosomes in a cultured coccoid magnetotactic bacterium. Proc. R. Sot. Lond. B 251, 231-236. Meldrum, F.C., Mann, S., Heywood, B.R., Frankel, R.B. and Bazylinski, D.A. (1993) Electron microscopy study of magnetosomes in a cultured vibrioid magnetotactic bacteria. Proc. R. Sot. Lond. B 251, 237-242. Moench, T.T. (1988) Bilophococcus magnetotacticus gen. nov. sp. nov. a motile magnetic coccus. Anton. v. Leeuwenhoek Int. J. Gen. Microbial. 54, 483-496. Moench, T.T. and Konetzka, W.A. (1978) A novel method for the isolation and study of a magnetotactic bacterium. Arch. Microbial. 119, 203-212. Oberhack, M., Siissmuth, R. and Frank, H. (1987) Magnetotactic bacteria from freshwater. Z. Naturforsch. 42c, 300306. Sakaguchi, T., Burgess, J.G. and Matsunaga, T. (1993) Magnetite formation by a sulfate-reducing bacterium. Nature 365, 47-49. Schleifer, K.H., Schtiler, D., Spring, S., Weizenegger, M., Amann, R., Ludwig, W. and Kohler, M. (1991) The genus Magnetospirillum, gen. nov. Description of Magnetospirillum gryphiswaldense and transfer of Aquaspirillum magnetotacticum to Magnetospirillum mugnetotacticum, comb. nov. Syst. Appl. Microbial. 14, 379-385. Sparks, N.H.C., Courtaux, L., Mann, S. and Board, R.G. (1986) Magnetotactic bacteria are widely distributed in sediments in the UK. FEMS Microbial. Lett. 37, 305-308. Spormann, A.M. and Wolfe, R.S. (1984) Chemotactic, magnetotactic and tactile behaviour in a magnetic spirillum. FEMS Microbial. Lett. 22, 171-177. Spring, S., Amann, R., Ludwig, W., Schleifer, K.H., van Gemerden, H. and Petersen, N. (1993) Dominating role of an unusual magnetotactic bacterium in the microaerobic zone of a freshwater sediment. Appl. Environ. Microbial. 59, 23972403. Spring, S., Amann, R., Ludwig, W., Schleifer, K.H. and Petersen, N. (1992) Phylogenetic diversity and idetification of non-culturable magnetotactic bacteria. Syst. Appl. Microbiol. 15, 116-122. Stolz, J.F., Chang, S.-B.R. and Kirschvink, J.L. (1986) Magnetotactic bacteria and single-domain magnetite in hemipelagic sediments. Nature 321, 849-851. Thomhill, R.H., Burgess, J.G., Sakaguchi, T. and Matsunaga, T. (1993) A morphological classification of bacteria containing bullet-shaped magnetic particles. FEMS Microbial. Lett. 115, 169-176. Wolfe, R.S., Thauer, R.K. and Pfennig, N. (1987) A ‘capillary racetrack’ method for isolation of magnetotactic bacteria. FEMS Microbial. Ecol. 45, 31-35. Wolin, E.A., Wolin, M.J. and Wolfe, R.S. (1963) Formation of methane by bacterial extracts. J. Biol. Chem. 238, 28822886.
Journal
of Microbiological
Methods
26 (1996)
Journal ofMicrobiological Methods
139-145
A novel me,thod for isolation of magnetic bacteria without magnetic collection using magnetotaxis Toshifumi
Sakaguchi,
qf Biotechnology,
Departtnertt
Received
Noriyuki
Tokyo University
16 August
Tsujimura,
of Agriculture
Tadashi Matsunaga”
and Technology,
Koganei.
Tobo
184, Japan
1995: revised 29 February 1996; accepted 2 March 1996
Abstract A novel method was developed for isolating magnetic bacteria without magnetic collection using magnetotaxis. The method consists of incubation of sediments, enrichment of bacteria in the medium, isolation of enriched bacteria by colony formation, and optimization of conditions for growth and synthesis of magnetic particles. The water column above natural sediment, incubated at 25°C under dim light, and containing many species of bacteria, was employed as the inoculum. Collection of magnetc bacteria using magnets was not carried out. Ferric quinate was used as the main iron source in the liquid isolation medium. Due to iron sulfide precipitation, formation of black crystals was observed in the enriched culture of magnetic bacteria. Magnetic bacteria were purified by colony formation from enriched cultures which formed the black crystals. Culturing condition was optimized by addition of appropriate nutrients which behave as electron acceptors or donors. This method allows isolation of non-motile and non- or weakly-magnetotactic bacteria, which would not accumulate in the presence of an applied magnetic field. A sulphate-reducing magnetic anaerobe. RS-1, which is weakly magnetotactic, was isolated by this method. In addition, the successful isolation of RS-1 by this method suggests the presence of magnetic bacteria which exist in a non-magnetic state in sediments. Keywords:
Enrichment;
Isolation;
Sediment;
Magnetic
bacteria; Sulfate-reducing
1. Introduction Magnetic intracellular
bacteria
have
nano-sized
the
ability
fine magnetic
cells are able to respond terrestrial or artificial
to synthesize particles.
The
and orient along the lines of magnetic fields. Therefore,
since the discovery of magnetic bacteria in 1975 [4], all isolation methods of magnetic bacteria have relied on harvesting with a magnet or artificial magnetic fields [ 1,7,18,20,25,28,35] and many types of magnetic bacteria of various morphologies, such as *Corresponding 857713
author.
0167-7012/96/$15.00
Tel.:
0
+81
423 887020:
fax:
+81
423
1996 Elsevier Science B.V. All rights reserved
PII SO167-7012(96)00905-O
bacteria
spirilla, vibrioids, cocci, rods, and multicellular bacteria [ 12,161 have been observed in various ecological niches and in enriched samples [1,5,6,11,13,15,17,25,26,29,30,33,34,36]. However, it is difficult to maintain magnetic bacteria in an artificial environment. The number of axenically cultured magnetic bacteria is extremely limited in comparison with the diversity in natural sediments. Axenic cultures of magnetic bacteria are typically obtained by magnetic collection using their magnetotactic properties [2,3,7,10,20-23,281. On the other hand, the presence of forms of magnetic bacteria which are non-magnetic or nonmotile as a result of environmental factors such as
140
T. Sakaguchi et al. I Journal of Microbiological
oxygen concentration or the carbon source, has been demonstrated in axenic culture [7,8,14,19,20,28]. It is impossible to distinguish magnetic bacteria in a non-magnetic state from other microorganisms without using molecular techniques [9,31,32]. Therefore, in natural environments also, we expect the existence of non-motile, non-magnetic, and weakly magnetotactic bacteria which can not be detected as magnetic bacteria. Thus, we report here a novel enrichment and isolation method for magnetic bacteria without magnetic collection using their magnetotaxis, which has been applied to the enrichment and isolation of a weakly magnetotactic sulfate-reducing bacterium, strain RS-1 [27].
2. Materials and methods 2.1. Collection
of sediment
Water and sulfide-rich sediment samples were taken from a waterway near Kameno River in Wakayama Prefecture, western Japan. Sediment was divided into lOOO-ml glass bottles with plastic caps, and water from that location or distilled water was poured into the bottle so that each contained sediment and water in a ratio of 1:2. These samples were incubated with the caps loose to allow gaseous exchange at room temperature (approximately 25°C). Changes in the samples were observed during incubation.
2.2. Observations of microorganisms in the water column above incubated sediment and crude enrichment culture Microorganisms in the water column (not the sediment) of sample vessels after incubation and in enrichment media added to this water, were observed using a phase-contrast microscope (Model BH-2PC, Olympus, Tokyo, Japan). To provide a magnetic field for microscope slide preparations, a rectangular samarium-cobalt (Sm-Co) magnet of size 17 X 15 X 15 mm (TDK, Tokyo, Japan) was placed on the stage of a microscope. The magnetic sensitivity of
Methods 26 (1996) 139-145
the microorganisms which appeared in water and enrichments was determined by observing rotation of cells when the Sm-Co magnet on the microscope was rotated by hand. To measure cell weight and growth in the water and enrichments, dry weights were determined, and optical densities of solutions were measured using a spectrophotometer (UV-2200, Shimadzu, Kyoto, Japan) at 660 nm. Cell concentrations were determined using a hemacytometer. Redox potential and dissolved oxygen concentration were measured using Ag-AgC1 and oxygen electrodes (BO-219, Able, Tokyo, Japan).
2.3. Preparation of medium for enrichment of magnetic bacteria and iron source optimization The composition of the medium for enrichment of magnetic bacteria was as follows (per liter of distilled water): 0.1 g of potassium dihydrogen phosphate, 0.06 g of ammonium nitrate, 0.05 g of yeast extract, 0.02 g of succinate, 2.0 ml of Wolfe’s mineral solution (ferrous sulfate was omitted), 0.05 g of sodium thioglycolate, and 2.0 ml of each different iron source solution. To determine the optimum iron source for synthesis of intracellular magnetic particles of magnetic bacteria in the medium, five 17 mM iron sources, ferric chloride, ferrous sulfate, ferric quinate, ferric gallate, and ferric citrate, were prepared. These solutions were made and used as principal iron sources in the medium [20]. Media containing different iron sources (34 PM) were adjusted to pH 6.9 with NaOH solution before autoclaving (120°C 2 atm, 10 min). After sterilization and cooling, 35 ml of each medium were poured into a 50-ml conical flask. The medium was sparged with 100% argon gas to establish anaerobic conditions, and the gas phase was replaced with 100% argon. The flasks were sealed with butyl gum stoppers, and inocula were injected using disposable syringes. Water (0.2 ml) above the sediment incubated at room temperature, containing non-magnetic microorganisms, was inoculated into 50-ml conical flasks containing 35 ml of the prepared medium and iron source to be tested. To enrich for magnetic bacteria, the flasks were incubated at room temperature (approximately 25°C) under dim light for 7 days after inoculation.
T. Sakaguchi et al. I Joumal
2.4. Investigation oj’ the effects of sulfur and nitrogen compounds on the growth of magnetic bacteria Sulfur compounds (sodium sulfide, sodium thioglycolate, cysteine and sodium sulfate) and nitrogen compounds (sodium nitrate, sodium nitrite) were used to investigate which optimum electron acceptor was required for griowth of enriched magnetic bacteria. Each compound was added separately at concentrations of 0.3-l.5 mM to the crude culture in which the rod-shaped magnetic bacteria occurred. Ferric quinate (32 I was used as the Fe source. After 5 days, cell concentration was measured using a hemacytometer.
2.5. Purification of enriched magnetic colony formation
bacteria by
Magnetic bacteria which were enriched in the crude culture by ad’dition of the appropriate nutrient (as an electron acceptor) into the enrichment medium were purified by colony formation on agar plates (0.7% w/v agar) in an anaerobic jar (Oxoid, Basingstoke, UK) [20]. Succinate was removed from the enrichment medium. Ferric quinate and sodium sulfate were used as Fe and S sources, respectively.
2.6. IdentiJcation of optimum growth of magnetic bacteria
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electron donor for
To maintain large numbers of axenic magnetic bacteria, the optimum electron donor (carbon source) for growth was determined. Solutions of each carbon source (100 n-M) and yeast extract (Difco) were sterilized by filtration through nitrate cellulose membranes (pore size 0.2 pm, Advantec Toyo, Tokyo, Japan). These sterilized solutions were added to the enrichment medium lacking a carbon source at concentrations of 0.75-4.5 mM. Ferric quinate (32 ,uM) and sodium sulfate (0.93 mM) were used as the Fe and S sources, respectively. After 5 days, cell growth in the presance of each test compound was measured using a hemacytometer. The effect of the addition was estimated by comparison with a control to which no test compound was added.
3. Results 3.1. Observations of microorganisms sediment ana’ water column
in natural
Grey sludge with an odor of hydrogen sulfide was collected from the bottom of a waterway (depth; approximately 30 cm). Morphologically diverse magnetic bacteria such as cocci, spirilla and rods were observed in natural sediment before incubation at room temperature. The number of magnetic bacteria was approximately lo’-10’ cells per cm3 of the sediment. Approximately 90% of these cells were similar to rapidly swimming motile cocci Bilophococcus magnetotacticus [24] and their diameter was 2-3 pm. After 7 days, many microorganisms appeared in the water column above sediment incubated at room temperature, and the solution became turbid. The turbidity (optical density at 660 nm> increased from 0.058 to 0.080. The solution contained lo*-lo9 cells and, 12 mg of dry cells per liter. The redox potential (Eh) was 241-250 mV at pH 8.05. The dissolved oxygen concentration in the water column was almost 0 ppm. Cells in the water column did not respond to the magnetic field of a Sm-Co magnet (did not show magnetotaxis). However, some of the non-magnetic microorganisms were morphologically similar to magnetic bacteria which were collected magnetically at the sampling site. Increase in bacterial turbidity could be repeatedly observed in several samples of sediments. Bacterial growth in the aquatic zone of the sample vessels was therefore reproducible. 3.2. Growth of magnetic bacteria from samples of non-magnetic microorganisms in enrichment media When ferric quinate was used as an iron source in the enrichment medium, the color of the culture changed from colorless to blackish 7 days after incubation, and rod-shaped magnetic bacteria were present (Fig. 1). The redox potentials of the culture decreased to about -20 mV (pH 7.18-7.23). The cultures smelled of hydrogen sulfide and contained non-magnetic bacteria, such as spirilla, rods and vibrioids. The optical density at 660 nm of the cultures was 0.095-0.125 (107-lo8 cells/ml). How-
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quinate enriched medium, growth of magnetic bacteria was observed. These microorganisms had similar morphology to the non-magnetic bacteria isolated from the aquatic part of the sediment samples. These magnetic bacteria grew at densities of 5 X lo5 to 1 X lo6 cells/ml. They were motile, with a swimming speed of approximately 20 pm/s. In addition, it was possible to maintain these bacteria in complex crude culture by continued subculturing in the laboratory. The rod shaped magnetic bacteria appeared 5-10 days after 10% inoculation of this crude culture into fresh enrichment medium. 3.3. Effect of sulfur and nitrogen compounds on growth of magnetic bacteria in the enrichment medium
Fig. 1. Blackish culture containing rod shaped magnetic bacteria (A: (1) inoculated sample, (2) before the inoculation) and a phase contrast photomicrograph of enriched rod-shaped magnetic cells in the blackish culture (B: bar = 100 pm).
ever, when ferric citrate was used as the iron source, no color change occurred and the culture contained fewer types of bacteria than the blackish cultures. When ferric chloride, ferric gallate and ferrous sulfate were used as iron sources, growth of black crystals was observed in the cultures but no growth of magnetic bacteria was observed. However, when the same microorganisms were grown in ferric Table 1 Effects of added sulfur compounds
on numbers
When sodium sulfate was added to the enrichment medium, the population of magnetic bacteria increased more than lo-fold (lo7 cells/ml) compared to other sulfur compounds (Table 1). The concentration of magnetic bacteria gradually increased with increasing sulfate concentration of the medium (Fig. 2). The color of the culture changed to blackish, and hydrogen sulfide was generated. On the other hand, other sulfur compounds (sodium thioglycolate, cysteine and sodium sulfide) did not noticeably enhance the concentration of magnetic bacteria. When nitrate or nitrite was added to the medium, the concentration of magnetic bacteria did not increase. 3.4. Isolation of magnetic formation
bacteria by colony
Magnetic bacteria which increased in the enrichment medium were spread on agar plates. A rodshaped magnetic bacterium (RS-1) formed white irregular round colonies on agar plates under an-
of rod shaped magnetic
bacterial
in complex
crude culture
Source
Concentration
No addition Sodium thioglycolate (0.88 mM) Cysteine (0.57 n&l) Sodium sulfide (0.31 mM) Sodium sulfate (0.42 mM)
Less than lo5 cells/ml 8.1 X 105-1.0 X lo6 cells/ml 1.3-5.2 X lo5 cells/ml 1.3-3.2 X lo6 cells/ml More than 10’ cells/ml
“After 5 day, each growth was measured,
and the initial cell concentration
of magnetic
of rod” shaned magnetic bacteria
cells was approximately
lo4 cells/ml.
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T. Sakaguchi et al. I Journal of Microbiological Methods 26 (1996) 139-145
u
0.0
0.4
0.8
1.2
Sulfate concentration
1.6 Carbon source concentration
(mM)
Fig. 2. Effect of sulfate concentration in the isolation medium on growth of rod-shaped magnetic bacteria. The original Fe salt in these media was ferric quinate (32 PM). Initial cell concentration: 5 X lo4 cells/ml.
aerobic conditions (Fig. 3), and was isolated as a pure strain. The micro-colonies appeared 14-20 days after the inoculation. Their diameter was less than 1.5 mm. Appears that choice is random. Cells in the colony were not magneto-sensitive, due to iron deficiency or a metabolic reason, but were the same size and morphology as the rod-shaped magnetic bacteria observed in the crude culture. Although the cells were not magneto-sensitive in colonial form, they recovered their ability to synthesize intracellular
(mM)
Fig. 4. Effects of carbon sources on growth of rod-shaped magnetic bacteria. Yeast extract 0.3 g/l, 0.93 mM of sodium sulfate and 32 /.LM of ferric quinate were added to each medium. Initial cell concentration: 2.5 X lo5 cells/ml, temperature 25°C. 5 days growth after the cell inoculation.
magnetic particles when cultured in the liquid enrichment medium to which 0.31 mM sodium sulfate and 0.3 g/l yeast extract (Difco) were added. They were therefore identified as magnetic bacteria. 3.5. Effect of carbon sources on growth of magnetic bacteria in the enrichment medium Addition of 0.3-0.5 g/l of yeast extract (Difco) increased the number of magnetic cells in enrichment media lacking carbon sources. Over 1.0 g/l of yeast extract inhibited growth. Lactate, malate, pyruvate, ethanol, and fumarate enhanced growth of the isolated rod-shaped magnetic bacteria (RS-l), and served as electron donors for growth under sulfatereducing conditions. These carbon sources were effective in increasing cell numbers of magnetic bacteria. As shown in Fig. 4, pyruvate was a particularly good carbon source, and the optimum concentration was over 1.5 mM. A final cell concentration of 1 X lo* cells/ml was reached in liquid culture when an initial cell concentration of 2.5 X lo5 cells/ml was employed. However, succinate and acetate did not enhance growth.
4. Discussion Fig. 3. Colony formaticn of enriched magnetic bacteria (RS-1) on enrichment medium containing agar (0.7% w/v). Bar indicates I .O cm.
This novel isolation method mainly consists of the following steps: (1) incubation of sediment, (2)
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enrichment of bacteria in the medium, (3) isolation of enriched bacteria by colony formation, (4) optimization of nutrients and conditions for growth and for synthesis of bacterial magnetic particles. In particular, artificial magnetic fields were not used for the collection of magnetic bacteria. Bar magnets were used only to identify cells rotated by the magnetic force as magnetic bacteria. The characteristic feature of this method was the use of a water column above sediment incubated at 25°C (room temperature) under dim light as the inoculum. This water was turbid and contained various non-magnetic microorganisms, which appeared to be from the sediment, and may have included a non-magnetic form of magnetic bacteria. When the culture was enriched for these bacteria, their color changed to blackish, due to precipitation of metal sulfide compounds by sulfate reduction. Therefore, it was possible to utilize this color change as an indicator of the presence of magnetic bacteria like RS-1. In fact when we succeeded in enriching rod-shaped magnetic bacteria from other sampling points (data not shown), each of the enrichments turned the color to blackish. For further enrichment of magnetic bacteria, growth optimization tests were carried out by addition or modification of an appropriate nutrient which was capable of acting as an electron acceptor or donor for growth. This is a valuable procedure for establishing a pure culture, determining the conditions under which magnetic inclusions are synthesized, investing the properties of isolated bacteria. In previous pure cultures of magnetic bacteria [3,7,20,23,28], magnetotaxis (swimming along magnetic field lines) was important for collection of the bacteria. Magnetic isolation is limited to microorganisms with strong and stable magnetotaxis. Magnetic cells of RS-1 showed different aerotaxis to magnetic microaerophiles in slide preparations. They accumulated at a single point in the center of the cover slip under artificial magnetic fields (Fig. 5). Although artificial magnetic fields elicited a magnetic response from RS-1 cells, these cells could not be collected, due to their strong anaerotaxis and weak magnetotaxis. Furthermore, the ratio of magnetic north to south seeking cells was approximately 1:l in a population of the cells, and they reverse their direction frequently. These observations indicate that RS1 is difficult to isolate and purify magnetically. This suggests that the range of magnetic bacteria which
___.~ -
-... lOOplIt
Fig. 5. Anaerotaxis of rod shaped magnetic bacteria (RS-I) towards a single point at the center of a microscope slide. Arrow represents direction of magnetic axis (bar = 100 pm).
can be collected using artificial magnetic fields is limited, and our method allows isolation of nonmotile and non- or weakly magnetotactic bacteria. This successful isolation of RS-1 shows that it is possible to isolate magnetic bacteria without use of magnetophoresis due to magnetotaxis, and implies the presence of magnetic bacteria which can not be detected as magnetic bacteria because of existing in a non-magnetic form in natural sediments.
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