Journal Of Microbiological Methods

4 downloads 0 Views 539KB Size Report
The cultures were cryopreserved in liquid nitrogen under anaerobic conditions using 5% dimethylsulfoxide as a cryoprotectant. For easy storage and transport, ...
ELSEVIER

Journal

of Microbiological Methods 35 ( 1999) 177-182

Journal OfMicrobiological Methods

Preservation of some extremely thermophilic chemolithoautotrophic bacteria by deep-freezing and liquid-drying methods Khursheed

A. Halik”

DSMZ-Deutsche Saumlung van Nikroorganismen und Zeilkulturen. GmbH. Nascheroder Weg lb, W-38124 Braunschweig, Germany

Received 11 September 1998; received in revised form 9 December 1998: accepted 11 December 1998

Abstract Long-term preservation methods for extreme thermophilic chemolithoautotrophic bacteria representing various species are described. The cultures were cryopreserved in liquid nitrogen under anaerobic conditions using 5% dimethylsulfoxide as a cryoprotectant. For easy storage and transport, the cultures were successfully liquid-dried, directly from the liquid phase without involving freezing under semiaerobic conditions using effective protective agents such as ethylenediamine and meso-inositol. The tested cultures showed good stability and survival rates after drying, after cryopreservation and on long-term storage. All tested strains were successfully preserved and reactivated within relatively short time. The viability, stability and ability of chemolithoautotrophic growth was not affected. Cryopreservation, liquid-drying and reactivation 0 1999 Elsevier Science B.V. All under microaerobic conditions proved very effective for these oxygen sensitive cultures. rights reserved. Keywords:

Chemolithoautotrophs:

Cryopreservation:

Hyperthermophile;

1. Introduction thermophilic and The hyperthermophilic chemolithoautotrophic HZ-oxidizers, such as Aquifer pyrophilus, genobacters,

“Hydrogenothermus “Thermocrinis

hirschii “, Hydroruber ”

and various unclassified chemolithoautotrophic bacteria can only be grown and maintained in selective media and under special conditions (Bonjour and Aragno, 1986; Huber et al., 1992, 1998; Kawasumi et al., 1984; Thomm, 1998). Based on their hyperthermophily and microaerophilly they are only well adapted to geothermal and hydrothermal systems in which oxygen is limited by its low solubility at high temperatures and

*Corresponding author. Fax: + 49-531-2616418 0167-7012/99/.$

- see front matter

PII: SO167-7012(98)00116-X

0

Liquid-drying,

Long-term

preservation

by the reduction caused by various volcanic gases. They have a very short subculturing interval and regular subculturing of their stocks is very time consuming and requires careful handling. The effective maintenance and preservation of such cultures is thus desirable in order to store these organisms for prolonged periods in a viable and stable state. Several microorganisms can be preserved with a good viability and stability by freezing in liquid nitrogen (- 196°C) (Kirsop and Doyle, 1991; Malik, 1984, 1991, 1998). There is, however a lack of cryopreservation data for the genus Aqui$caZes, Hydrogenothermus, Hydrogenobacter and other hyperthermophilic and microaerophilic hydrogen oxidizers (Bonjour and Aragno, 1986; Huber et al., 1992, 1998; Kawasumi et al., 1984; Thomm, 1998). Similarly no data exist about the successful liquid-

1999 Elsevier Science B.V. All rights reserved

K.A. Halik I Journal of Microbiological Methods 35 (1999) 177-182

178

drying and freeze-drying of such sensitive cultures although it is desirable to preserve these through drying because of several advantages. In view of this, studies were conducted for the effective long-term preservation of these extreme thermophilic chemolithoautotrophic bacteria. This study presents data on successful cryopreservation and liquid-drying of such fragile bacteria for longterm storage.

2. Material

were from the German Collection and Cell Cultures (DSMZ).

of Microorganisms

2.2. Media and cultivation procedure The cultures were grown under appropriate conditions in the specified media as described in the literature, the DSMZ Catalogue of Strains or in the catalogues of strains of major culture collections. The cultures were grown until they showed good growth. Old cultures were avoided.

and methods 2.3. Cryopreservation

2.1. Microorganisms Cryopreservation was performed in mini-screw cap glass ampoules as described previously (Malik, 1984, 1991, 1998). For harvesting, the chemolithoautotrophically grown thick cultures were centrifuged for 30 mitt at 4000 X g in the bottles or tubes. The supematant was removed anaerobically under a stream of nitrogen gas (Malik, 1984, 1991). The pellet was resuspended carefully in ice cold sterile cryoprotectant solution (5% v/v in H,O of dimethylsulfoxide, DMSO). In the case of cultures which do not form a pellet, thick cell suspensions (preferably above lo6 cells/ml) were mixed in equal quantities with the double concentrated cryoprotec-

Twelve selected strains of thermophilic and hyperthermophilic chemolithoautotrophic Hz-oxidizer from various species were used throughout this work (these are listed in Tables 1 and 2). Their optimum growth temperatures were from 55°C to 85°C. The strains which are not validly published are marked with inverted commas. Unclassified chemolithoautotrophic bacteria DSM 12045 and 12047 are strictly chemolithoautotrophic H,-oxidizers growing optimally at 85°C and 70°C whereas DSM 12046 is a facultative chemolithoautotrophic Hz-oxidizer growing optimally at 65°C (Thomm, 1998). All bacteria

Table 1 Survival of various hyperthermophilic

HZ-oxidizers

Species and DSM number

Aquifex pyrophilus; 6858 Bacillus schliegelii; 2000 Bacillus tusciae; 2912 Calderobactenum hydrogenophilum; 2913 Hydrogenobacter acidophilus, 1125 1 Hydrogenobacter thennophilus; 6534 Hydrogenothermus hirschii 11420 “Pseudomonas Hydrogenothertnophila” 6765 “Thermocrinis ruber”; 12173 Unclassified bacterium; 12045 Unclassified bacterium; 12046 Unclassified bacterium; 12047

during storage in liquid nitrogen Logarithmic

value of countsa

Before freezing

After freezing

Storage (2-3 years)

4.5 5.5 4.5 5.5 6.5 6.5 6.5 6.5 3.5 4.0 3.5 4.5

4.5 5.5 4.5 5.5 6.5 6.5 6.5 6.5 3.5 3.5 3.5 4.5

4.5 5.0 4.5 5.5 6.5 6.5 6.5 6.5 NTh NT NT NT

“Serial tenfold dilutions were prepared and the highest dilution n to yield visible growth gave total viable counts of 10” cells ml-’ (or n log when expressed as logarithmic counts). Losses of viability were thus expressed as log value reductions. For more details on storage see Section 2. bNot tested.

K.A. Halik I Journal of Microbiological Methods X5 (1999) 177-182 Table 2 Survival of various hyperthermophilic

Hz-oxidizers

after liquid-drying

Species and DSM number

Aquijex pyrophilus; 6858 Bacillus schliegelii; 2000’ Bacillus tusciae; 2912d Calderobacterium hydrogenophilum; 29 13 Hydrogenobacter acidophilus; 1125 1 Hydrogenobacter thermophilus; 6534 “Hydrogenothermus hirschii”: 11420 “Pseudomonas Hydrogenothermophila” 6765” “Thermocrinis ruber”; 12173 Unclassified bacterium; 12045 Unclassified bacterium; 12046 Unclassified bacterium; 12047

179

and storage

Logarithmic value of counts’ Liquid-drying Before

After

Storage

4.5 6.0 4.5 5.5 5.5 6.5 4.5 6.5 3.5 4.5 4.0 4.5

4.5 5.5 4.5 5.5 5.5 6.5 4.5 6.5 3.5 4.5 4.0 4.5

NT’ (NT) 5.0 4.5 5.5 (NT) 5.0 (NT) 6.5 (NT) 4.5 (NT) 6.5 NT (NT) NT (NT) NT (NT) NT (NT)

(3.5?

(3.5) (4.5) (NT) (4.5) (3.5) (3.5) (3.5) (4.5)

(3.0)

(3.5) (4.5) (NT) (4.5) (3.5) (3.5) (3.5) (4.5)

“Serial tenfold dilutions were prepared and the highest dilution a to yield visible growth gave total viable counts of 10” cells ml -’ (or n log when expressed as logarithmic counts). Losses of viability were thus expressed as log value reductions. Storage at 9°C for 2-3 years. ‘The results in brackets were obtained using ethylenediamine (Protective mixture B). ‘Not tested. “Results after freeze-drying.

tive agents. The cells were allowed to equilibrate for 1.5 min with the cryoprotectant DMSO in an ice bath. An aliquot of 1.5 ml of cell suspension was dispensed in to each glass ampoule. A slight over pressure resulted due to the injected cell suspension, which was advantageous to avoid sucking in of liquid nitrogen during freezing. As the aspirated liquid nitrogen in the ampoules (due to under pressure and leakage) might cause explosion when such ampoule are taken out for thawing. Immediately afterwards, the filled glass ampoules were clamped onto labeled aluminum canes. The cell suspension in ampoules was first cooled at a rate of about 1°C min _’ to about - 30°C. This was accomplished by placing the filled ampoules in a deep-freezer at - 30°C for about 1 h or in the gas phase of liquid nitrogen for a few min. Thereafter, the ampoules were frozen by direct immersion in liquid nitrogen or in the gas phase of liquid nitrogen (Malik, 1984, 1991, 1998). For reactivation, the frozen ampoules were kept in the gas phase for a few min in order to allow any liquid nitrogen that might have entered the ampoules, due to leakage or the build-up of a vacuum, to escape. The details of the reactivation procedure and the refreezing of the partially thawed contents of the ampoule have already been described in details (Malik, 1998). The inoculated growth

medium was incubated under appropriate growth conditions. For the estimation of viable cell counts, 1.O ml of inocula was transferred from the unfrozen (for cell counts before freezing) and from the thawed cell suspension (for cell counts after freezing) into 9.0 ml medium and 3-6 serial decimal dilutions were prepared depending upon the thickness of the cell suspension. After incubation, the number of viable cells was determined using the most probable number method (MPN). For comparison, the viable cell counts before freezing and after freezing were recorded and percentage survival was calculated. 2.4. Preservation 24.1.

Preparation

by liquid-dying of thin discs of carrier

material

For double vial preparation, ampoules of neutral glass (45 X 10 mm) were filled with 0.1 ml of 20% (w/v) skim milk (Bacto, Difco 0032) containing 1% (w/v) neutral activated charcoal and 5% (w/v) mesoinositol. The activated charcoal used was of medicinal grade (obtained from Caelo, 4010 Hilden, FRG) but any other bacteriological activated charcoal of comparable quality can also be used. The ampoules were loosely plugged with non-absorbent cotton wool and sterilized at 115°C for 13 min. These were

180

K.A. Hdik

I Journal

of Microbiological

frozen at about - 40°C for few hours and then freeze-dried in bulk for 8-12 h using a standard freeze-drying technique (Malik, 1988). This resulted in a thin disc of carrier material in each ampoule.

2.4.2. Preparation of protective media Solutions of protective medium A = 5% mesoinositol containing 1% activated charcoal and B = 40 mM ethylenediamine dihydrochloride, 3% mesoinositol containing 1% activated charcoal were prepared in 0.1 M phosphate buffer (pH 7.0) or buffered mineral medium H, (Malik, 1988) in a screw-cap bottle. The pH was controlled (6.8-7.2) and the solution was bubbled with nitrogen gas and the bottle was closed tightly and autoclaved at 115°C for 13 min. The solutions were stored at 4°C.

2.4.3. Preparation of a cell suspension Lmd the liquid-drying procedure The cells were harvested by aseptic centrifugation under anaerobic conditions (see Section 2.3). The pellet was suspended in a protective medium (A or B) to yield a heavy cell suspension and the cells were equilibrated for about 20 min at 20°C whilst maintaining anaerobiosis. For liquid-drying under semi-aerobic conditions, more details have already been described (Malik, 1990, 1992, 1998). The first step in the liquid-drying process was continued for about 2 h at lo-30 mbar and second step drying was conducted under 0.1 to 0.01 mbar vacuum for about l-2 h. Details of the procedure have been described previously (Malik, 1990, 1992). For prolonged storage, the liquid-dried cultures (in small glass vials) were quickly transferred to soft glass tubes ( 130 X 15 mm, containing silica gel and cotton plugs), were constricted and subjected to secondary drying for 2-3 h. Afterwards, the ampoules were sealed under vacuum as is usually done for the double-vial preparation (Malik, 1988). The viability counts were checked before liquid-drying, immediately after liquid-drying and after storage. For comparison, the viable cell counts were calculated and percentage survival was determined. For estimation of viability counts a definite volume (0.05 ml) of cell suspension was liquid-dried. For revival and reactivation about 1.5 ml of medium was added to the liquid-dried ampoule and, from this, serial ten-

Methods 3.5 (1999) 177-182

fold dilutions were prepared in appropriate liquid media. The revived cultures were observed for chemolithoautotrophic growth, change in doubling time, change in cell morphology and for signs of mutation. Normal growth usually appeared after second transfer into fresh medium, as, in a few cases, growth was inhibited by the high concentration of protective mixture used during the liquid drying procedure. .

3. Results and discussion Cryopreservation of microorganisms ensures longterm stability and the post-thaw viability is generally independent of storage time (Kirsop and Doyle, 199 1). For effective cryopreservation, penetrating cryoprotective agent dimethylsulphoxide (DMSO) is generally used (Kirsop and Doyle, 1991; Malik, 1984, 1991, 1998). It provides good protection to the sensitive Cultures and so far almost all facultative and obligately hydrogen oxidizers have successfully been cryopreserved with this at the DSMZ. The survival results after cryopreservation of few selected thermophilic hyperthermophilic and chemolithoautotrophic H,-oxidizers from various species are shown in Table 1. Normally, no loss in viability of preserved cells occurs during long-term storage in liquid nitrogen. However, some loss in viability of cells was observed after repeated freezethaw cycles when a single ampoule was used repeatedly. In spite of this, the final concentration of living cells in the remaining cell suspension was high enough to provide an adequate source of inoculum. Previously a large collection (almost all species accessioned at the DSMZ) of normal facultative and obligately aerobic chemolithoautotrophic hydrogen bacteria were successfully preserved with a newly developed freeze-drying method (Malik, 1976, 1988). All these resulting in good survival and with full retention of plasmids after lyophilization. The Spore forming thermophilic Bacillus schlegelii DSM 2000 and Bacillus tusciue DSM 2912 which are facultative chemolithoautotrophic H,oxidizers (growing optimally at 70°C and 55°C) (Bonjour and Aragno, 1984, 1986) also showed good survival after

freeze-drying and during storage using this method (Malik, 1988). Similarly the thermophilic cultures of Hydrogenobacter thermophilus DSM 6534 and closely related Calderobacterium hydrogenophilum DSM 2913 (Kawasumi et al., 1984; Kryukov et al., 1983) which are strictly chemolithoautotrophic H2oxidizers (growing optimally at 70°C) also survived after lyophilization with this method but the percentage survival was low. However, the cultures of thermophilic hyperthermophilic other and chemolithoautotrophic H?-oxidizer failed to survive freeze-drying. Liquid-drying has several advantages over freeze-drying and it has been effectively used at the DSMZ for preserving large collections of fragile microorganisms which were difficult to lyophilize (Malik, 1990, 1992, 1998). The new liquid-drying method developed by the author (Malik, 1990) was modified for the effective preservation of these oxygen sensitive hyperthermophilic chemolithoautotrophic H,-oxidizers. All tested cultures of Aqufex “H~drogenothermus hirschii “. Hydropyrophilus. genobacters, “Thermocrinis ruber *’ and various newly isolated unclassified chemolithoautotrophic bacteria were successfully dried under reduced conditions using this modified liquid-drying method. These proved viable after drying with a good percentage survival and during storage no drastic losses were observed. The survival results after liquid-drying and during subsequent storage are shown in Table 2. In the case of recently accessioned and preserved strains no survival results after prolonged periods of storage were available., However. their shelf life was good enough as estimated from the results of accelerated storage test at 37°C. It has been observed that the survival values of the dried specimens after longterm storage (in years) at 5-9°C generally corresponded to those estimated from the results of the short-term storage (in weeks) at 30-37°C. The comparative effect of two protective media (A 5 B) on the viability of few selected cultures during liquid-drying is shown in Table 2. It is presumed that ethylenediamine binds the cells and total amount of polyamines in the cells did not change during desiccation (Sakane et al., 1992). Therefore, the use of ethylenediamine has been reported to be effective fol L-drying of some fragile bacteria (Sakane et al., 1992). However, in. our case no superiority was

observed in the percentage viability results after drying with the protective mixture (B) containing ethylenediamine. Only in few cases morphologically, better cells were observed after reactivation from the specimens L-dried in the presence of ethylenediamine. All the dried cultures proved stable and no mutation with respect to the change in cell morphology, colony shape. motility or loss in the ability of chemolithoautotrophic growth was detected. Although most are facultative aerobes but under chemolithoautotrophic growth conditions mostly they tolerate only low oxygen concentrations and cultures grown under such conditions are semiaerobic. This is because most of these have been isolated from geothermal and hydrothermal environment, in which presence of oxygen is limited by its low solubility at high temperatures and by the reduction caused by volcanic gases like H,S. Therefore, it has been observed that the cryopreservation, liquid-drying and reactivation of such bacteria in the absence of oxygen proved very effective. The long-term storage of such sensitive hyperthermophilic chemolithoautotrophic H,-oxidizers is an attractive advantage, since cultures so preserved are stable and can be stored without further attention. The liquid-dried cultures are not damaged during transportation. as otherwise these are fragile, show quick lysis and are difficult to keep and transport as active cultures.

Acknowledgements I am thankful to Andrea Schiitze and Sabine Welnitz for their technical assistance. I also thank Prof. Dr. M. Thomm, Institut fiir Allgemeine Mikrobiologie. C.-A.-Universitk Kiel, Germany for providing information on his newly isolated strains DSM 12045. 12046 and 12047.

References Bonjour, F.. Aragno, M., 1984. Bacillus tusciae. a new species of thermophilic. facultatively chemolithoautotrophic hydrogen-oxidizing spore former from a geothermal area. Arch. Microbial. 139, 397-401,

182

K.A. Halik 1 Journal

qf Microbiological Methods 35 (1999) 177-182

Bonjour, F., Aragno, M., 1986. Growth of thermophilic, obligatorily chemolithoautotrophic hydrogen-oxidizing bacteria related to Hydrogenobacter with thiosulfate and elemental sulfur as electron and energy source. FEMS Microbial. Lett. 35, 1 l-15. Huber, R., Wilharm, T., Huber, D., Trincone. A., Burggraf, S., Konig, H., Rachel, R., Rockinger, I., Fricke, H., Stetter, K.O., 1992. Aquifex pyrophilus gen. nov. sp. nov.. represents a novel group of marine hyperthermophilic hydrogen-oxidizing bacteria. System. Appl. Microbial. 15, 340-351. Huber, R., Eder, W., Heldwein, S., Wanner, G., Huber, H., Rachel, R., Stetter, K.O., 1998. Thermocrinis tuber gen. nov., sp. nov., represents a “pink filament”-forming hyperthermophilic bacterium isolated from Yellowstone National Park. Appl. Environ. Microbial (in press). Kawasumi, T., Igarashi, Y., Kodama, T., Minoda, Y., 1984. Hydrogenobacter thermophilus gen. nov., sp. nov., an extremely thermophilic, aerobic, hydrogen-oxidizing bacterium. hit. .I. Syst. Bact. 34, S-10. Kirsop, B.E., Doyle, A., 1991. Maintenance of Microorganisms and Cultured Cells. Academic Press. London. Kryukov, VR., Savelyeva, N.D., Pusheva. M.A.. 1983. Calderobacterium hydrogenophilum gen. nov., sp. nov., an extreme thermophilic bacterium and its hydrogenase activity. Microbiologya. 52, 781-788. Malik, K.A., 1976. Preservation of Knallgas bacteria. In: Dellway.

H. (Eds.), Proceeding of Fifth International Fermentation Symposium. Westkreuz Druckerei and Verlag, Bonn and Berlin, pp. 180. Malik, K.A., 1984. A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions. J. Microbial. Methods 2. 41-47. Malik, K.A., 1988. A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria. J. Microbial. Methods 8, 259-271. Malik. K.A.. 1990. Simplified liquid-drying method for the preservation of microorganisms sensitive to freezing and freezedrying. J. Microbial. Methods 12. 125-132. Malik, K.A., 1991. Cryopreservation of bacteria with special reference to anaerobes. World J. Microbial. Biotechnol. 7, 629-632. Malik, K.A., 1992. A new method for preservation of microorganisms by liquid-drying under anaerobic conditions. J. Microbial. Methods 14, 239-245. Malik, K.A., 1998. Preservation of Chloroflexus by deep-freezing and liquid-drying methods. J. Microbial. Methods 32. 73-77. Sakane, T., Fukuda, I., Itoh, T., Yokota, A., 1992. Long-term preservation of halophilic archaebacteria and thermoacidophilic archaebacteria by liquid drying. J. Microbial. Methods 16. 239-245. Thomm, M., 1998. Unpublished data (Personal communication).