A study of a bacterial immobilization substratum for use in the bioremediation of crude oil in asaltwater system

An immobilization matrix, Drizit, was examined for use in the bioremediation of oil in asaltwater system. The support was examined by scanning electron microscopy, cell-loadingcapacity, absorption of Ekofisk crude oil, the effect on cell recoverability of dryingDrizit-immobilized cells at room temperature, 55°C or freeze drying and storage for up to 3months, and the capability of immobilized Pseudomonas fluorescens to enhance biodegradation ofpetrol (Slovene diesel) in a saltwater system. Results showed that Drizit is an ideal immobilizationsubstratum for use in bioremediation, and the substratum was found to have a good cell-loadingcapacity (3·75 mg protein g⁻¹ substratum) and a high absorbency of oil(7·49 g Ekofisk g⁻¹ substratum). After drying the immobilized cells at roomtemperature, 55°C or freeze drying and subsequent storage, cells were not recoverable after 2weeks, 1 week or 2 months, respectively, and a significant decrease was seen in the number ofcells recovered after drying and 1 week's storage in all cases. Enhanced biodegradation of C₁₃–C₁₈ pristane and phytane, in petrol (Slovene diesel) occurred with theimmobilization of Ps. fluorescens to Drizit. An average degradation of 73·8%occurred in the immobilized system in comparison with the free system which resulted in anaverage degradation of 39·8%. When the two systems were supplemented with nitrates andphosphates, degradation in the free supplemented system increased to an average of 72·4%.However, no significant difference in biodegradation was found between the immobilized systemand the immobilized system supplemented with nitrates and phosphates which achieved anaverage degradation of 74.15%.     

Survival of Freeze Dried Bacterial Cultures

The survival of 100 strains of bacteria, representing 15 genera, on freeze drying and during storage in the freeze dried state for 10 years has been assessed. Gram-positive organisms tended to survive better than Gram-negative. Viable organisms were recovered from all but one of the cultures.     

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.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

冷冻干燥保藏曲霉菌种的效果

本文报告了用冷冻干燥法保藏曲霉属（Aspergillus）5种8株曲霉的效果,并分别对这些曲霉菌株的糖化酶活力进行检测。这些曲霉菌株经过冷冻干燥保藏8年后全部保持生活能力,其培养及形态特征除一株生长稍差外,其余菌株均保留原有形状,测定其糖化酶活力未有明显变化。     

The Effect of Freeze Drying on the Viability of Chromobacterium lividum

Old well-pigmented cultures of Chromobacterium lividum withstood freeze drying and subsequent storage better than young unpigmented cultures. Recovery from the dried state was greater when the recovery medium contained blood.     

Preservation of encysted polytomella.

Cysts of Polytomella parva and Polytomella caeca were recovered after 7 days storage at cryogenic temperatures following drying on shredded filter paper, silica gel or without added substrate. Accelerated storage testing, by exposing dried material to elevated temperatures, indicated that shredded filter paper was the best of the substrates tested. Polytomella parva was recovered after 5 years storage at -30 degrees C when dried on filter paper but not when dried on silic agel. Determinations of the number of cysts recovered indicated that viable cysts survived all conditions of storage tested. However, excystment following storage was delayed, the extent depending on storage conditions and the substrate used for drying. Most rapid recovery occurred when cysts were rehydrated immediately after drying, and after storage on filter paper at below -70 degrees C.     

Influence de l'âge de la culture sur la survie de Rhizobium meliloti à la lyophilisation et à la conservation après lyophilisation

Summary Survival after freeze drying of Rhizobium meliloti grown on different media was higher in young cultures when cells were in their logarithmic phase than in the old which were in their stationary phase. On the contrary the ability of the freeze dried organisms to survive during storage at 30°C was better for cells from old cultures than from young ones.     

State transitions and physicochemical aspects of cryoprotection and stabilization in freeze-drying of Lactobacillus rhamnosus GG (LGG)

Aims: The frozen and dehydrated state transitions of lactose and trehalose were determined and studied as factors affecting the stability of probiotic bacteria to understand physicochemical aspects of protection against freezing and dehydration of probiotic cultures. Methods and Results: Lactobacillus rhamnosus GG was frozen (–22 or –43°C), freeze‐dried and stored under controlled water vapour pressure (0%, 11%, 23% and 33% relative vapour pressure) conditions. Lactose, trehalose and their mixture (1 : 1) were used as protective media. These systems were confirmed to exhibit relatively similar state transition and water plasticization behaviour in freeze‐concentrated and dehydrated states as determined by differential scanning calorimetry. Ice formation and dehydrated materials were studied using cold‐stage microscopy and scanning electron microscopy. Trehalose and lactose–trehalose gave the most effective protection of cell viability as observed from colony forming units after freezing, dehydration and storage. Enhanced cell viability was observed when the freezing temperature was ?43°C. Conclusions: State transitions of protective media affect ice formation and cell viability in freeze‐drying and storage. Formation of a maximally freeze‐concentrated matrix with entrapped microbial cells is essential in freezing prior to freeze‐drying. Freeze‐drying must retain a solid amorphous state of protectant matrices. Freeze‐dried matrices contain cells entrapped in the protective matrices in the freezing process. The retention of viability during storage seems to be controlled by water plasticization of the protectant matrix and possibly interactions of water with the dehydrated cells. Highest cell viability was obtained in glassy protective media. Significance and Impact of the Study: This study shows that physicochemical properties of protective media affect the stability of dehydrated cultures. Trehalose and lactose may be used in combination, which is particularly important for the stabilization of probiotic bacteria in dairy systems.     

Survival of microorganisms after drying and storage

Bacteria, yeasts and fungi suspended in a dextran solution were added to ampoules containing strips of filter paper which were dried without vacuum conditions. The ampoules were sealed and stored in the dark at room temperature. Viability counts were made of the original suspension immediately after drying and after storage periods of 3–48 months. Although bacterial cultures of many genera did not show much resistance against dry conditions, bacteria of 13 other genera had survived well or moderately after 4 years of storage. Most of the dried yeast cultures had survived after this period. Of the 16 fungal genera tested, species of 6 genera exhibited growth after 4 years. Results of this study were compared with those of two other preservation methods by which the same microorganisms were used.     

Polysaccharide production benefits dry storage survival of the biocontrol agent Pseudomonas fluorescens S11:P:12 effective against several maladies of stored potatoes

Pseudomonas fluorescens S11:P:12 (NRRL B-21133) is a biological control agent able to suppress several potato diseases and sprouting. Notably, it produces a polysaccharide during liquid cultivation, and the objective of this work was to determine the role of this material in the bio-control process. First, the polysaccharide was isolated, purified and identified as marginalan, which accumulated to ~3.3 g/L in cultures. The bioactivity of isolated marginalan applied alone or in combination with washed cells of strain S11:P:12 was tested in potato bioassays of dry rot and pink rot suppressiveness and sprout inhibition. Since the formulation and storage of a dried biocontrol product is preferred for commercial use, the impact of marginalan on cell survival during drying and storage was also studied. Washed bacteria formulated with 0–6.6 g/L polysaccharide were either applied to Hyflo granules, then slowly dried for 24 h with airflow at 50–60% relative humidity, or in 1-µL droplets placed in replicate wells of a micro-plate, then quickly dried for 1 h in a biohazard hood. Both Hyflo and micro-plate dry storage results indicated that marginalan significantly reduced cell death after drying, such that the final stable viable cell density was 2.5–5 orders of magnitude greater, respectively, than if no marginalan were included with cells. Marginalan had no significant impact on disease or sprout suppression by strain S11:P:12, and its main benefit to biocontrol was viable cell preservation during drying and storage. When marginalan was formulated with other selected P. fluorescens strains, its benefits to drying and storage survival were again evident (especially after 4°C instead of 25°C storage), but its effects were more subtle than for strain S11:P:12, and dry rot suppression was not impacted.     

Formulation development of the biocontrol agent Bacillus subtilis strain CPA-8 by spray-drying

Aims: To prepare commercially acceptable formulations of Bacillus subtilis CPA‐8 by spray‐drying with long storage life and retained efficacy to control peach and nectarine brown rot caused by Monilinia spp. Methods and Results: CPA‐8 24‐h‐ and 72‐h‐old cultures were spray dried using 10% skimmed milk, 10% skimmed milk plus 10% MgSO₄, 10% MgSO₄ and 20% MgSO₄ as carriers/protectants. All carriers/protectants gave good percentages of powder recovery (28–38%) and moisture content (7–13%). CPA‐8 survival varied considerably among spray‐dried 24‐h‐ and 72‐h‐old cultures. Seventy‐two hours culture spray dried formulations showed the highest survival (28–32%) with final concentration products of 1·6–3·3 × 10⁹ CFU g^?1, while viability of 24‐h‐old formulations was lower than 1%. Spray‐dried 72‐h‐old formulations were selected to subsequent evaluation. Rehydration of cells with water provided a good recovery of CPA‐8 dried cells, similar to other complex rehydration media tested. Spray‐dried formulations stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and viability was maintained or slightly decreased by 0·2–0·3‐log. CPA‐8 formulations after 4‐ and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on nectarines and peaches resulting in a 90–100% reduction in disease incidence. Conclusions: Stable and effective formulations of biocontrol agent B. subtilis CPA‐8 could be obtained by spray‐drying. Significance and Impact of the Study: New shelf‐stable and effective formulations of a biocontrol agent have been obtained by spray‐drying to control brown rot on peach.     

Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113

Sugarbeet seeds used by farmers are often pelleted using an EB^TM-based mix. During the pelleting process, the seeds are dried immediately after application of the mix. In this work, the effects of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113Rif were investigated using a 1:1 EB^TM/vermiculite mix and sugarbeet seeds pelleted with this material. Growing F113Rif for 3 d (28 °C) within the EB^TM/vermiculite mix amended with nutrients (sucrose asparagine broth), instead of adding the cells to the unamended mix immediately before drying the mix or the pelleted sugarbeet seeds, resulted in improved survival of the strain in the mix or on the seeds, respectively, during subsequent storage. A slower drying (20 h instead of 3 h) of the F113Rif-inoculated EB^TM/vermiculite mix to 11% w/w water content enhanced strain survival in the mix during storage, but the drying conditions studied had no effect on inoculant survival on the seed during storage when pelleted seeds were dried to 10% w/w water content. Biological control of damping-off disease of sugarbeet (caused by Pythium spp.) in soil microcosms was achieved when F113Rif was inoculated in the unamended mix 3 d before pelleting the seeds, but not when nutrient-amended mix was used. Inoculum preparation and drying of the formulation are key factors to consider when optimizing the use of a commercial EB^TM/vermiculite seed formulation for delivery of a biocontrol Pseudomonas inoculant.     

Long-term preservation of yeast cultures by liquid-drying

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

Effect of protective solutes on leakage from and survival of immobilized Lactobacillus subjected to drying, storage and rehydration

When lactic acid bacteria are used industrially as fermentation starters it is important to obtain stable and highly viable bacterial cultures. Six strains of Lactobacillus encapsulated in Ca-alginate gel beads were investigated to determine whether dehydration, storage and rehydration may inflict injury. A negative relationship between leakage of lactate dehydrogenase and survival rates was found. Mesophilic lactobacilli showed only negligible leakage compared with thermophilic strains when dehydrated at 30 °C to a level of 0·11 g H₂0 (g dry wt)⁻¹. The choice of an appropriate suspending medium to be introduced before drying was therefore very important for thermophilic lactobacilli in order to increase the survival rates during dehydration, storage and rehydration. The osmoregulatory solutes tested were adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS). Less injury was inflected during dehydration for Lactobacillus helveticus with adonitol, glycerol and NFMS. Survival rates for the strains subjected to immobilization, dehydration, storage and rehydration varied with the strain and the protective solute when fluidized-bed drying was used at 5 °C to a level as high as 0·34 g H₂0 (g dry wt)⁻¹. Non-fat milk solids gave the best protection for thermophilic lactobacilli, while adonitol and NFMS were best for mesophilic lactobacilli.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

The synergistic preservative effects of the essential oils of sweet basil (Ocimum basilicum L.) against acid-tolerant food microflora

Essential oils extracted by hydrodistillation from five different varieties of Ocimum basilicum L. plants (Anise, Bush, Cinnamon, Dark Opal and a commercial sample of dried basil) were examined for antimicrobial activity against a wide range of foodborne Gram-positive and -negative bacteria, yeasts and moulds by an agar well diffusion method. All five essential oils of basil showed antimicrobial activity against most of the organisms tested with the exception of Flavimonas oryzihabitans and Pseudomonas species. The inhibitory effect of Anise oil, in comparison with mixtures of the predominant components of pure linalool and methyl chavicol, against the acid-tolerant organisms, Lactobacillus curvatus and Saccharomyces cerevisiae , was examined in broth by an indirect impedance method. Synergistic effects between Anise oil, low pH (pH 4·2) and salt (5% NaCl) were determined. The antimicrobial effect of Anise oil was also assessed in a tomato juice medium by direct viable count, showing that the growth of Lact. curvatus and S. cerevisiae was completely inhibited by 0·1% and 1% Anise oil, respectively. The results of the current study indicate the need for further investigations to understand the antimicrobial effects of basil oils in the presence of other food ingredients and preservation parameters.     

SURVIVAL OF BACTERIA AFTER TWENTY-ONE YEARS IN THE DRIED STATE.

Miller, Ruth E. (Woman's Medical College of Pennsylvania, Philadelphia) and Loraine A. Simons. Survival of bacteria after twenty-one years in the dried state. J. Bacteriol. 84:1111-1114. 1962.-Cultures dried in a vacuum over calcium chloride at room temperature and stored in the refrigerator at 10 C were rehydrated after 21 years in this condition. Only 13 of 202 cultures, representing 67 different species, failed to grow. Nine of these were members of four different species, several other strains of which grew on rehydration. Failure to grow may have been due to a small inoculum before drying. Of 32 dried cultures removed from vacuum and stored in the refrigerator exposed to air, 9 survived for periods varying from 37 to 43 weeks. Cultures which survived generally retained the properties they had before drying. In this study, the grampositive organisms survived drying better than gram-negative bacteria. Since the number of organisms in the original inoculum before drying was not determined, quantitative studies could not be done. It is concluded that this procedure is a very satisfactory method for the preservation of even the most delicate organisms for long periods of time.     

Inactivation mechanisms of lactic acid starter cultures preserved by drying processes

The preservation of lactic acid starter cultures by drying are of increased interest. A further improvement of cell viability is, however, still needed, and the insight into inactivation mechanisms of the cells is a prerequisite. In this present work, we review the inactivation mechanisms of lactic acid starter cultures during drying which are not yet completely understood. Inactivation is not only induced by dehydration inactivation but also by thermal- and cryo-injuries depending on the drying processes employed. The cell membrane has been reported as a major site of damage during drying or rehydration where transitions of membrane phases occur. Some drying processes, such as freeze drying or spray drying, involve subzero or very high temperatures. These physical conditions pose additional stresses to cells during the drying processes. Injuries of cells subjected to freezing temperatures may be due to the high electrolyte concentration (solution effect) or intracellular ice formation, depending on the cooling rate. High temperatures affect most essential cellular components. It is difficult to identify a critical component, although ribosomal functionality is speculated as the primary reason. The activation during storage is mainly due to membrane lipid oxidation, while the storage conditions such as temperature moisture content of the dried starter cultures are important factors.     

Drought tolerance by lentil rhizobia (Rhizobium leguminosarum) from arid and semiarid areas of Pakistan

Ten strains of lentil rhizobia (Rhizobium leguminosarum ) were evaluated for drought tolerance by exposing them to soil moisture potentials of −0·03, −1·0 and −1·5 MPa. Water availability, rhizobial strain and time of exposure to drought had a significant ( P ≤ 0·001) effect on the number of surviving rhizobia g⁻¹ of soil. Highest cell counts were observed at −0·03 MPa, followed by soil maintained at −1·0 and −1·5 MPa. Five strains originating from saline areas showed significantly ( P ≤ 0·05) better survival under low water potential after 35 days. Two strains exhibited greatest survival under low water potential and produced viable cell counts of more than 10⁷ rhizobia g⁻¹ of soil. These strains could probably be used successfully as inoculants for lentil production in arid and semi-arid environments.