Calcium-Induced Alteration of Cellular Morphology Affecting the Resistance of Lactobacillus acidophilus to Freezing

Examination of factors affecting the resistance of Lactobacillus acidophilus NCFM culture concentrates to freeze injury induced during frozen storage at -20°C revealed that calcium supplementation of the growth medium contributed to the storage stability of cells prepared in static culture. Culture concentrates of L. acidophilus NCFM were prepared from cells propagated in MRS broth or MRS broth supplemented with 0.1% calcium carbonate, calcium chloride, or calcium phosphate. After 28 days of frozen storage at -20°C, concentrated cells (3.2 × 10⁹ colony-forming units per ml) prepared from MRS broth cultures showed an 84% reduction in viable cells. Of the remaining viable cells, 88% were sublethally injured and unable to form colonies on MRS agar supplemented with 0.15% bile. Cells prepared in calcium-supplemented MRS broths demonstrated more resistance to frozen storage. Viability and injury losses in the frozen concentrates were limited to 10 to 39% and 3 to 23%, respectively. It was observed that calcium supplementation of MRS medium resulted in a morphological transition of L. acidophilus NCFM from filamentous to bacilloid rods, and the bacilloid cells were more resistant to freezing and storage at conventional freezer temperatures. The results suggest that the morphology of the L. acidophilus cell may be an important consideration in the preparation of freeze-stable culture concentrates.     

Storage of brewing yeasts by liquid nitrogen refrigeration

Many yeast strains are difficult to maintain in culture in a stable state, and long-term preservation by lyophilization, which has proved useful for other fungi, has given poor results with brewing yeasts. As an alternative to continuous subculture, which maximizes strain variability, various methods of cryogenic storage were investigated. Yeast strains were frozen with or without cryoprotectants (such as glycerol or inositol) and stored at -196 C. Recovery after warming was estimated from plate counts, and survivors were screened to detect changes in the frequency of morphological types, respiratory-deficient mutants, and glycerol-sensitive mutants. Strains varied in their sensitivity to freezing, and survival was modified by the growth medium, the freezing munstrua, and the freezing conditions. Suspension of cells in 10% (vol/vol) glycerol, cooled at 1 C/min, warmed rapidly and plated on malt-yeast extract-glucose-peptone agar produced the highest percentage of viable colonies with a minimal change in metabolic characteristics. In two of the strains tested, no significant increase in mutation rate was detected under any of the treatments; the strains were maintained in a stable state and were metabolically comparable to unfrozen strains. In one strain of Saccharomyces uvarum after some freezing treatments, the percentage of respiratory-deficient mutants increased markedly, the fermentation rate declined, and a loss of flocculation occurred. The freezing parameters which increased the level of respiratory-deficient cells should be avoided in maintaining this strain. Maintenance of cultures of brewing yeasts by cryogenic storage has several advantages over other preservation techniques: the method is simple and reproducible, the cultures have remained stable over a 3-year test period, and the viability is high.     

Bacterial Culture Preservation in Frozen and Dry-Film Methylcellulose

Forty-seven of 61 bacterial cultures, including strains of Pseudomonas, Xanthomonas, Erwinia, Agrobacterium, Corynebacterium, Serratia, Klebsiella, and Escherichia, remained viable after storage in frozen methylcellulose or in dried methylcellulose for up to 38 months. Pathogenicity remained intact for those strains tested. Bacteria were grown on a solid medium and then removed and placed in 1.0% methylcellulose (cellulose methyl ether) to make a final suspension of 10⁸ colony-forming units (CFU) per ml. For storage in dried form, the bacteria-methylcellulose suspension was placed in a petri dish and dried in a forced-air incubator. After 24 h of storage at 25°C, viable populations of 10⁵ CFU/mg (equivalent to 10⁶ CFU/ml) were recovered. Populations of 10² to 10⁴ CFU/mg were recovered after storage of up to 38 months. Similar results were obtained in frozen methylcellulose. Survival was greatly enhanced when the growth medium for the bacteria was potato dextrose peptone rather than nutrient agar, yeast dextrose calcium carbonate peptone, or King's medium B. Addition of 0.1 M MgSO₄ to the methylcellulose suspension and to the resuspending liquid also increased survival and recovery from storage for some strains. Methylcellulose storage should be a simple, inexpensive, and reliable method of maintaining cultures for short or long periods of time.     

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All teste cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae range from 70–100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1–2 log₁₀ counts was observed. No further loss in viability occurred after 1–2 years of storage.The small size of the ampoules and the use of single ampoule for 15–20 repeated retrievals proved economical with respect to storage space and costs.The system is compact and suitable for the preservation of anaerobic phototropic bacteria and other fragile anaerobic microorganisms.     

Isolation of Halotolerant Thermus spp. from Submarine Hot Springs in Iceland

Thermophilic, aerobic bacteria of the genus Thermus were isolated from submarine alkaline hot springs in Iceland. Five submarine hot springs were sampled, and all had viable counts of Thermus spp. of about 10³ CFU/ml. All submarine strains grew in the presence of NaCl at 3% or higher, but no strains from terrestrial hot springs would grow at concentrations higher than 1% NaCl. The growth rate of submarine Thermus strains was not stimulated by NaCl and was reduced at NaCl concentrations higher than 1%. The pattern of growth of these isolates on single carbon sources was similar to that of terrestrial isolates.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

Numbers and viability of bacteria in ornithogenic soils of Antarctica

Summary Bacteria in ornithogenic soils from Ross Island, Antarctica, were counted by direct observation, and the percentages of viable organisms were assessed by incubation with ³H-glucose and by enumerating numbers of colony-forming units. The effects of incubation times and temperatures, and of storage of the samples, on the uptake of ³H-glucose were determined. Direct counts showed that large total numbers of bacteria were present in samples from occupied penguin colonies and recentlyabandoned sites. The percentages of bacteria metabolizing ³H-glucose increased when incubation was extended from 2 h to 8 h at field (average 4–5°C) or laboratory (average 18.5°C) temperatures to a maximum of 22%; storage of the samples for 31 days had no significant effect. The numbers of colony-forming units (CFU) were less than 0.058% of the direct counts. There were 77 times as many CFU in samples from the abandoned site compared to the inhabited colony. About 10% of the CFU were cocci compared with about 48% visible by direct microscopy. The glucose utilization data indicated that far more of the bacteria were viable than were cultured.     

Impact of Mount St. Helens Eruption on Bacteriology of Lakes in the Blast Zone

Lakes lying within the blast zone of Mount St. Helens showed dramatic increases in heterotrophic bacterial numbers after the eruption of 18 May 1980. The total microscopic counts of bacteria in some of the most severely affected lakes were more than 10⁷ cells per ml, an order of magnitude above the counts in outlying control lakes. Likewise, the numbers of viable bacteria reached levels of more than 10⁶ cells per ml, compated with fewer than 10⁴ cells per ml in control lakes. The CPS medium used for enumeration provided growth of up to 81.5% of the bacteria during sampling of one of the blast zone lakes. The high numbers of bacteria and the efficacy of the viable enumeration procedure are evidence that the lakes have been transformed rapidly from oligotrophy to eutrophy due to the eruption and its aftermath. Organic material leached from the devastated forest vegetation is thought to be responsible for the enrichment of heterotrophs. Total coliform bacteria were found in all of the blast zone lakes, and some lakes contained fecal coliform bacteria. Klebsiella pneumoniae was the predominant total coliform and was also identified as one of the fecal coliform bacteria, although Escherichia coli was the predominant species in that category. Our data indicate that bacterial populations peaked in the outer blast zone lakes in the summer of 1980 and in most of the inner lakes during the summer of 1981.     

Recovery of a marine dinoflagellate following controlled and uncontrolled freezing

A free-living, marine dinoflagellate, Crypthecodinium cohnii, was successfully preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a 1 °C/min cooling rate. After storage for a minimum of 7 days at ?150 °C material frozen by this method demonstrated a 47.7% mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at ?55 °C for 1 hr. This material demonstrated a mean recovery of 30.8% with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after uncontrolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 68% recovery following controlled freezing.The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cause.     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.     

Survival of Cold-Stressed Campylobacter jejuni on Ground Chicken and Chicken Skin during Frozen Storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4°C, freezing at −20°C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log₁₀ CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log₁₀ CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and −20°C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×10⁶ mL⁻¹ and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl^® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl^® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl^®, Andromed^®, Bioxcell^®, and UL with 8% LDL (low-density lipoproteins). Triladyl^® and Andromed^® performed better than Bioxcell^® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl^® and Andromed^® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.     

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

Identity and Behavior of Xylem-Residing Bacteria in Rough Lemon Roots of Florida Citrus Trees

An aseptic vacuum extraction technique was used to obtain xylem fluid from the roots of rough lemon (Citrus jambhiri Lush.) rootstock of Florida citrus trees. Bacteria were consistently isolated from vascular fluid of both healthy and young tree decline-affected trees. Thirteen genera of bacteria were found, the most frequently occurring genera being Pseudomonas (40%), Enterobacter (18%), Bacillus, Corynebacterium, and other gram-positive bacteria (16%), and Serratia (6%). Xylem bacterial counts fluctuated seasonally. Bacterial populations ranged from 0.1 to 22 per mm³ of root tissue (about 10² to 2 × 10⁴ bacteria per g of xylem) when bacterial counts were made on vascular fluid, but these numbers were 10- to 1,000-fold greater when aseptically homogenized xylem tissue was examined similarly. Some of the resident bacteria (4%) are potentially phytopathogenic. It is proposed that xylem bacteria have an important role in the physiology of citrus.     

Comparison of bacteria from deep subsurface sediment and adjacent groundwater

Samples of groundwater and the enclosing sediments were compared for densities of bacteria using direct (acridine orange direct staining) and viable (growth on 1% PTYG medium) count methodology. Sediments to a depth of 550 m were collected from boreholes at three sites on the Savannah River Site near Aiken, South Carolina, using techniques to insure a minimum of surface contamination. Clusters of wells screened at discreet intervals were established at each site. Bacterial densities in sediment were higher, by both direct and viable count, than in groundwater samples. Differences between direct and viable counts were much greater for groundwater samples than for sediment samples. Densities of bacteria in sediment ranged from less than 1.00×10⁶ bacteria/g dry weight (gdw) up to 5.01 ×10⁸ bacteria/gdw for direct counts, while viable counts were less than 1.00×10³ CFU/gdw to 4.07×10⁷ CFU/gdw. Bacteria densities in groundwater were 1.00×10³–6.31×10⁴ bacteria/ml and 5.75–4.57×10² CFU/ml for direct and viable counts, respectively. Isolates from sediment were also found to assimilate a wider variety of carbon compounds than groundwater bacteria. The data suggest that oligotrophic aquifer sediments have unique and dense bacterial communities that are attached and not reflected in groundwater found in the strata. Effective in situ bioremediation of contaimination in these aquifers may require sampling and characterization of sediment communities.     

Microbiological quality changes in the intestine of hybrid tilapia (Oreochromis niloticus x Oreochromis aureus) in fresh and frozen storage condition

AIMS: The goal of this study was to monitor the quantitative and qualitative bacterial flora in the intestine of hybrid tilapia in fresh fish and fish kept in frozen storage conditions for 1 year. METHODS AND RESULTS: Quantitative and qualitative analyses of the bacterial flora associated with the intestine of hybrid tilapia (Oreochromis niloticus x Oreochromis aureus) in fresh fish and fish kept in frozen storage conditions for 1 year were carried out. In fresh and frozen fish, aerobic plate count (APC) ranged from 1.6 +/- 1.2 x 10(8) to 1.5 +/- 0.9 x 10(5) CFU g(-1) in the intestine of tilapia collected from pond 1, 8.7 +/- 2.3 x 10(7) to 6.5 +/- 3.8 x 10(4) CFU g(-1) in the intestine of tilapia from pond 2, and 1.9 +/- 2.9 x 10(8) to 6.2 +/- 2.8 x 10(4) CFU g(-1) in the intestine of tilapia from pond 3. APC for all the groups of fish decreased c. 2-log cycles after 1 months frozen storage; thereafter, counts slowly declined during frozen storage for 1 year. Altogether, 16 bacterial genera were identified: Gram-negative rods (67%) dominated. Both in fresh and frozen conditions, four bacterial species viz. Shewanella putrefaciens, Corynebacterium urealyticum, Aeromonas hydrophila and Flavobacterium sp. were always present, with a prevalence of 10% in most cases. Shewanella putrefaciens was the most dominant organism (15% of the total isolates) throughout the studied period. During frozen storage some of the bacteria were not recovered, but most of the bacteria survived after prolonged freezing. CONCLUSIONS: This study describes the aerobic heterotrophic microflora found in the intestine of fresh and frozen tilapia. The unique aspect of this study concerns the data revealing the micro-organisms, which are viable after prolonged freezing. Contamination of edible portions of fish could originate from gastrointestinal sources. SIGNIFICANCE AND IMPACT OF THE STUDY: The present results may enhance knowledge in controlling the storage life of fish, and fish product quality. Bacterial activity is by far the most important factor influencing fish quality, so bacterial numbers can be used as an index of quality. Storage of frozen tilapia without evisceration could be avoided.     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

A generally applicable cryopreservation method for nitrite-oxidizing bacteria

Nitrite-oxidizing bacteria are key members of the global nitrogen cycle but their study is hampered by their limited availability in culture, mostly due to laborious cultivation procedures and the lack of stable preservation methods. In this study, it was demonstrated that long-term cryopreservation of nitrite-oxidizing bacteria assigned to the genera Nitrobacter, Nitrospina, Nitrococcus, Nitrotoga and Nitrospira was possible using a simple and rapid protocol. Their survival was tested with different cryoprotecting agents, DMSO and Hatefi, and in various carbon-rich preservation media, ten-fold diluted TSB, and ten-fold diluted TSB supplemented with 1% trehalose, and 1% sucrose. Optimal preservation conditions were strain-dependent and marine strains appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide with or without ten-fold diluted trypticase soy broth as a preservation medium allowed successful preservation of all tested strains.     

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.