Biochemical aspects of basidiospore maturation in Agaricus bisporus at various temperatures

Phosphatidylethanolamine is the main phospholipid of Agaricus bisporus basidiospores obtained under sterile conditions from young basidiomes with closed partial veils. Storing the basidiospores for five months at room temperature resulted in a complete loss of their germinating capacity. Conversely, storing them at a low temperature increased their germination rate by 15-20%. At both temperature levels, the phosphatidylcholine ratio significantly increased during storage to the level found in mature basidiospores. In addition, a drastic (8-10-fold) decrease in trehalose content occurred after two months of storage at room temperature. The trehalose content decreased only 1.5-fold at low temperatures. The involvement of trehalose and lipids in the retention of spore viability is discussed.     

Germination of Basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores were studied. Basidiospores failed to germinate on starvation agar and required the presence of carbon and nitrogen sources (asparagine and/or glucose) in the medium. Upon 3-week storage, basidiospores germinated after 4–5 days. Heat shock (20 min at 45°C) and decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus basidiospores differing from zygospores of Mucorales. Basidiospores contained 17–19% lipids with a composition of fatty acids differing from those of the pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike basidiospores stored at 2°C, basidiospores stored for 5 months at 20°C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

Gaseous environments modify reserve carbohydrate contents and cell survival in the brewing yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The use of H₂, He and O₂ during batch fermentation of Saccharomyces cerevisiae BRAS291 increased the final intracellular glycogen contents of the cells from 2-fold to 10-fold compared with a gas-free condition, and this depended on the gas applied. Differently, the intracellular trehalose contents increased from 2-fold to 10-fold in reducing conditions compared with more oxidizing conditions. During storage at 4°C, the viability of cells cultivated with gas was twice that of cells cultivated without gas. These results could be explained by the intracellular carbohydrate contents as well as yeast ultrastructural modifications observed previously.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Effects of pH,NH<Subscript>4</Subscript>-N concentration,temperature, and storage period on basidiospore germination in an ectomycorrhizal ammonia fungus <Emphasis Type="Italic">Hebeloma vinosophyllum</Emphasis>

Basidiospore germination in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum was stimulated by 10–500 mM NH₄Cl aqueous solution at pH 4.5–9.0, but not by pure water. The basidiospores germinated at 10°–35°C with an optimum at 25°–30°C. The highest germination percentage (83.0%) was observed in 100 mM NH₄Cl aqueous solution adjusted to pH 8.0 by KOH, when the basidiospores were incubated at a density of 10⁶ spores/ml at 30°C for 14 days. The percent germination value decreased with the increased duration of storage under both dry and wet conditions. Humidity and temperature affected the longevity of H. vinosophyllum basidiospores. The basidiospores maintained their germination ability longer under a dry condition than under a wet condition. The greatest longevity was accomplished by storage at 15°C under a dry condition.     

Germination of basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores (BS) were studied. BS failed to germinate on starvation agar and required the presence of carbon and nitrogen (asparagine and/or glucose) sources in the medium. Upon 3-week storage, BS germinated after 4-5 days. Heat shock (20 min at 45 degrees C) and the decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus BS differing from zygospores of Mucorales. BS contained 17-19% lipids with a composition of fatty acids differing from those of pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike BS stored at 2 degrees C, the BS stored for 5 months at 20 degrees C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

Preservation of H2 production activity in nanoporous latex coatings of Rhodopseudomonas palustris CGA009 during dry storage at ambient temperatures

To assess the applicability of latex cell coatings as an ‘off‐the‐shelf’ biocatalyst, the effect of osmoprotectants, temperature, humidity and O₂ on preservation of H₂ production in Rhodopseudomonas palustris coatings was evaluated. Immediately following latex coating coalescence (24 h) and for up to 2 weeks of dry storage, rehydrated coatings containing different osmoprotectants displayed similar rates of H₂ production. Beyond 2 weeks of storage, sorbitol‐treated coatings lost all H₂ production activity, whereas considerable H₂ production was still detected in sucrose‐ and trehalose‐stabilized coatings. The relative humidity level at which the coatings were stored had a significant impact on the recovery and subsequent rates of H₂ production. After 4 weeks storage under air at 60% humidity, coatings produced only trace amounts of H₂ (0–0.1% headspace accumulation), whereas those stored at < 5% humidity retained 27–53% of their H₂ production activity after 8 weeks of storage. When stored in argon at < 5% humidity and room temperature, R. palustris coatings retained full H₂ production activity for 3 months, implicating oxidative damage as a key factor limiting coating storage. Overall, the results demonstrate that biocatalytic latex coatings are an attractive cell immobilization platform for preservation of bioactivity in the dry state.     

Protocols for dry DNA storage and shipment at room temperature

The globalization of DNA barcoding will require core analytical facilities to develop cost‐effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry‐state DNA stabilization systems: commercial Biomatrica^® DNAstable^® plates, home‐made trehalose and polyvinyl alcohol (PVA) plates on 96‐well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at ?20 °C. PCR and selective sequencing were performed over a 4‐year interval to test the condition of DNA extracts. Biomatrica^® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica^® at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at ?20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long‐term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities.     

Changes in germinability,lipid peroxidation,and antioxidant enzyme activities in pear stock (<Emphasis Type="Italic">Pyrus betulaefolia</Emphasis> Bge.) seeds during room- and low-temperature storage

The aim of this study was to determine if loss of germinability in Pyrus betulaefolia seeds stored at 4°C and at room temperature is associated with a loss of membrane lipid peroxidation or changes in antioxidant enzyme activities. The results indicated that germination percentage clearly decreased when seeds were stored at room temperature rather than at 4°C from 6 to 12 months. Room-temperature storage of the pear stock seed for 12 months decreased germination to 15.52%, but germination percentage was not changed when seed was stored at 4°C for 12 months. MDA, a marker for membrane lipid peroxidation, increased significantly under room-temperature storage conditions. Antioxidant enzyme (SOD, POD, and CAT) activities were a good indicator of germination percentage in pear stock seeds. Antioxidant enzyme activities of pear stock seeds at 4°C were higher than antioxidant enzyme activities in seeds stored at room temperature from 6 to 12 months. Antioxidant enzyme activities of the pear stock seed decreased markedly under conditions of room-temperature storage from 6 to 12 months. The results of this study showed that long-term room-temperature storage was detrimental for maintaining the vigor of P. betulaefolia seeds. The mechanisms responsible for this outcome are a higher level of membrane lipid peroxidation and a lower level of activity of antioxidant enzymes.     

Extremophile extracts and enhancement techniques show promise for the development of a live vaccine against Flavobacterium columnare

The effects of temperature, ionic strength, and new cryopreservatives derived from polar ice bacteria were investigated to help accelerate the development of economical, live attenuated vaccines for aquaculture. Extracts of the extremophile Gelidibacter algens functioned very well as part of a lyophilization cryoprotectant formulation in a 15-week storage trial. The bacterial extract and trehalose additives resulted in significantly higher colony counts of columnaris bacteria (Flavobacterium columnare) compared to nonfat milk or physiological saline at all time points measured. The bacterial extract combined with trehalose appeared to enhance the relative efficiency of recovery and growth potential of columnaris in flask culture compared to saline, nonfat milk, or trehalose-only controls. Pre-lyophilization temperature treatments significantly affected F. columnare survival following rehydration. A 30-min exposure at 0 °C resulted in a 10-fold increase in bacterial survival following rehydration compared to mid-range temperature treatments. The brief 30 and 35 °C pre-lyophilization exposures appeared to be detrimental to the rehydration survival of the bacteria. The survival of F. columnare through the lyophilization process was also strongly affected by changes in ionic strength of the bacterial suspension. Changes in rehydration constituents were also found to be important in promoting increased survival and growth. As the sodium chloride concentration increased, the viability of rehydrated F. columnare decreased.     

A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

Long-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ∼22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells.     

The Serum-Resistant Transfection Evaluation and Long-Term Stability of Gene Delivery Dry Powder Based on Mesoporous Silica Nanoparticles and Polyethyleneimine by Freezing-Drying

Mesoporous silica nanoparticles (MSNs) with large surface area, tunable pore size, and low toxicity can act as suitable vehicles for drug and gene delivery. An MSN/DNA/PEI complex delivery system was prepared by using MSNs to hold plasmid DNA coated with polyethyleneimine (PEI), and the dry powder formulation was produced by freeze-drying with trehalose as lyoprotectant. The MSN/DNA/PEI complexes successfully enhanced the gene expression with about 1.5-fold higher efficiency as compared with the control, and even better effects and lower toxicity were achieved at lower content of PEI. Also, this gene delivery system showed nearly sixfold higher efficiency in the serum-containing condition than the control, so further application of these vehicles in vivo is highly appreciated. Besides, the trehalose containing lyophilized formulation could hold the availability for at least 4 months of storing at room temperature, presenting the potential for industrial production and transportation of gene therapy.     

Enhancing long-term storage and stability of engineered living materials through desiccant storage and trehalose treatment

Engineered living materials (ELMs) have broad applications for enabling on-demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast-laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1-year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ-CT and confocal microscopy indicate that cells form spherical colonies within F127-BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on-demand, cold-chain independent bioproduction.     

The influence of processing and storage on hydrogen cyanide and tannin contents of para-rubber seed and its products

Para-rubber seed and its products, including the autoclaved and fermented oil meals, were assayed for HCN content at post-harvest intervals from 1 week to 9 months of storage at room temperature. The tannin content of all these products was also estimated after 3 months of storage.Rubber seed and its kernels contained 638 and 749 mg HCN/kg, respectively, 1 week after harvest; these values gradually diminished to 25.3 and 26.7 mg/kg, respectively, after 9 months storage. The rate of reduction in HCN levels was fast for the first 2 months of storage and slower later. The HCN levels in other rubber seed products also declined during storage. Thus, storage at room temperature for a minimum period of 2 months appeared to be an effective method of reducing the HCN content of rubber seed and its products to safe levels.The tannin levels in rubber seed and its products were low (0.42–0.53%) and within the safety levels for incorporation in livestock feeds. Moreover, the tannins were confined to the shell portion of the rubber seeds. Thus decortication appeared to be a satisfactory method for eliminating the tannins in rubber seeds, but increased the HCN levels slightly.Oil extraction and autoclaving failed to reduce the HCN and tannin levels, but fermentation successfully reduced both HCN and tannin levels in the rubber seed and its products.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Comparison of Keeping Quality between Pressure-processed Jam and Heat-processed Jam: Changes in Flavor Components,Hue, and Nutrients during Storage

Products of strawberry jam prepared by a high hydrostatic pressure-processing method and a conventional heat-processing method were examined with regard to the changes of quality such as volatile flavor components, anthocyanins, browning index, furfural, sucrose contents, and vitamin C contents during storage at 5°C and 25°C for 1–3 months. The quality of pressure-processed jam immediately after manufacturing was superior to that of heat-processed jam in preserving the fresh flavor and natural color of raw fruits. The superior quality was maintained under low temperature storage for two to three months. However, room temperature storage resulted in rapid deterioration including off-flavor, discoloration, browning, and decomposition of sucrose and vitamin C. So, the commercial value of pressure-processed jam decreased within a brief period. On the other hand, heat-processed jam did not change in quality under room temperature storage for three months. It is supposed that dissolved oxygen and enzyme systems retained are the causative factors of rapid deterioration in pressure-processed jam.     

Characterization of a <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strain from tomato rhizosphere and its use for integrated management of tomato damping-off

A highly antagonistic Pseudomonas fluorescens strain was isolated from tomato rhizosphere and characterized for its in vitro and in vivo biocontrol potential against Pythium aphanidermatum. The identified Pseudomonas fluorescens strain (PfT-8) was capable of producing high levels of chitinase, β-1,3-glucanase, cellulase, fungitoxic metabolites and siderophores. Seven different carrier formulations including a talc-based powder, lignite-based powder, peat-based powder, lignite + fly ash-based powder, wettable powder, bentonite-paste and polyethylene glycol (PEG) paste were prepared utilizing PfT-8. Shelf life was evaluated for up to 6 months of storage at ambient room temperature (28 °C). Biocontrol efficacy of formulations was studied under greenhouse and field conditions. The formulations were stable up to at least 2 months of storage at ambient room temperature. Among the formulations, peat, lignite, lignite+fly-ash and bentonite paste based formulations maintained higher propagule number than others and also showed greater biocontrol potential. However, propagule number gradually decreased with time. Several organic amendments including farm yard manure (FYM), leaf-compost, poultry manure, press mud, vermi-compost and neem cake were incorporated into soil to study their influence on P. fluorescens colonization in the rhizosphere and on potential disease control. Soil incorporation of organic amendments and specifically poultry manure and FYM, significantly reduced damping-off incidence and also augmented the rhizosphere population of the marked␣P.␣fluorescens strain that was resistant to streptomycin and rifampicin. An integrated␣approach of damping-off management employing dual inoculation of PfT-8 in seed and soil coupled with soil application of organic amendments including poultry manure or␣FYM was evaluated under field conditions. Under these conditions, damping-off incidence substantially reduced by up to 90% and further the healthy plant stand, plant biomass and plant rhizosphere population of P. fluorescens increased significantly.     

Dependence of the viability and virulence of siberian isolates of the entomopathogenic fungus Beauveria bassiana (Bals-Griv.) Vuill. on the period of their storage at positive temperatures

A study of Siberian isolates of Beauveria bassiana (Bals-Griv.) Vuill. has shown the dependence of their viability and virulence on the storage period and temperature. The isolates remain viable and virulent for a longer period (up to 3 years) when they are stored at a low positive temperature (+8°C) than when stored at room temperature (+18°C). According to our correlation analysis, when stored at room temperature, the virulence of the studied isolates towards the test insects depends on the storage period (57–72%) and, to a lesser extent (28–43%), on other unaccounted factors.     

Control of electron transfer by protein dynamics in photosynthetic reaction centers

Abstract

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample. While much work has been carried out to elucidate a mechanistic view of how each of these matrices interact with proteins to provide stability, rarely have the effects of these two independent systems been directly compared to each other. This review aims to compile decades of research on how different glassy matrices affect two types of photosynthetic proteins: (i) the Type II bacterial reaction center from Rhodobacter sphaeroides and (ii) the Type I Photosystem I reaction center from cyanobacteria. By comparing aggregate data on electron transfer, protein structure, and protein dynamics, it appears that the effects of these two distinct matrices are remarkably similar. Both seem to cause a “tightening” of the solvation shell when in a glassy state, resulting in severely restricted conformational mobility of the protein and associated water molecules. Thus, trehalose appears to be able to mimic, at room temperature, nearly all of the effects on protein dynamics observed in low temperature glycerol glasses.     

Sodium–Sulfur Flow Battery for Low‐Cost Electrical Storage

A new sodium–sulfur (Na–S) flow battery utilizing molten sodium metal and flowable sulfur‐based suspension as electrodes is demonstrated and analyzed for the first time. Unlike the conventional flow battery and the high‐temperature Na–S battery, the proposed flow battery system decouples the energy and power thermal management by operating at different temperatures for the storage tank (near room temperature) and the power stack (100–150 °C). The new Na–S flow battery offers several advantages such as easy preparation and integration of the electrode, low energy efficiency loss due to temperature maintenance, great tolerance of the volume change of the metal anode, and efficient utilization of sulfur. The Na–S flow battery has an estimated system cost in the range of $50–100 kWh^?1 which is very competitive for grid‐scale energy storage applications.