Antibacterial polyphenols from olive oil mill waste waters

Olive oil vegetation waters (VW) were highly toxic to both phytopathogenic Pseudomonas syringae (Smith, Yung et al. ) pv. savastanoi (Gram-negative) and Corynebacterium michiganense (Gram-positive) and showed bactericidal activity in their original concentration (in raw form). Among the main polyphenols, present in the waste waters, methylcatechol proved to be the most toxic to Ps. savastanoi at 10⁻⁴ mol 1⁻¹, and also demonstrated bactericidal activity, while on Coryne. michiganense it was only slightly active; catechol and hydroxytyrosol were less active on Ps. savastanoi , but inactive on Coryne. michiganense ; tyrosol and its synthetic isomers 1,2- and 1,3-tyrosol were completely inactive on both bacteria. Among the derivatives of VW polyphenols considered, acetylcatechol and guaiacol were selectively toxic for Ps. savastanoi , while o -quinone was strongly toxic for both bacteria. The minor carboxylic polyphenols of VW at 10⁻⁴ mol 1⁻¹ were all inactive on the bacteria. VW, catechol, 4-methylcatechol and the less abundant carboxylic polyphenols proved to be toxic on Hep2 human cells. Finally the possibility of using the active polyphenols in agriculture in an integrated pest management program for the protection of the olive plant is discussed.     

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

Evaluation of a countercurrent biosorption system for the removal of lead and copper from aqueous solutions

Abstract: An automated bench-scale countercurrent biosorption system (CBS) has been designed for the removal of metals from aqueous effluents. The system has been tested with activated sludge microorganisms as a biosorbent and lead and copper as model metals. Nearly 5 1 of a lead nitrate solution at 100 mg l⁻¹ of lead have been treated down to a final concentration of 0.1 mg l⁻¹ (99.9% removal) by using 4.8 g of dry biosorbent. Under similar conditions, copper chloride solutions at 100 mg 1⁻¹ of copper were treated down to a final concentration of 35–45 mg l^−l representing 60% removal. The advantage of the CBS is to maximize metal concentration in the biosorbent, from which the metal may thereby be recovered if desired. In addition, the CBS minimizes metal concentration in the treated effluent, which is the first objective of the treatment.     

Determination of dissolved carbon dioxide by coulometric titration in modified atmosphere systems

The solubility of carbon dioxide (CO₂) in microbiological media at different pH values, water activities ( a_w ), temperatures, buffering capacities and ratios of headspace to media volumes was determined by using a coulometer. Buffering capacity and ratio of headspace to media volume were shown to be the major factors influencing the solubility of CO₂ in modified atmosphere model systems. The growth inhibitory effects of different dissolved CO₂ concentrations (0–50 μmol ml^-1) were determined for Pseudomonas fragi at 8°C and 22 C. Pseudomonas fragi was shown to be strongly affected by the CO₂ concentration in the media. A carbon dioxide concentration of 40 μmol ml^-1 was needed to inhibit Ps. fragi at 8°C. The importance of measuring dissolved CO₂ concentrations in modified atmosphere packaging applications was shown and the coulometer proved to be an excellent tool for this purpose.     

Isolation of a novel pentachlorophenol-degrading bacterium, Pseudomonas sp. Bu34

A pentachlorophenol (PCP)-degrading bacterium was isolated from possible PCP-contaminated soil from Pusan, Korea and identified as a member of the genus Pseudomonas. It used PCP as its sole source of carbon and energy. This micro-organism was capable of degrading PCP more effectively, certified by the increase in cell density and the decrease in PCP substrate. Pseudomonas sp. Bu34 was able to degrade a much higher concentration of PCP (4000 mg l⁻¹) than any previously reported PCP-degrading bacteria and fungi and to grow in mineral salts solution containing one of a variety of chlorophenols. In non-acclimated strain Bu34, the cell number decreased from 87 to 99·9% in 75–4000 mg l⁻¹ PCP at 24 h. In the acclimated strain the PCP toxic effect did not appear with 75 mg l⁻¹ PCP treatment, but 1000–4000 mg l⁻¹ PCP decreased the cell number of strain Bu34 by 25% to 24 h and then the cell number slightly increased at 48 h. Therefore, it suggested that the maximum resistance of acclimated strain Bu34 to PCP was 4000 mg l⁻¹ PCP. We suggest that strain Bu34 could be used as a micro-organism for the bioremediation of highly PCP-contaminated soils, water or wood products.     

Effect of High Concentrations of Carbon Dioxide on Growth Rate of Pseudomonas fragi, Bacillus cereus and Streptococcus cremoris

The maximum specific growth rates of Pseudomonas fragi, Bacillus cereus and Streptococcus cremoris were studied over a wide range of carbon dioxide concentrations. The growth rate compared with a control was reduced to 50% in Ps. fragi at 0–5 atm CO₂, in B. cereus at 1—3 atm and in Strep, cremoris at 8–6 atm. B. cereus and Strep, cremoris were completely inhibited at 3 and 11 atm CO₂, respectively. The growth rate of the aerobic Ps. fragi at 0–99 atm CO₂ (0–01 atm oxygen) was reduced to about 20% of that in air. The growth rate of Ps. fragi was decreased at oxygen concentrations lower than 0–01 atm.
When Ps. fragi was grown at oxygen limitation (0.0025 atm oxygen) and exposed to 0.99 atm CO₂, the inhibiting effect of the CO₂ was added to that of the oxygen limitation. No indications of a synergistic effect between CO₂ inhibition and oxygen limitation were noted.
B. cereus and Strep, cremoris were tested under anaerobic conditions.     

The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes , Flavimonas oryzihabitans , and three species of Pseudomonas . The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0·125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10³ to 10⁶ cfu ml⁻¹. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens . The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0·1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0·1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.     

Application of bioluminescence-based microbial biosensors to the ecotoxicity assessment of organotins

The toxicity of two common organotin pollutants and their initial breakdown products (tributyltin, dibutyltin, triphenyltin and diphenyltin) were assessed using two different bioluminescent microbial biosensors: Microtox and lux -modified Pseudomonas fluorescens pUCD 607. The organotins were made up as standards, and tested both in double-deionized water and in extracted soil solution, the latter representing a realistic matrix for terrestrial contamination. Microtox was especially sensitive to the organotins, with 50% effective concentration (EC₅₀) values (15 min) for tributyltin as low as 21·9 μg l⁻¹ in pure water, and 0·118 μg l⁻¹ in soil extract. The sensitivity of Microtox was increased by an order of magnitude in soil extract. The Ps. fluorescens was less sensitive, with EC₅₀ values (30 min) of around 800 μg l⁻¹ in pure water. The toxicity to Ps. fluorescens was decreased by around an order of magnitude in soil extract. The two biosensors showed different response patterns, with Microtox being more sensitive to the triorganotins and Ps. fluorescens being more sensitive to the diorganotins.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

Fish production in the Jarama River, Central Spain

Fish production was estimated at three sites on the Jarama River, a small, typical upland river in Central Spain. Population estimates were made by the successive removal method of electrofishing. The same six species were recorded at each of the sites: Chondrostoma polylepis, Barbus barbus bocagei, Leuciscus cephalus pyraenaicus, Salmo trutta m. fario, Gobio gobio and Cobitis paludicola , with the first three species always dominant. Density, biomass, production (assuming that N_o is the total number of eggs spawned), and available production were, respectively: 13502-85776 ind. ha⁻¹, 178.6–221.3 kg ha⁻¹, 221.7–583.6 kg ha⁻¹ yr⁻¹, 118.1–271.9 kg ha⁻¹ yr⁻¹. Production estimates based on mortality curves were 7.9–19.5% (mean: 13.7) lower than those based on estimated from the number of eggs laid. Production per unit of area was highest at the widest and deepest site. Brown trout production contributed only 2–4% of the total production for all sites.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.     

Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India

Phenol is an industrially important compound which has a wide range of applications. Being highly soluble in water, it appears as the major pollutant in waste waters arising from both phenol manufacturing and from industrial units that utilise phenol. Because of its toxicity, bioremediation of phenol is necessary. Since some of the phenol-bearing industrial waste waters are alkaline in nature, use of alkaliphilic bacteria for bioremediation of phenol was investigated. Alkaliphilic bacteria were isolated from sediments of an alkaline lake in Lonar, Dist. Buldhana, Maharashtra State, India, by phenol enrichment at pH 10.0 and phenol concentration of 500 mg/l. The lake (lat. 19°58'45", long. 76°34') is known to be a unique inland saline lake in Asia. It has a circular periphery and diameter of 2 km around the top of the banks and 1.2 km at the bottom. The lake has a high saline level (～ 2649 mg/l sodium chloride) and a high level of alkalinity (～ 2605 mg/l calcium carbonate). Alkaliphilic strains of Arthrobacter spp., Bacillus cereus, Citrobacter freundii, Micrococcus agilis and Pseudomonas putida biovar B were capable of removing phenol from waste waters arising from industries manufacturing methyl violet (using phenol as one of the major raw materials) and cumene-phenol. The waste waters from both these units were alkaline in nature (pH ～ 9.95-10.1) and had a high phenol content (368-660 mg/l). The alkaliphilic bacteria being studied removed 100% of the phenol from the industrial waste waters within 48 h of incubation under shake culture conditions and at an ambient temperature of 28 ± 2 °C. Bioremediation of phenol by alkaliphilic strains of Arthrobacter spp., B. cereus, C. freundii and M. agilis seems to be the first report.     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: numbers of the bacterium and symptom development on mushrooms grown in various environments after artificial inoculation

The recovery of Pseudomonas tolaasii applied to peat, limestone and mushroom caps, is very difficult, recovery rates being 0.2–16.0%. Without Agaricus bisporus mycelium, inoculated Ps.tolaasii disappears in the casing layer. As mushroom primordia grew in size on inoculated mushroom beds, the number of detectable cells of the pathogen increased. Symptoms of blotch disease became visible when 5.4 times 10⁶ cfu were detectable, when the mushroom primordia were 6 mm in diameter; 60% of mushrooms showed symptoms before they were 15 mm in diameter. Application of Ps.tolaasii cells as low as 20 cfu/cm² of bed gave epidemics of this severity. Neither size nor age of mushrooms affects their susceptibility. When Ps.tolaasii was placed directly onto caps, 6 times 10⁷ cfu were necessary to produce a blotch lesion (though only 3.5 times 10⁶ cfu could be recovered). Changes in r.h. and temperature did not affect the numbers of cells of Ps.tolaasii on inoculated caps; very frequent watering did so. Increased severity of the disease was seen only on over-watered mushrooms; this occurred by increase in the size of lesions seen at the primordium stage. The number of cells of Ps.tolaasii present on the early primordial stages of mushroom growth controls the extent of blotch disease seen at harvesting, whereas variations in r.h. or temperature during growing do not do so. An illustrated disease symptom measurement key (of general application for assessing severity of blotch disease) is included in the text.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Direct induction of homozygous diploidization in the fission yeast Schizosaccharomyces pombe by pressure stress

Abstract Hydrostatic pressure stress and a dye plate method were first used to investigate the direct induction of homozygous diploids from the haploid yeast Schizosaccharomyces pombe . Above 100 MPa at 25 °C for 10 min, pressure stress greatly inactivated the haploid strains of JY1 (L972 h ⁻) JY3 (L975 h ⁹⁰) and JY334 ( ade 6-M216 leul h ⁺). At the same time, when pressure stressed cells of these strains at more than 100–200 MPa were spread on a dye plate, some pressure-effected visible colonies were stained violet (variant colonies); the rest were stained pink, similar to colonies originating from haploid cells that were not pressure-stressed. Based on the cell size, DNA content, crosses, and random spore analyses for the segregation of mating types or auxotrophic markers, variant cells originating from color changed colonies of JY1 after pressure stress were very stable and found to be homozygous diploid with an h− / h− genotype at the mating-type locus. From these results we conclude that pressure stress in combination with a dye plate is a simple and useful method for direct induction of homozygous diploid cells with very high stability.     

Studies on the Production of Extracellular Proteinases by a Non-pigmented Strain of Chromobacterium lividum Isolated from Abattoir Effluent

A highly proteolytic bacterium isolated from abattoir effluent was identified as a non-pigmented strain of Chromobacterium lividum. Ferrous or ferric ions at concentrations between 1·8 × 10^-5 and 9 × 10^-4 g ions/1, which is 2–3 orders of magnitude greater than that required for growth, were essential for extracellular proteinase production in aerated but not in static culture. Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ ions could not replace iron. Four proteinases (I-IV) were produced in static culture, but only proteinase I was formed in significant quantities in aerated culture. With both forms of culture amino nitrogen was essential for proteinase production; glucose inhibited formation in aerated, but not static, cultures. Growth occurred over the range 1–33 °C, whereas proteinase production ceased at 27 °C, with maximum activity at 13 °C. Proteinase production appeared to be controlled by an interaction between iron, oxygen tension and glucose.     

NEW GENERA OF FRESHWATER CRYPTOMONADS FROM COLORADO

Given their rapid growth and nutrient assimilation rates, Porphyra spp. are good candidates for bioremediation. The production potential of two northeast U.S. Porphyra species currently in culture ( P. purpurea and P. umbilicalis ) was evaluated by measuring rates of photosynthesis (as O₂ evolution) of samples grown at 20° C. Gametophytes of P. umbilicalis photosynthesized at rates that were 80% higher than those of P. purpurea over 5–20° C at both sub-saturating and saturating irradiances (37 and 289 μmol photons m⁻² s⁻¹). Porphyra umbilicalis was both more efficient at low irradiances (higher alpha) and had a higher P_max than did P. purpurea (23.0 vs. 15.6 μmol O₂ g⁻¹ DW min⁻¹), suggesting that P. umbilicalis is a better choice for mass culture where self-shading may be severe. The photosynthesis-irradiance relationship for the Conchocelis stage of P. purpurea was also examined. Tufts of filaments, grown at 10, 15, and 20° C, were assayed at growth temperatures at irradiances ranging from 0–315 μmol photons m⁻² s⁻¹. Tufts were slightly more productive at 15° than at 10° C, but only ca. 4–6% as productive as gametophytes. Maximum rates of net photosynthesis were reduced by 66–74% in tufts grown at 20° C (only about 2% of gametophytes). The Conchocelis stage, however, need not limit mariculture operations; once Conchocelis cultures are established, they can be maintained over the long-term as ready sources of spores for net seeding.     

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium

Aims:  To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633.
Methods and Results:  Pseudomonas putida were grown with TTAB in the presence or absence of AlCl₃, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl₃, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl₃, indicating that the PC level is dependent upon activation of this enzyme.
Conclusions:  The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al³⁺ and should be viewed as a temporary reservoir of available Al³⁺ to allow a more efficient utilization of TTAB by Ps. putida .
Significance and Impact of the Study:  The changes in the PL of Ps. putida in the presence of TTAB and AlCl₃ indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.     

Bacteriological stability and growth kinetics of Pseudomonas aeruginosa in bottled water

Bacteriological stability of water bottled in plastic containers and the growth kinetics of Pseudomonas aeruginosa were determined. Samples of water from the source, water to be bottled, finished product and sterile water bottled in non-returnable and returnable containers were analysed for aerobic colony count, coliforms, Escherichia coli and Ps. aeruginosa. The samples were examined for up to 30 d storage. Aerobic colony count increased 6 d after bottling to between 10³ and 10⁵ cfu ml⁻¹. Coliforms and E. coli were not found in any sample. Pseudomonas aeruginosa was isolated from commercial products bottled in returnable plastic containers due to the contamination from the containers and the subsequent multiplication utilizing trace nutrients. The predominant Ps. aeruginosa strains showed high doubling time (26 h) due to competition from the accompanying flora. In the absence of competing flora Ps. aeruginosa reached higher density than the maximum reached by aerobic flora, with a doubling time of only 3·6 h. After 30 d storage, this micro-organism was predominant.