Occurrence and Thermoresistance of Spores of Psychrophilic and Psychrotrophic Aerobic Sporeformers in Soil and Foods

Spores of psychrotrophic (able to grow at 5°C) aerobic sporeformers occurred in soil in high numbers (2 × 10³-5 × 10⁶/g), whereas psychrophilic (able to grow at 0°C) spores were present at significantly lower levels (500–10⁵/g). Psychrotrophic spores were absent in herbs and spices: in pasteurized meals prepared industrially their numbers varied from <10 to 1000/g. For spores harvested from Trypticase Soy Agar (TSA), the heat resistance of the cold-tolerant sporeformers was low with D _90°C-values from 1–11 min. The recovery of heated psychrophilic spores on this medium at 5°C was equal to their recovery at 20°C. However, the recovery of heated psychrotrophic spores was lower at 5°C than at 20°C, whereas unheated spores gave the same counts at both temperatures. The heat resistance of naturally occurring spores of cold-tolerant sporeformers washed from soil was comparable with the resistance of spores formed on TSA.     

Difference in sporogenous bacterial populations in thermophilic (55 degrees C) and mesophilic (35 degrees C) anaerobic sewage digestion

Spores, sporeforming vegetative cells, and asporogenous populations were enumerated in two semicontinuous anaerobic fermentors digesting municipal primary sludge at 35 and 55 degrees C for more than 87 days. In the 35 degrees C fermentor, the anaerobic total population was 312.5 X 10(6)/ml, with 25.0 X 10(6)/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 23.1 X 10(6), 59.2 X 10(6), 26.2 X 10(6), and 7.3 X 10(6)/ml, respectively, with 2.8 X 10(6), 6.7 X 10(6), 3.4 X 10(6), and 1.5 X 10(6)/ml being sporogenous, respectively. The sporeformers accounted for 8.0 to 20.0% of each of the respective populations. In the 55 degrees C fermentor, the anaerobic total population was 512.5 X 10(6)/ml, with 336.6 X 10(6)/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 97.7 X 10(6), 190.7 X 10(6), 75.8 X 10(6), and 11.2 X 10(6)/ml, respectively, with 47.8 X 10(6), 110.6 X 10(6), 43.3 X 10(6), and 5.1 X 10(6)/ml, respectively, being sporogenous. The sporeformers represented 45.5 to 65.7% of each of the respective populations. The numbers of thermophilic sporeforming vegetative cells in the 55 degrees C fermentor were 9.0 to 19.8 times higher than their counterparts in the 35 degrees C fermentor. Most sporeformers were in the vegetative state in the 35 and 55 degrees C fermentors. After 18 days of fermentation at 55 degrees C, sporeformers carried out most of the digestion; however, the digestion was shared by both sporeformers and asporogenous bacteria after 87 days of fermentation. In the 35 degrees C fermentor, asporogenous bacteria digested most of the sludge. During the 18- and 87-day experimental periods, sporeformers were never predominant.     

Difference in sporogenous bacterial populations in thermophilic (55 degrees C) and mesophilic (35 degrees C) anaerobic sewage digestion.

Spores, sporeforming vegetative cells, and asporogenous populations were enumerated in two semicontinuous anaerobic fermentors digesting municipal primary sludge at 35 and 55 degrees C for more than 87 days. In the 35 degrees C fermentor, the anaerobic total population was 312.5 X 10(6)/ml, with 25.0 X 10(6)/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 23.1 X 10(6), 59.2 X 10(6), 26.2 X 10(6), and 7.3 X 10(6)/ml, respectively, with 2.8 X 10(6), 6.7 X 10(6), 3.4 X 10(6), and 1.5 X 10(6)/ml being sporogenous, respectively. The sporeformers accounted for 8.0 to 20.0% of each of the respective populations. In the 55 degrees C fermentor, the anaerobic total population was 512.5 X 10(6)/ml, with 336.6 X 10(6)/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 97.7 X 10(6), 190.7 X 10(6), 75.8 X 10(6), and 11.2 X 10(6)/ml, respectively, with 47.8 X 10(6), 110.6 X 10(6), 43.3 X 10(6), and 5.1 X 10(6)/ml, respectively, being sporogenous. The sporeformers represented 45.5 to 65.7% of each of the respective populations. The numbers of thermophilic sporeforming vegetative cells in the 55 degrees C fermentor were 9.0 to 19.8 times higher than their counterparts in the 35 degrees C fermentor. Most sporeformers were in the vegetative state in the 35 and 55 degrees C fermentors. After 18 days of fermentation at 55 degrees C, sporeformers carried out most of the digestion; however, the digestion was shared by both sporeformers and asporogenous bacteria after 87 days of fermentation. In the 35 degrees C fermentor, asporogenous bacteria digested most of the sludge. During the 18- and 87-day experimental periods, sporeformers were never predominant.     

Growth temperature preferences of fungal strains from Victoria Land,Antarctica

Thirty-five strains of microfungi, isolated from various sites in Victoria Land, Antarctica, were grown at eight temperatures ranging from 0 to 45°C. Only 1 strain (Chaetomium sp. from hot soil) was a thermotolerant mesophile; other strains were psychrophilic (2 strains) or psychrotrophic (32 strains). The fitness of different species to thermal instability is discussed, based on the width of the growth rate curves.     

Dry-heat resistance of selected psychrophiles.

The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.     

Dry-heat resistance of selected psychrophiles.

The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.     

Incidence of Mesophilic Clostrodium Spores in Raw Pork, Beef, and Chicken in Processing Plants in the United States and Canada

The anaerobic film pouch technique was used to quantitate and isolate clostridial spores in 2,358 samples of raw meat (1,078 of chicken, 624 of beef, 656 of pork). Of 19,727 putrefactive anaerobic (PA) sporeformers isolated, 1 was confirmed by mouse protection testing to be Clostridium botulinum type C. This isolate was obtained from a Western Canada chicken sample which contained 5.33 clostridia per gram. These data indicate a very low incidence of botulinal contamination in raw meats at the packing-plant level (0.042% of 2,358 samples) and an almost 20,000:1 ratio of nonbotulinal PA sporeformers to mesophilic C. botulinum spores. The mean level of PA contamination was 2.8 PA sporeformers per gram of meat; 77% of the samples contained three or less PA sporeformers per gram. Small but statistically significant differences in the incidence of clostridial spores were noted for season, geographical region, and type of meat.     

Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens

A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 X 10-2 to 3.3 X 10-6 per g. The organisms most frequently isolated were Clostridium subterminate, C. sordelii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridium. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content.     

The core proteins of 35S hnRNP complexes. Characterization of nine different species

Ribonucleoprotein complexes (hnRNP) containing fragments of heterogeneous nuclear (hn)RNA and sedimenting at 35-40 S were isolated from the nuclei of HeLa S3 cells using the pH 8.0/diffusion technique. These hnRNP complexes are thought to be part of the hnRNA processing apparatus. The major protein components (core proteins) were identified by their constant ratios in native particles and in 35S hnRNP particles reconstituted in vitro. All of the core proteins, with one exception, show an increase in Mr on sodium dodecylsulfate (NaDodSO4)/polyacrylamide gels containing 8 M urea, indicative of secondary structure elements resistant to denaturation by NaDodSO4. The nine core proteins found by us are: A1 [Mr(NaDodSO4) 31 X 10(3)/Mr (urea) 38 X 10(3), apparent isoelectric point, pIapp 9.3], A2 (32.5 X 10(3)/39 X 10(3), 8.4), B1a (35.5 X 10(3)/41 X 10(3), 8.8), B1b (35.5 X 10(3)/44 X 10(3), 8.3), B1c (35.5 X 10(3)/43 X 10(3), 5.7) B2 (37 X 10(3)/42 X 10(3), 9.15), C1 (39 X 10(3)/46 X 10(3), 9.2), C2 (40.5 X 10(3)/45 X 10(3), 5.55) and C3 (38.5 X 10(3)/37 X 10(3), 4.8). Individual proteins were electroeluted from two-dimensional gels and their amino acid composition determined. Difference indices were calculated and show a group of closely related basic proteins (A1, A2, B1a, B1b, B2, C1), two related slightly acidic proteins (B1c, C2) and a distinct acidic member (C3). Two-dimensional analysis of tryptic fragments and one-dimensional separation of peptides after V8 protease treatment support these data. Peptide mapping of the proteins A1 and A2 from bovine and human cells yields identical fragments indicating a high degree of cross-species conservation. An additional protein (D4: 44 X 10(3)/55 X 10(3), greater than 9.5) was found, which preferentially associates with heavier, oligomeric hnRNP structures. Only traces of actin are present in the 35S hnRNP fraction. All core proteins are modified by charge. A large part of the charge isomers arises by phosphorylation, which has been shown by labeling with 32PO4 in vivo and with [gamma-32P]ATP in vitro. In vitro the phosphate transfer is mediated by an endogenous protein kinase associated with the 35S hnRNP complexes. The major core protein A1 exists in two conformeric forms (A1 and A1x) of which only A1x serves as phosphate acceptor in vivo.     

Selective Process for Efficient Isolation of Soil Bacillus spp

We were able to isolate Bacillus thuringiensis from environmental samples with a background of 10 bacteria per g of soil. Our selection process differed significantly from classical selection methods which permit only the desired organism to grow. In our process, germination of B. thuringiensis spores was selectively inhibited by sodium acetate, while most of the undesired sporeformers germinated. Next, all of the nonsporulated microbes were eliminated by heat treatment at 80 degrees C for 3 min. The surviving spores were then plated on a rich agar medium and allowed to grow until they sporulated. Of random colonies picked from agar, 20 to 96% were crystal-forming Bacillus species. B. thuringiensis and B. sphaericus were routinely selected by this method.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes' milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 X 10(2) Enterobacteriaceae/ml, 3.9 X 10(2) coliforms/ml and 2.0 X 10(2) faecal coliforms/ml, whereas the respective mean counts were 6.2 X 10(3)/ml, 5.4 X 10(3)/ml and 1.3 X 10(3)/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Effects of temperature on the growth of psychrophilic bacteria from glaciers.

Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.     

Isolation and characterization of psychrophilic yeasts producing cold-adapted pectinolytic enzymes

AIMS: The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold-active pectinolytic enzymes produced by the isolated psychrophilic yeasts. METHODS AND RESULTS: Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic-psychrophilic yeast (PPY) strains PPY-3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY-3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY-5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5 degrees C, and showed the activities of several cold-active pectinolytic enzymes. CONCLUSION: The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. Significance and Impact of the Study: It is possible that the cold-active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5 degrees C.     

Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater.

Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.     

Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater.

Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.     

Nocardia in soils of southeastern Spain: abundance, distribution, and chemical characterisation

Actinomycetes belonging to the genus Nocardia were isolated from the surface horizon of 15 out of 46 soil samples examined. All the nocardiae strains isolated contained mycolic acids and saturated and unsaturated straight chain fatty acids (from 12 to 18 carbon atoms) and tuberculostearic acid and were biochemically identified as members of the Nocardia asteroides complex. Nocardiae were detected in alluvial, brown, and serosem great soil groups, but not in calcic brown, solontchack, and regosol great soil groups. Numbers of nocardiae isolated varied from 5.12 X 10(2) to 1.21 X 10(4) colony-forming units per gram of dry weight, and they were statistically correlated with the carbon content (percent C) of the soil. Soil samples were, in general, very dry.     

Occurrence, growth, and suppression of salmonellae in composted sewage sludge

Composted sewage sludge may be used to improve soil quality, but there remains some doubt concerning the microbiological safety of the product. Sewage sludge composts from 30 municipalities were sampled, and four samples (12%) contained salmonellae (two contained fewer than 0.3/g, and the other two had 21/g and 1.7 X 10(4)/g). All 30 composts were inoculated with salmonellae; the populations decreased at a specific death rate of about 0.15 h-1 over 24 h at 36 degrees C. In irradiation-sterilized composts inoculated with salmonellae, the salmonellae grew at a rate of 0.65 doublings per h for over 24 h. Growth and death rates were found to be moisture and flora associated. The growth or death rates for antibiotic-resistant salmonellae were not different from those of nonresistant strains. It was concluded that the active indigenous flora of compost establishes a homeostatic barrier to colonization by salmonellae, and in the absence of competing flora, reinoculated salmonellae may grow to potentially hazardous densities. The active microflora of moist composts eliminated contaminating salmonellae (10(5)/g) after 6 weeks.     

Occurrence, growth, and suppression of salmonellae in composted sewage sludge.

Composted sewage sludge may be used to improve soil quality, but there remains some doubt concerning the microbiological safety of the product. Sewage sludge composts from 30 municipalities were sampled, and four samples (12%) contained salmonellae (two contained fewer than 0.3/g, and the other two had 21/g and 1.7 X 10(4)/g). All 30 composts were inoculated with salmonellae; the populations decreased at a specific death rate of about 0.15 h-1 over 24 h at 36 degrees C. In irradiation-sterilized composts inoculated with salmonellae, the salmonellae grew at a rate of 0.65 doublings per h for over 24 h. Growth and death rates were found to be moisture and flora associated. The growth or death rates for antibiotic-resistant salmonellae were not different from those of nonresistant strains. It was concluded that the active indigenous flora of compost establishes a homeostatic barrier to colonization by salmonellae, and in the absence of competing flora, reinoculated salmonellae may grow to potentially hazardous densities. The active microflora of moist composts eliminated contaminating salmonellae (10(5)/g) after 6 weeks.     

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)