Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.     

寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。     

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.     

8种不同方法保藏病原菌效果的对比观察

采用８种不同的菌种保藏方法,对１８种常见病原菌的保藏时间及生物学特性的影响进行了对比观察。结果表明：冷冻干燥法保藏菌种时间最长（本实验室已保存１５年）;肉浸汤琼脂平板法保藏菌种时间最短（２～３月）。保藏时间由长到短依次为：冷冻干燥法＞半固体冷冻法≥半固体斜面加液体石蜡覆盖法＞半固体斜面法＞肉浸汤加液体石蜡覆盖法＞血琼脂平板法＞肉浸汤法＞肉浸汤琼脂平板法。且相同方法对不同菌种保藏时间不同。保藏时间在１年以内的菌种,其生物学特性无明显变异;而经冷冻干燥法保藏时间较长的白喉棒状杆菌、金黄色葡萄球菌、甲型副伤寒沙     

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.     

Natural biopolymer for preservation of microorganisms during sampling and storage

Stability of microbial cultures during sampling and storage is a vital issue in various fields of medicine, biotechnology, food science, and forensics. We have developed a unique bacterial preservation process involving a non-toxic, water-soluble acacia gum polymer that eliminates the need for refrigerated storage of samples.The main goal of this study is to characterize the efficacy of acacia gum polymer for preservation of pathogenic bacteria (Bacillus anthracis and methicillin-resistant Staphylococcus aureus—MRSA) on different materials, used for swabbing and filtration: cotton, wool, polyester, rayon, charcoal cloth, and Whatman paper.Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5-45 °C for MRSA and 40-90 °C for B. anthracis). Our results showed higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum. Addition of acacia gum polymer to swabbing materials or filters will increase efficacy of sample collection and identification of pathogenic bacteria from locations such as hospitals or the environment. Proposed approach can also be used for long-term storage of culture collections, since acacia gum contributes to viability and stability of bacterial cultures.     

The Preservation of Bacteria and Fungi on anhydrous Silica Gel: an Assessment of Survival over Four years

The preservation method of Perkins (1962) using suspensions in skim-milk was used to preserve 33 bacteria and 22 fungi on anhydrous silica gel. During storage at room temperature, 64% of the bacteria and 77% of the fungi survived 1 year or more. Storage at 4° often increased the survival period c. 2- to 3-fold: 73% of the bacteria and all 12 of the fungi tested at 4° survived > 1 year. At the last testing, 60% of the bacteria and 36% of the fungi were still viable after storage at 4° for periods between 3 and 4 years. The Gram positive bacilli tended to survive the silica gel preservation process better than most Gram negative bacilli. Some factors influencing survival after preservation on silica gel are discussed; the results support the use of a closed storage tube.     

Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus

AIMS: The efficiency of trehalose, sucrose and maltose to protect Lactobacillus bulgaricus during drying has been evaluated in bacteria grown at low water activity. METHODS AND RESULTS: Bacteria were grown in MRS (control), and in MRS supplemented with sucrose (MRS-sucrose) or with polyethyleneglycol (PEG) (MRS-PEG) as low water activity media. The growth in low water activity media (MRS-sucrose and MRS-PEG) prior to drying enhanced the effectiveness of trehalose as thermoprotectant during drying. The efficiency of sucrose was improved when bacteria were grown in MRS-sucrose. On the other hand, the growth in both low water activity media did not affect the efficiency of maltose. The damage produced during dehydration has been evaluated by means of growth kinetics in milk. The preservation of bacteria dehydrated with sucrose, after growing them in MRS-sucrose, appears to be as efficient as the dehydration with trehalose. CONCLUSIONS: The growth of L. bulgaricus in low water activity media enhances the protective action of trehalose and sucrose. SIGNIFICANCE AND IMPACT OF THE THE STUDY: These results may aid the dairy industry to improve the recovery of the starters at low costs after preservation processes.     

Biomachining: Preservation of Acidithiobacillus ferrooxidans and treatment of the liquid residue

Biomachining has become a promising alternative to micromachining metal pieces, as it is considered more environmentally friendly than their physical and chemical machining counterparts. In this research work, two strategies that contribute to the development of this innovative technology and could promote its industrial implementation were investigated: preservation of biomachining microorganisms (Acidithiobacillus ferrooxidans) for their further use, and making valuable use of the liquid residue obtained following the biomachining process. Regarding the preservation method, freeze‐drying, freezing, and drying were tested to preserve biomachining bacteria, and the effect of different cryoprotectants, storage times, and temperatures was studied. Freezing at –80°C in Eppendorf cryovials using betaine as a cryoprotective agent reported the highest bacteria survival rate (40% of cell recovery) among the studied processes. The treatment of the liquid residue in two successive stages led to the precipitation of most of the total dissolved iron and divalent copper (99.9%). The by‐products obtained (iron and copper hydroxide) could be reused in several industrial applications, thereby enhancing the environmentally friendly nature of the biomachining process.     

甲烷氧化混合菌的保藏方法研究

【目的】甲烷氧化混合菌是自然界中吸收甲烷的关键微生物,在甲烷氧化混合菌的研究和应用中,首先要解决其长期稳定保藏的问题,保藏方法应能有效保持菌群结构和功能的完整性、稳定性。【方法】以从煤矿土壤富集得到的两种结构稳定的甲烷氧化混合菌为实验体系,研究对比了冷藏法、低温冷冻法、石蜡油冷冻法、甘油冷冻法4种保藏方法,考察保藏前后混合菌的生长状况、MMO活性、菌群结构等。【结果】保藏6个月后,除甘油冷冻法以外,经其它3种方法保藏的混合菌,都具有与保藏前相当的细胞密度、甲烷氧化能力、MMO酶活以及传代稳定性,且DGGE图谱显示保藏前后的菌群结构变化不大。【结论】这3种保藏方法都可以有效的保持甲烷氧化混合菌功能和菌群结构的稳定性。     

冷风干燥制备高活菌的恶臭假单胞菌菌粉

【目的】获得高活菌恶臭假单胞菌菌粉,提高菌体干燥及保藏存活率。【方法】选用冷风干燥法制备活菌粉,并优化吸附载体与保护剂。【结果】冷风干燥制备恶臭假单胞菌菌粉干燥存活率普遍达到65%以上,显著优于喷雾干燥(24%);对载体与保护剂进行正交试验优化,确定了载体为混合的硅藻土和碱处理玉米芯粉,混合比为1:2,保护剂(质量比)为甘露醇7%、谷氨酸钠5%、甘油1%,制得菌粉活菌数为1.03×1011 CFU/g,室温保藏30 d和4 °C保藏60 d存活率分别达到40.54%和71.67%。【结论】冷风干燥温度相对较低(10?40 °C),对菌体损伤小,碱处理玉米芯粉、甘露醇和谷氨酸钠是提高菌粉保藏存活率的重要因子,此法克服了革兰氏阴性菌菌粉不易制备和不耐保藏的瓶颈。     

Microflora of the aerobic preservation of directly brined green olives from Hojiblanca cultivar

New procedures for the preservation stage of ripe olives from Hojiblanca cultivar were studied. An aerobic fermentative process was used with initial pH correction (0.3% acetic acid) and various NaCl concentrations: 6, 3 and 0% (w/v) in tap water. Treatments were carried out at industrial level and the spontaneous changes monitored. At initial salt concentrations of 6 and 3% (w/v) NaCl, pH rose progressively, reaching 4.3 at equilibrium maintaining during this period a constant free lactic acidity of around 0.4% (w/v). When the initial solution was tap waste, however, the pH decreased rapidly to stabilize at about 3.7, and lactic acidity increased continuously to reach values over 1% (w/v) at the end of the preservation process. In all treatments aeration effectively purged the carbon dioxide from the preservation brines, preventing shrivelling of olives. The microbial growth was strongly influenced by the initial NaCl concentration. At 6 and 3%, only yeasts grew, the most abundant being Pichia membranaefaciens, P. vini, P. fermentans and Hansenula polymorpha. However, when there was no NaCl, lactic acid bacteria colonized the solution. Lactobacillus plantarum and Pediococcus inopinatus were the only species found. In this case there was a co-existence between yeasts and lactic acid bacteria. As the treatment that supported lactic acid bacteria achieved the best final pH and acidity for olive stability, it may help to overcome the obstacles to a lactic fermentative process during the preservation stage of ripe olives from the Hojiblanca cultivar.     

A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria

A simple effective and compact freeze-drying method involving skim milk 20% (w/v) and glutamate 5% or meso-inositol 5% or honey 10% or raffinose 5% for the long-term preservation of bacteria is described. As a case example more than 160 strains representing 36 species of nitrogen-fixing bacteria, 11 species of chemolithorutotrophic bacteria and five species of Aquaspirillum were successfully preserved. All tested strains proved viable and showed about 10–100% survival after freeze-drying and during 2–3 years of storage at +9°C. In such lyophilized cultures no loss in plasmids or other desirable characters was observed. The method is also suitable for the preservation of other fragile and difficult microorganisms as several other strains including bacteria with introduced plasmids could equally survive well and retained plasmids after lyophilization with this method.     

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts.     

An evaluation of five preservation techniques and conventional freezing temperatures of -20 degrees C and -85 degrees C for long-term preservation of Campylobacter jejuni

AIMS: This study aimed to identify a simple, inexpensive preservation technique that will allow a quick and reliable recovery of Campylobacter jejuni following long-term periods of preservation. METHODS AND RESULTS: Preservation techniques include (i) Cryobank microbial preservation system using hypertonic 'cryopreservative solution' and glass beads, (ii) Cryobank microbial preservation system using defibrinated lysed horse blood and glass beads, (iii) FBP medium, (iv) 15% glycerol/85% nutrient broth no. 2 culture, and (v) 50% glycerol/50% nutrient broth no. 2 culture. Each preservation technique was evaluated over a 1-year period at conventional freezing temperatures of -20 degrees C and -85 degrees C. Replacement of 'cryopreservative fluid' in commercially prepared vials of glass beads with lysed horse blood increased the duration of preservation of Camp. jejuni by up to 6 months. CONCLUSIONS: FBP medium proved the most successful preservation technique with 100 and 80% recovery after 1 year at -85 degrees C and -20 degrees C, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a simple inexpensive preservation method for long-term storage of Camp. jejuni.     

The effect of step changes in sucrose concentration on the growth of Salmonella typhimurium LT2

Water activity is a method of preservation that can affect microbial growth in foods and that may fluctuate during their processing, distribution and storage. Sucrose has been used to change the water activity of microbiological culture media. Suspensions of Salmonella typhimurium LT2 in the exponential phase of growth have been subjected to step changes in sucrose concentration at 20°C. The changes in the numbers of viable bacteria were measured with time and the experimental growth curves compared with predictions based on growth data obtained at constant sucrose concentrations. Steps down in sucrose concentration showed some apparent loss of viability after the step followed by growth at a rate close to the expected value. Steps up in sucrose concentration resulted in a greater apparent loss of viability after the step and either growth or the inducement of lag, depending on the final concentration of sucrose. A series of small steps up in sucrose concentration to 45% (w/v) was able to sustain growth where it was not possible by inoculation directly into this concentration. Improved recovery of bacteria subject to osmotic stress was possible with a medium containing sodium chloride.     

Character of interactions of saprophytic soil microflora via gaseous metabolites

The character of interaction between saprophytic soil bacteria via gaseous metabolites was studied. It was established that, at the metabolic level, a diverse character of interspecies interrelationships between bacteria exist, directly influencing their reproduction and preservation in soil. Volatile compounds produced by bacteria are able to act as both intra-and interspecies regulators of microbial communities. The soil microbiocenosis composition may be therefore regulated by volatile products of metabolism of saprophytic soil bacteria. Methanol released by bacteria into the environment plays an important role in this process.     

Safeguarding bacterial resources promotes biotechnological innovation

Environmental research delivers valuable bacterial resources for biotechnology. We believe that systematic long-term preservation of bacteria will promote future biotechnological innovations, by safeguarding the accessibility of bacteria already recognized to have interesting features and providing a “pool” of bacterial resources for novel applied research. To this end, we want to advocate the incorporation of preservation tests in environmental or applied microbiological research. This paper introduces non-specialists to different preservation methods for bacteria. Several parameters that influence long-term storage of bacterial resources are explained and practical tips and guidelines are formulated. Also, the vital role of public culture collections is highlighted and the state-of-the-art of preservation of non-pure cultures is described.     

Optimization of preservation conditions of As (III) bioreporter bacteria

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4°C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.     

Character of interactions of saprophytic soil microflora via gaseous metabolites

The character of interaction between saprophytic soil bacteria via gaseous metabolites was studied. It was established that, at the metabolic level, a diverse character of interspecies interrelationships between bacteria exist, directly influencing their reproduction and preservation in soil. Volatile compounds produced by bacteria are able to act as both intra- and interspecies regulators of microbial communities. The soil microbiocenosis composition may be therefore regulated by volatile products of metabolism of saprophytic soil bacteria. Methanol released by bacteria into the environment plays an important role in this process.