Long-term preservation of yeast cultures by liquid-drying

Various selected strains from about 20 species of yeasts, which are reported to be sensitive to freeze-drying and liquid-drying, were successfully dried directly from the liquid phase without freezing using a simplified liquid-drying method. All tested cultures proved viable and the majority of the tested strains showed good survival rates after drying. However, different survival levels for different yeasts were observed; generally the sensitivity to drying appeared to be strain-specific. After 1 years' storage at 9°C, no further loss in viability was observed. Accelerated storage testing, for 1 week at 45°, resulted in further loss of viability to various degrees. Yeasts that were filamentous, osmotolerant or psychrophilic appeared to be sensitive to liquid-drying and had relatively lower survival levels than the others. Growth and liquid-drying under microaerobic conditions resulted in improved survival. The dried yeast cultures proved stable and no mutation or loss in desirable characters was detected. The method can be used for the drying and long-term preservation of nearly all yeast genera.K.A. Malik and P. Hoffmann are with the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, W-3300 Braunschweig, Germany     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

Preservation by freezing of potentially probiotic strains ofLactobacillus rhamnosus

The aim of this work was to detect the best conditions to preserve by freezing potentially probiotic strains ofLactobacillus rhamnosus isolated from food. Four strains isolated from Parmigiano Reggiano cheese, the commercial strainLactobacillus GG and the type strain ATCC 7469T were used in the present study. Two different pre-incubation times (5 and 24 h), three protective media (Skim milk, Skim milk plus glucose and MRS plus glycerol) and two storage temperatures (?20 and ?80 °C) were used for a preservation period of 90 days. A sensible loss of survival of the strains was detected and the acidifying activity decreased depending on the different factors analysed. Moreover, plate counts performed in MRS plus bile salts evidenced that a considerable percentage of cells suffers damages deriving from cold. This study showed that the growth phase of the cells plays an important role for the resistance to the storage by freezing. Finally, Skim milk had the best protective action, showing the highest activity at ?80 °C.     

Ethanol and glycerol production under aerobic conditions by wild-type,respiratory-deficient mutants and a fusion product of Saccharomyces cerevisiae

Summary Experiments were performed to investigate growth, ethanol and glycerol production by wild-type strains (RHO) and respiratory-deficient (rho) mutants of Saccharomyces cerevisiae. Furthermore protoplasts were fused in order to enhance the fermentation capacity of a flocculent strain. At high substrate conditions, 150 g/l of saccharose, there is no difference in cell growth. However, at a glucose concentration of 10–20 g/l the mutants grow much slower. After 3 days of incubation at 28° C in a complete medium the viability of the two strains is the same. In minimal medium on the other hand the number of viable cells of the mutant is 100-fold reduced. All mutants tested showed a higher specific activity of alcohol dehydrogenase (ADH I) and an enhanced production of glycerol compared with the wild-type strain. By protoplast fusion a modified flocculent strain was obtained with higher specific activity of ADH I and a reduced biosynthesis of glycerol. However, the yields of ethanol (75–78%) are about the same for the wild-type strain and the rho mutants under aerobic conditions in absence of catabolite repression.     

Freezing Resistance and Cryopreservation of Cell Strains of Rhaponticum carthamoides and Thalictrum minus

Effects of long-term (few months) culturing and short pregrowth (up to 7 days prior to deep freezing) in the presence of mannitol (5–6%), ABA (5.0–7.5 × 10^–5 M), or both substances on cryogenic resistance of leusea (Rhaponticum carthamoides, strains Rhs-2 and Rhs-8) and meadow rue (Thalictrum minus L., strain B-233) cell suspension cultures were studied. Cryoprotective capacities of 48 solutions were studied at slow (0.33°C/min) freezing to the temperature of liquid nitrogen with an automatic initiation of crystallization. Cells were stored in liquid nitrogen for several days or months. ABA had a cryoprotective effect, provided that subculturing intervals were 12–14 days. At more frequent subculturing (every 7 days), pregrowth on ABA-containing medium did not increase survival percentage compared to pregrowth with mannitol. Successful cryopreservation of these strains has been achieved due to strict standardization of 7-day subculturing regime, pregrowth in the presence of mannitol prior to freezing, and cryopreservation with dimethyl sulfoxide, sucrose, trehalose, and glycerol. The cell survival rates after thawing were 60% (Rhs-8), 80% (Rhs-2), and 70% (B-233). The cell growth resumed on the third to seventh day. The growth indices and protoberberine synthesizing activity in B-233 strain reached their control values at the ninth subculturing after a post-thaw recovery.     

Segregational Respiratory-Deficient Mutants of a “Petite Negative” Yeast Schizosaccharomyces pombe 972h−

No viable respiratory-deficient mutants of Schizosaccharomyces pombe 972h(-) could be obtained by acriflavine and ethidium bromide treatments. These mutagens induce 15 to 70% of microcolonies which, after a growth-lag of a few days, further develop into normal, respiratory-competent colonies. These results suggest that unstable petites were induced. Segregational respiratory-deficient mutants resistant to cobalt sulfate inhibition were isolated. Some of these strains are deficient in cytochrome a + a(3) and respire at low rates. The morphology of their mitochondrial membranes is modified: either the cristae are absent or they show aberrant concentric or tubular structures. Segregational mutants resistant to the respiratory inhibitors, 2,4-dinitrophenol or decamethylene diguanidine, were obtained. Neither mitochondrial structure nor function seems to be modified in these mutants. A segregational mutant resistant to benzimidazole inhibition does not grow on glycerol, although neither growth on glucose nor respiration appear to be affected.     

液氮保存曲霉菌种的效果

本文报告了以蒸馏水、10%甘油和5%二甲基亚砜(DMSO)为保护剂,用液氮冻结法保存5种8株曲霉的效果,并检测了这些菌分别产生的亚甲基丁二酸、柠檬酸、蛋白酶和糖化酶的生理活性。这些菌在液氮气相(接近-150℃)中保存180天全部保持着生活能力,它们的培养特征和形态特征保留原来的形状。所测定的液氮保存8株菌种的生理活性,除两株糖化酶活力稍有降低外,其它菌株没有明显的变化。     

Low-cost and low maintenance preservation of Agaricus brasiliensis cultures

Agaricus brasiliensis cultures quickly lose viability when stored at cool temperatures, even for a short period of time. We evaluated several low-cost preservation methods using varied substrates, preservation solutions, and storage temperatures. Agaricus brasiliensis was intolerant to freezing temperatures, making liquid nitrogen use and deep-freezing methods impossible for its preservation. The best preservation conditions for the A. brasiliensis CS1 strain tested in this study were obtained by using rice as substrate and water as preservation solution, with storage at room temperature or when using soil, mushroom cultivation compost, or rice and stored at 10 °C without preservation solution. Those cultures that were reactivated showed the same productivity attributes as the control. In addition, no effect on productivity or biological efficiency was observed through successive subculturing of the strain (CS1). Parboiled rice was successfully used for other A. brasiliensis strains (CS2, CS5, CS7, CS9, and CS10), and also for Pleurotus ostreatus, P. sajor-caju, and Lentinula edodes.     

Susceptibility of Saccharomyces spp. and Schwanniomyces spp. to the aminoglycoside antibiotic G418.

Industrially useful polyploid yeasts such as the brewing yeasts do not possess any auxotrophic genetic markers and hence are not easily amenable to plasmid-mediated DNA transformations. In an attempt to obtain genetic markers, a number of useful Saccharomyces sp. strains and some amylolytic Schwanniomyces sp. strains were tested for their susceptibility to the antibiotic Geneticin G418 , a 2-deoxystreptamine reported to be active against bacteria, yeasts, and plant and animal cells. All of the Saccharomyces sp. strains, including the brewing strains, were found to be susceptible to G418 in the concentration range of 150 to 500 micrograms/ml. Of the three Schwanniomyces species investigated, only Schwanniomyces castellii (strain 1402) was found to be resistant to G418 at concentrations up to 1 mg/ml. Resistance was exhibited both in liquid media and on glycerol-peptone-yeast extract agar plates. This finding is interesting in view of the possibility of using this strain as a DNA donor for transformations aimed at introducing the amylolytic capability into brewing yeasts.     

Proceedings: Preservation of bacteria by freezing at moderately low temperatures

In order to get some basic information for the development of a long-term preservation method by freezing at moderately low temperatures, the viability of 259 strains belonging to 32 genera and 135 species was measured. Cells were suspended in 10% glycerol and stored at ?53 °C for 16 months. About 93%, 88%, and 74% of aerobic bacteria gave viable cell counts higher than 10⁵/ml, 10⁶/ml, and 10⁷/ml, respectively. About 10% of gram-positives and 3% of gram-negatives gave viable cell counts lower than 10⁵/ml. There seemed to be some species—and genus—specificity with respect to viability after frozen storage and liquid paraffin-seal storage. Strains of coryneform bacteria, genera of the family Enterobacteriaceae, and the genus Pseudomonas were generally resistant. Pseudomonas putrefaciens proved to be specifically sensitive. Lactic acid bacteria were subject to sublethal injury, requiring special recovery media. Psychrophilic bacteria were very susceptible to frozen storage. All the tested strains of acetic acid bacteria survived frozen storage well both in 10% glycerol and in 10% honey at ?28 °C for 4.5 years. Honey proved to be a better adjuvant for frozen storage than glycerol. It was suggested from the results that for many kinds of bacteria, long-term preservation by freezing at moderately low temperatures might be possible when appropriate procedures are applied.     

Viability of fastidious Phytophthora following different cryopreservation treatments

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the −1 °C min⁻¹ freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.     

Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs.

Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts. During storage of frozen doughs, strains of T. delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days. Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain. Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323. These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions. The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S. cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T. delbrueckii (IGC 5321 and IGC 5323). However, the strains of S. cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.     

Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: a novel approach for developing protistan cryopreservation strategies

The development of cryoconservation methods for the long-term storage of algal cultures is important for the ex situ preservation of biological diversity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogenic storage, this study aims to evaluate the role of oxidative stress in cryoinjury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatments. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl radical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were identified as preparative centrifugation and sub-zero freezing treatments. Poststorage survival in E. gracilis was significantly (P < 0.05) enhanced when the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggesting that the additive was capable of ameliorating oxidative stress. The potential of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in recalcitrant unicellular algae is discussed.     

The use of protoplast fusion in demonstrating chromosomal and mitochondrial inheritance of respiratory-deficiency in Kluyveromyces lactis, a petite-negative yeast

Summary Protoplast fusion has provided us with a means of genetical analysis in respiratory-deficient mutants of the petite-negative yeast, Kluyveromyces lactis. Demonstration of mitochondrial-inheritance in respiratory-deficient mutants of petite-negative yeasts has not previously been possible due to the inviability of petite/microcolony cells.     

Long-term preservation of anammox bacteria

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.     

Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at -70 degrees C.

We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.     

液氮冻结法保藏毛霉目菌种的效果

本文报告了用液氮冻结法保藏毛霉目菌种的效果。对14属26种32株进行了液氮保藏试验,其结果慢速冻结孢子成活率高于快速冻结。用10%的甘油、10%的二甲基亚砜和蒸馏水作保护剂制成的孢子悬液,经慢速冻结后储存在液氮罐气态中二年,以甘油作保护剂的孢子成活率为90%,二甲基亚砜的为85%,蒸馏水的为79%。并检测了某些菌株的反丁烯二酸、蛋白酶、α-半乳糖苷酶、脂肪酶、果胶酶、葡萄糖苷酶等生理活性,结果冻结后的活性与冻结前相比,无明显差异。     

Helicobacter pylori in Liquid Culture: Evaluation of Growth Rates and Ultrastructure

This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (0.1% wt/vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140–200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 120 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and the cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at −80°C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order). Received: 10 November 1997 / Accepted: 29 January 1998     

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

Summary Heat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.