Exploring <Emphasis Type="Italic">in vitro</Emphasis> germplasm conservation options for sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrids) in South Africa

In vitro plantlets of sugarcane cultivar NCo310 were maintained in slow growth conditions at both 18 and 24°C and on four semi-solid media: SG1—Murashige and Skoog (MS) salts and vitamins with 20 g L^?1 sucrose, SG2—½ MS with 10 g L^?1 sucrose, SG3—MS with 20 g L^?1 sucrose and 1 mg L^?1 abscisic acid (ABA), and SG4—½ MS with 10 g L^?1 sucrose and 1 mg L^?1 ABA. After 8, 12, 24, 36, and 48 mo shoot multiplication rates were recorded, shoots were removed from storage and subcultured every 2 wk on SG1 with 0.015 mg L^?1 kinetin and 0.1 mg L^?1 benzyl aminopurine for 2 mo. At 18°C, all media supported storage for 48 mo with subculturing every 12 mo. Shoot multiplication post-retrieval was significantly higher on the SG2 medium compared with the non-stored control (362 ± 84 and 126 ± 26 shoots per recovered shoot after 2 mo, respectively). In addition, shoots could be maintained for 48 mo on SG2 medium with one subculture without compromising post-storage multiplication ability. At 24°C, storage on all four media supported recovery and multiplication of shoots for 8 mo and only SG2 medium facilitated survival for 12 mo. There was no advantage to incorporating ABA into the storage media, regardless of the temperature and storage time. Cryopreservation of cultivar NCo376 in vitro-derived shoot meristems using the V-cryo-plate method demonstrated that the sucrose concentration in the loading solution (0.8–1.8 M) had no significant effect on survival of the meristems, which ranged from 41.7 ± 4.8 to 69.4 ± 10%.     

Formulation of the biocontrol agent <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> CPA-8 using different approaches: liquid,freeze-drying and fluid-bed spray-drying

The present work focuses on the assessment and comparison of three different formulation technologies and the effect of protectants on cell viability, storage stability and antagonistic activity of the biocontrol agent Bacillus amyloliquefaciens CPA-8. Cultures were concentrated with different protective substances such as MgSO₄, sucrose and skimmed milk (SM) and subjected to liquid formulation, freeze-drying and fluid-bed spray-drying. Results showed that CPA-8 freeze-dried cells without protectants or amended with SM suffered the highest losses in cell viability (0.41?0.48 log). Moreover, the cell viability of the tested freeze-dried products decreased after four months of storage at both tested temperatures (4 and 22 °C). Otherwise, liquid and fluid-bed spray-dried products were stable for four months at 4 °C and for 12 months at 22, 4 and ?20 °C, respectively, and no effect of the protectants was observed. The most suitable CPA-8 products were then tested against Monilinia laxa and M. fructicola in artificially wounded nectarines and in all cases the antagonistic activity was maintained similar to fresh cells. The efficacy results revealed that the formulation process did not affect the biocontrol potential of CPA-8. This work led us to conclude that effective formulations with final concentrations ranging from 1.93 × 10⁹–2.98 × 10⁹ CFU ml^?1 and from 4.76 × 10⁹–1.03 × 10¹⁰ CFU g^?1 were obtained for liquid and dried products, respectively. Additionally, the suitability of the fluid-bed spray drying technology should be taken into account to develop a stable and effective CPA-8 product for practical applications to control brown rot in stone fruit.     

Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability

Abstract

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22?±?1.35% with the viable cell number of 9.5?±?0.07?×?10⁹ CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6?×?10⁹ CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k_?18?=?8.04?×?10^?6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k₄?=?4.48?×?10^?4 and k₂₅?=?9.72?×?10^?3 under 4 and 25?°C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.     

Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization

Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l^?1), yeast extract (25.93 g l^?1), and corn steep liquor (10.45 g l^?1) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW^?1) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet^?1 h^?1. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.     

Bacterial aerosols in an urban nursery school in Gliwice,Poland: a case study

This work presents the results of the study of airborne bacteria in a kindergarten in Gliwice, Upper Silesia, Poland. In this study, the samples of bioaerosols were collected using six-stage Andersen cascade impactor (with aerodynamic cutoff diameters 7.0, 4.7, 3.3, 2.1, 1.1, and 0.65 μm). The level of culturable bacterial aerosols indoors was about 3000 CFU m^?3—six to eight times higher than outdoors. In the classrooms, respirable bacterial particles, <4.7 µm, contributed up to 85 % of the total number of culturable bacteria, increasing the possible adverse health effects due to their inhalation. The identification of the bacterial species showing the dominance of gram-positive cocci in the indoor environment and non-sporing gram-positive rods in the outdoor air indicates that most of the bacteria present in the studied kindergarten are human origin. Using the obtained data, the nursery school exposure dose (NSED) of bioaerosols was estimated for the children and personnel of this kindergarten (nursery school). The highest value of NSED was obtained for younger children (930 CFU kg^?1) compared to older children (about 600 CFU kg^?1) and to the kindergarten staff (about 300 CFU kg^?1). This result suggests the elevated risk of adverse health effects in younger children exposed to the bioaerosols in the kindergarten, including infections.     

Production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells of recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Recombinant Escherichia coli, expressing the oleate hydratase gene of Stenotrophomonas maltophilia, was permeabilized by sequential treatments with 0.125 M NaCl and 2 mM EDTA. The optimal conditions for the production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells were 35 °C and pH 7.0 with 0.1 % (v/v) Tween 40, 50 g permeabilized cells l^?1, and 17.5 g α-linolenic acid l^?1. Under these conditions, permeabilized cells produced 14.3 g 10-hydroxy-12,15(Z,Z)-octadecadienoic acid l^?1 after 18 h, with a conversion yield of 82 % (g/g) and a volumetric productivity of 0.79 g l^?1 h^?1. These values were 17 and 168 % higher than those obtained by nonpermeabilized cells, respectively. The concentration, yield, and productivity of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid obtained by permeabilized cells are the highest reported thus far.     

Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii

High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L^?1 yeast extract and 300 g L^?1 sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h^?1, FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L^?1 was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days.     

Molecular Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in High-Temperature Petroleum Reservoirs

Anaerobic ammonium-oxidizing (anammox) process plays an important role in the nitrogen cycle of the worldwide anoxic and mesophilic habitats. Recently, the existence and activity of anammox bacteria have been detected in some thermophilic environments, but their existence in the geothermal subterranean oil reservoirs is still not reported. This study investigated the abundance, distribution and functional diversity of anammox bacteria in nine out of 17 high-temperature oil reservoirs by molecular ecology analysis. High concentration (5.31–39.2 mg l^?1) of ammonium was detected in the production water from these oilfields with temperatures between 55°C and 75°C. Both 16S rRNA and hzo molecular biomarkers indicated the occurrence of anammox bacteria in nine out of 17 samples. Most of 16S rRNA gene phylotypes are closely related to the known anammox bacterial genera Candidatus Brocadia, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia, while hzo gene phylotypes are closely related to the genera Candidatus Anammoxoglobus, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia. The total bacterial and anammox bacterial densities were 6.4?±?0.5?×?10³ to 2.0?±?0.18?×?10⁶ cells ml^?1 and 6.6?±?0.51?×?10² to 4.9?±?0.36?×?10⁴ cell ml^?1, respectively. The cluster I of 16S rRNA gene sequences showed distant identity (<92%) to the known Candidatus Scalindua species, inferring this cluster of anammox bacteria to be a new species, and a tentative name Candidatus “Scalindua sinooilfield” was proposed. The results extended the existence of anammox bacteria to the high-temperature oil reservoirs.     

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1–0.9 h^?1) and initial glucose concentrations (20–60 g l^?1). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h^?1 dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml^?1 and 9,450 IU ml^?1 h^?1, respectively) with the combination of a 0.9-h^?1 dilution rate and a 40-g l^?1 initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.     

Estimation of carbon biomass and community structure of planktonic bacteria in Lake Biwa using respiratory quinone analysis

The relationship between bacterial respiratory quinone (RQ) concentration and biomass was assessed for Lake Biwa bacterial assemblages to evaluate the utility of bacterial RQ concentration as an indicator of bacterial carbon. The biomass estimated from the RQ concentration correlated well with that from cell volume, indicating that RQ concentration is an appropriate indicator of bacterial biomass. The estimated carbon content per unit of RQ (carbon conversion factor) of bacteria was 0.67 mg C nmol RQ^?1. Bacterial carbon biomass, which was estimated from the RQ concentration using the conversion factor, ranged between 0.008 and 0.054 mg C L^?1 (average 0.025 mg C L^?1) at 5 m depth and between 0.010 and 0.024 mg C L^?1 (average 0.015 mg C L^?1) at 70 m depth. Ubiquinone-8-containing bacteria dominated the epilimnion and hypolimnion. Compared to conventional image analysis, bacterial RQ analysis is a less laborious method of simultaneously determining bacterial biomass and community.     

Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock ‘Gisela<Superscript>®</Superscript>5’

The present study investigated the effects of gas-tight and gas-permeable culture containers and different sucrose concentrations, as well as sucrose and mannitol combinations on the development of an effective in vitro slow growth storage protocol (at 4 °C, in darkness) for ‘Gisela^®5’ shoot cultures. ‘Gisela^®5’ is the most widely used cherry rootstock in Europe. This dwarf triploid hybrid has many advantages over the conventional cherry rootstocks. Optimizations for the cold storage of ‘Gisela^®5’ in vitro shoot cultures included use of storage medium supplemented with 10, 20, 30, 45, and 60 g L^?1 sucrose and sucrose (15, 30 g L^?1) and mannitol (15 g L^?1) combinations, contained in gas-tight glass jars and gas-permeable ‘Star Pac?’ bags. Cold storage was prolonged to 12 months, during which in every 3 months, cultures were evaluated. Possibility of 16 month-cold storage in gas-tight glass jars was also explored, during which gas chromatographic analysis was performed for the detection of CO₂ and ethylene accumulation for the first 5 months of cold storage. Our results showed that both the 12- and 16-month conservations were possible, especially when 45 or 60 g L^?1 sucrose was supplemented to storage medium, contained in glass jars. Mannitol inclusion to the storage medium was also effective to reduce the metabolic activity of the shoot cultures during storage; however, it did not have a significant positive influence on shoot quality in post-conservation.     

Association of Bacteria with Marine Invertebrates: Implications for Ballast Water Management

Bacteria associated with plankton are of importance in marine bioinvasions and the implementation of ship’s ballast water treatment technologies. In this study, epibiotic and endobiotic bacteria associated with zooplankton, including barnacle nauplii, veliger larvae, and adults of the copepod Oithona sp., were characterized and quantified. Barnacle nauplius and veliger larva harbored ~4.4 × 10⁵ cells ind^?1 whereas Oithona sp. had 8.8 × 10⁵ cells ind^?1. Computation of bacterial contribution based on biovolume indicated that despite being the smallest zooplankton tested, veliger larvae harbored the highest number of bacteria, while barnacle nauplii, the largest of the zooplankton, tested in terms of volume contributed the least. Pulverization of zooplankton led to an increase in bacterial numbers; for example, Vibrio cholerae, which was initially 3.5 × 10³, increased to 5.4 × 10⁵ CFU g^?1; Escherichia coli increased from 5.0 × 10² to 1.3 × 10⁴ CFU g^?1; and Streptococcus faecalis increased from 2.1 × 10² to 2.5 × 10⁵ CFU g^?1, respectively. Pulverized zooplankton was aged in the dark to assess the contribution of bacteria from decaying debris. Aging of pulverized zooplankton led to emergence of Chromobacterium violaceum, which is an opportunistic pathogen in animals and humans.     

Enhanced production of ATP-binding cassette protein exporter-dependent lipase by modifying the growth medium components of Pseudomonas fluorescens

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l^?1, 40 g Tween 80 l^?1 and 30 g peptone l^?1, TliA was produced at a level of 2,200 U ml^?1 in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml^?1) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production.     

Accumulation of rosmarinic, chlorogenic and caffeic acids in in vitro cultures of Eryngium planum L.

Eryngium planum L. cell and organ cultures were maintained on Murashige and Skoog media (MS), supplemented with exogenous hormones of different types and various concentrations for high biomass growth. The callus and cell suspension cultures were treated with increased sucrose concentration and/or elicited by methyl jasmonate for the enhancement of selected phenolic acids accumulation. Three phenolic acids, rosmarinic acid (RA), chlorogenic acid (CGA) and caffeic acid (CA), were detected by HPLC-DAD in those cultures. The sum of their content in the dry material was found to be higher in the shoot culture (3.95 mg g^?1), root culture (7.05 mg g^?1), callus (6.20 mg g^?1) and cell suspension (2.04 mg g^?1) than in the leaves (1.87 mg g^?1) and roots (0.76 mg g^?1) of intact plants. The major compound of in vitro cultures was always rosmarinic acid. The content of RA could be increased approximately threefold (16.24 mg g^?1) in the callus culture and approximately twofold (3.91 mg g^?1) in the cell suspension culture by elicitation with 100 μM methyl jasmonate (MeJA). The higher concentration of sucrose (S) in the medium (5, 6 %) led to over a twofold increase of CGA content in the callus culture (2.54 mg g^?1). The three mentioned phenolic acids have been found in E. planum undifferentiated and differentiated in vitro cultures for the first time.     

Cell dualism: presence of cells with alternative membrane potentials in growing populations of bacteria and yeasts

It is considered that all growing cells, for exception of acidophilic bacteria, have negatively charged inside cytoplasmic membrane (Δψ^? - cells). Here we show that growing populations of microbial cells contain a small portion of cells with positively charged inside cytoplasmic membrane (Δψ⁺ - cells). These cells were detected after simultaneous application of the fluorescent probes for positive membrane potential (anionic dye DIBAC^-) and membrane integrity (propidium iodide, PI). We found in exponentially growing cell populations of Escherichia coli and Saccharomyces cerevisiae that the content of live Δψ^- - cells was 93.6?±?1.8 % for bacteria and 90.4?±?4.0 % for yeasts and the content of live Δψ⁺ - cells was 0.9?±?0.3 % for bacteria and 2.4?±?0.7 % for yeasts. Hypothetically, existence of Δψ⁺ - cells could be due to short-term, about 1 min for bacteria and 5 min for yeasts, change of membrane potential from negative to positive value during the cell cycle. This change has been shown by the reversions of K⁺, Na⁺, and Ca²⁺ ions fluxes across the cell membrane during synchronous yeast culture. The transformation of Δψ^-- cells to Δψ⁺ - cells can be explained by slow influx of K⁺ ions into Δψ^-- cell to the trigger level of K⁺ concentration (“compression of potassium spring”), which is forming “alternative” Δψ⁺-cell for a short period, following with fast efflux of K⁺ ions out of Δψ⁺-cell (“release of potassium spring”) returning cell to normal Δψ^- state. We anticipate our results to be a starting point to reveal the biological role of cell dualism in form of Δψ^- - and Δψ⁺ - cells.     

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens immobilized on Ca2+ alginate beads and under resting cell condition

Pseudomonas nitroreducens MILB-8054A isolated from petroleum-contaminated soil, immobilized on calcium alginate beads, and under resting cell condition, produced biosurfactants. Immobilized cells gave a best yield of 5.6 g rhamnolipid l^?1 using sucrose as carbon source. Time course study using resting cells showed that 2 % v/v of palm oil (preculture carbon source) and 10 % diesel (carbon source) gave the best rhamnolipid yield of 5.1 g l^?1 at pH 8 and temperature of 30 °C. Carbon utilization by resting cells was compared with that of growing cells. The best biosurfactant recovery procedure was acetone extraction.     

Hepatopancreatic cells of a stone crab <Emphasis Type="Italic">Menippe frontalis</Emphasis> from Perú: separation,viability study,and evaluation of lipoperoxidation against cadmium contamination

Crustaceans are frequently used as bioindicators, and changes in their metabolism at the hepatopancreas (HP) level are often followed in these studies. The HP is the site of digestion, absorption, nutrient storage, and toxic metal detoxification, enabling crabs to survive in metal contaminated regions. Cellular damage and high lipid peroxidation (LPO) levels have been found in crab populations under high cadmium (Cd) concentrations. The aim of this work was to separate and characterize the HP cells of the stone crab Menippe frontalis from the Pacific Ocean, Perú (5° 5′ 21″ S–81° 6′ 51″ W) and to measure the cellular viability and LPO after exposure to the non-essential metal Cd. The HP cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at concentrations of 10, 20, 30, and 40%. We found the same cell types that were described for other species (e.g., Ucides cordatus, Atlantic Ocean, Brazil). High cellular viability against 1 mmol L^?1 of Cd was observed for resorptive (R) cells in 20% sucrose layer (88 ± 8%, *P < 0.05, ANOVA), and blister (B) cells in the 40% sucrose layers (92 ± 7%, *P < 0.05, ANOVA). Cd (1 mmol L^?1) caused an increase in LPO levels, suggesting that crabs from polluted areas can be affected by toxic metals, generating a physiological stress. The gradient sucrose methodology can be used for different species and results in a similar separation, viability, and cellular identification. The results are a starting point for toxic metal studies for species distributed across different geographic coordinates.     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.     

Total bacterial number concentration in free tropospheric air above the Alps

Over a period from June to October 2010, we carried out four short campaigns on the northern alpine ridge (High Altitude Research Station Jungfraujoch, 3,450 m above sea level) to determine bacterial number concentrations by collecting aerosol with liquid impingers, followed by filtration, fluorescent staining and counting with a microscope. Impinger liquid was also subjected to drop freeze tests to determine the number of ice nucleators. Parallel measurements of ²²²Rn enabled us to distinguish air masses with no, or little, recent land surface contact (free troposphere, ²²²Rn ≤ 0.50 Bq m^?3) from air masses influenced by recent contact with land surface (²²²Rn > 0.50 Bq m^?3). In free tropospheric air, concentration of total bacteria was on average 3.4 × 10⁴ cells m^?3 (SD = 0.8 × 10⁴ cells m^?3). When wind conditions preceding sampling were calm, or when the station was in clouds during sampling, there was no detectable difference in bacterial number concentrations between free tropospheric air and air influenced by recent land surface contact. One campaign was preceded by a storm. Here, recent land surface contact had enriched the air in bacterial cells (up to 7.5 × 10⁴ cells m^?3). Very few of these bacteria may act as ice nucleators in clouds. The median ratio of ice nucleators to the number of bacterial cells in our study was 1.0 × 10^?5. We conclude that injection of bacterial cells into the free troposphere is an intermittent process. Conditions controlling the release of bacteria into near surface air are probably more of a limiting factor than vertical transport and mixing of near surface air into the free troposphere.     

Storage stability and sourdough acidification kinetic of freeze-dried Lactobacillus curvatus N19 under optimized cryoprotectant formulation

In this study, the response surface methodology was used to optimize the cryoprotective agent (skimmed milk powder, lactose and sucrose) formulation for enhancing the viability of Lactobacillus curvatus N19 during freeze-drying and storage stability of cells freeze-dried by using optimum formulation was evaluated. Our results showed that the most significant cryoprotective agent influencing the viability of L. curvatus N19 to freezing and freeze-drying was sucrose and skim milk, respectively. The optimal formulation of cryoprotective agents was 20 g/100 mL skim milk, 3.57 g/100 mL lactose and 10 g/100 mL sucrose. Using the optimum formulation during freeze-drying, the cell survival was found more than 98%. Under the optimal conditions, although only storage of the cells at 4 °C for 6 month retained the maximum stability (8.85 log cfu/g), the employed protectant matrix showed promising results at 25 °C (7.89 log cfu/g). The storage stability of cells under optimized conditions was predicted by accelerated storage test, which was demonstrated that the inactivation rate constant of the freeze-dried L. curvatus N19 powder was 9.74 × 10⁻⁶ 1/d for 4 °C and 2.08 × 10⁻³ 1/d for 25 °C. The loss of specific acidification activity after the storage at 4 and 25 °C was determined.