Studies on the biodegradation of nonionic surfactants applied in the polyester fiber industry. I. Activated sludge bacteria degrading the surfactants.

The paper presents characteristics of 76 strains of bacteria capable of utilizing nonionic surfactants Cirrasol FP, Cirrasol SF 200 and Cirrasol TCS as the source of carbon. The strains were isolated from two activated sludges adapted to the purification of wastes containing the above compounds at concentration 150--200 mg/l. The isolated strains belonged to the genera: Achromobacter, Alcaligenes, Arthrobacter, Flavobacterium, Mycobacterium, Nocardia, Pseudomonas and Xanthomonas. With load 0.11 mg surfactant/mg d.w./day bacteria belonging to Alcaligenes were dominating. With load 0.18--0.31 mg surfactant/mg d.w./day microorganisms were dominated by Pseudomonas. The highest intensity of degradation of the studied surfactant was shown by species: Alcaligenes viscolactis, Nocardia blackwellii and Pseudomonas rathonis.     

The Decomposition of Uric Acid in Built Up Poultry Litter

The decomposition of uric acid in built up poultry litter appears to be brought about almost exclusively by the action of aerobic bacteria. Organisms decomposing uric acid usually comprised about one quarter of the bacterial population. They were strains of Corynebacterium and less frequently strains of Nocardia, Streptomyces, Pseudomonas, Alcaligenes and Achromobacter. Uric acid was converted to ammonia by some of the organisms but only to urea by the majority. Hydrolysis of urea to ammonia could be brought about by strains of Corynebacterium, Micrococcus, Alcaligenes, Achromobacter and Cytophaga which had no action on uric acid. It is suggested that the ammoniacal smell and high alkalinity of built up poultry litter result largely from the decomposition of uric acid. The identity of the bacteria concerned is discussed.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Identification of dimethyl disulfide-forming bacteria isolated from activated sludge

Twenty-four strains with high dimethyl disulfide (DMDS)-forming ability were isolated from activated sludge and identified to the genus level. These bacteria were classified into four groups (A, B, C, and D) by the API ZYM System (API System S.A., Montalieu, France). Group A (three strains) was identified as genus Lactobacillus by the API 20B System, by the method of Cowan and Steel, and by production of lactic acid as confirmed by gas-liquid chromatography. Group B (eight strains) was identified as genus Corynebacterium by API 20B and the Cowan and Steel method. Group C (one strain) was suggested to belong to genus Corynebacterium by the API 20B System. Group D (12 strains) was identified as genus Pseudomonas or Alcaligenes by the API 20B System, as genus Alcaligenes by the Cowan and Steel method, and as Achromobacter group Vd by the API 20NE System. However, on the basis of guanine-plus-cytosine contents in DNA and form of flagella, these strains were identified as genus Pseudomonas. Formation of DMDS from DL-methionine and S-methyl-L-cysteine was tested. DMDS-forming bacteria isolated from activated sludge formed DMDS from both precursors. In genus Pseudomonas, P. aeruginosa could not form DMDS from either precursor, but P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, and P. testosteroni formed DMDS. In genus Alcaligenes, A. denitrificans subsp. xylosoxydans, A. denitrificans subsp. denitrificans, A. faecalis, and A. odorans formed DMDS from both precursors. Achromobacter group Vd formed DMDS from S-methyl-L-cysteine, but could not from DL-methionine.     

THE USE OF GLUCOSE INORGANIC SALTS MEDIA IN THE CLASSIFICATION OF THE COLI-AEROGENES BACTERIA. I. THE METHYL RED AND VOGES-PROSKAUER REACTIONS

SUMMARY: A chemically defined glucose ammonium phosphate medium gave methy red and Voges-Proskauer reactions which were superior to those in glucose peptone phosphate. Optimum condition for both tests require a starting pH of 6·8 and incubation for 3 days at 30°. The medium gave a negative correlation between the two reactions with 34 out of 35 strains that were positive to both tests in the peptone medium. The addition of glutamic acid helped to eliminate doubtful positive M. R. reactions but seemed to suppress acetylmethylcarbinol production by weakly V.-P. positive strains.     

Nonfermentative gram-negative bacteria encountered in clinical specimens

The characteristics of 1192 gram-negative nonfermentative bacteria isolated from human clinical specimens and 32 reference strains representing six genera (Pseudomonas, Alcaligenes, Achromobacter, Acinetobacter, Moraxella, Flavobacterium) encountered in clinical bacteriology are presented. Salient features for their identification in the routine diagnostic laboratory are summarized.     

THE EFFECT OF THE TETRACYCLINE COMPOUNDS ON THE STORAGE LIFE AND MICROBIOLOGY OF CHILLED EVISCERATED POULTRY

SUMMARY: Chlortetracycline, oxytetracycline and tetracycline, incorporated at 10 p/m in the ice slush used for cooling, were equally efficient in extending the storage life of eviscerated poultry which was subsequently frozen, thawed and stored at 1°. There was complete correlation between the sensory assessment of 'off'odour and the total numbers of micro-organisms present when these were determined by taking bulk samples of skin from the area under the wing and of the surface tissue of the visceral cavity near the vent. Direct microscopical counts confirmed that the latter area was the most heavily contaminated part of the visceral cavity. Non-pigmented strains of Pseudomonas were the main spoilage organisms of untreated chickens but Achromobacter strains and yeasts were responsible for the spoilage of the chlortetracycline treated chickens. Fluorescence was only observed in spoiled chickens where pigmented strains of Pseudomonas occurred in significant numbers. Spoilage in both control and antibiotic treated poultry was accompanied by a rise in pH in the contaminated muscles, but for various reasons this change could not be used as a direct measure of the extent of the microbial contamination.     

Physical evidence for plasmids in autotrophic,especially hydrogen-oxidizing bacteria

Agarose gel electrophoresis of crude lysates from 23 species of autotrophic bacteria revealed plasmids of various sizes in 12 species. The plasmid pattern varied considerably. While the majority of the plasmid-bearing species harbored one or two plasmids, one species, Alcaligenes latus, exhibited more than six ccc-DNA bands. With one exception the molecular masses of the plasmids were 50×10⁶ or higher. In Achromobacter carboxydus, Alcaligenes latus, Derxia gummosa and three strains of Paracoccus denitrificans large plasmids of molecular masses higher than 300×10⁶ were resolved. The examination of Thiobacillus A2 resulted in the discovery of two plasmids while Pseudomonas oxalaticus was apparently free of resident plasmid DNA. So far these plasmids can only be characterized as cryptic. Future studies may allow to correlate them with specific metabolic activities of their hosts such as the ability to grow on carbon monoxide or thiosulfate, to fix molecular nitrogen and to form soluble NAD-reducing and/or membrane-bound hydrogenases.     

Studies of Pseudomonas aeruginosa Infections Among Hospitalized Patients

A rapid identification method of glucose nonfermentative gram-negative rods was established and 320 strains isolated were divided into five groups according to their characteristics in pigmentation, acid from glucose, cytochrome oxidase activity and motility. Further characterization of the strains in each group resulted in the identification that the strains in group I were Pseudomonas aeruginosa, strains in group II, P. aeruginosa and Pseudomonas putida. Achromogenic strains of P. aeruginosa were classified into group III, Pseudomonas maltophilia, Pseudomonas alcaligenes and Alcaligenes faecalis into group IV and Acinetobacter calcoaceticus (Acinetobacter anitratus and Achromobacter lwoffii) in group V. When fluorescent pigment production was taken as a standard, 259 out of 263 chromogenic strains were identified as P. aeruginosa and the remaining four were P. putida. Whereas forty-five achromogenic strains included twenty-four A. calcoaceticus, eight P. aeruginosa, six A. faecalis, five P. maltophilia and two P. alcaligenes. From May 1970 to June 1971, 368 strains of glucose nonfermentative rods were isolated from clinical specimens sent to the Central Laboratories of Tohoku University Hospital and three fourth (286/368) of the isolates were P. aeruginosa     

The Properties and Classification of the Predominant Bacteria in Rotten Eggs

A collection of 226 strains of bacteria was assembled from eggs which had rotted on the premises of the producer and from others which had been allowed to rot in the laboratory at 10, 20 or 30°. A majority of the eggs had a mixed infection. All but 8 of the isolates were Gram negative rods. The predominant types were Alcaligenes faecalis, Aeromonas liquefaciens, Proteus vulgaris, Cloaca spp., Citrobacter sp. and Pseudomonas fluorescens. A nonpigmented pseudomonad could not be identified with any of the species included in Pseudomonas.     

Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon.

All the genes we examined that encoded biphenyl/polychlorinated biphenyl (PCB) degradation were chromosomal, unlike many other degradation-encoding genes, which are plasmid borne. The molecular relationship of genes coding for biphenyl/PCB catabolism in various biphenyl/PCB-degrading Pseudomonas, Achromobacter, Alcaligenes, Moraxella, and Arthrobacter strains was investigated. Among 15 strains tested, 5 Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on the XhoI 7.2-kilobase fragment corresponding to that of Pseudomonas pseudoalcaligenes KF707. More importantly, the restriction profiles of these XhoI 7.2-kilobase fragments containing bphABC genes were very similar, if not identical, despite the dissimilarity of the flanking chromosomal regions. Three other strains also possessed bphABC genes homologous with those of KF707, and five other strains showed weak or no significant genetic homology with bphABC of KF707. The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well to the DNA homology. On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1, lacked genetic as well as immunological homology with any of the other 15 biphenyl/PCB degraders tested. The existence of the nearly identical chromosomal genes among various strains may suggest that a segment containing the bphABC genes has a mechanism for transferring the gene from one strain to another.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria.

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

The amino acid sequence of the blue copper protein of Alcaligenes faecalis

The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonas aeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.     

Morphological and Biochemical Differentiation of Achromobacter and Moraxella (Debord''s Tribe Mimeae)

To determine the most useful laboratory tests for the differentiation of Achromobacter anitratus, Achromobacter lwoffii, and Moraxella duplex (DeBord's tribe Mimeae), 157 strains of these bacteria, isolated from clinical specimens, were examined for their morphological and biochemical characteristics. There were several differences between these nonfermentative, gram-negative diplococci: Moraxella was nonglucolytic in either infusion base or synthetic base, oxidase-positive, and sensitive to penicillin, whereas Achromobacter produced variable carbohydrate activity, and was oxidase-negative and resistant to penicillin. A. anitratus was distinguished from A. lwoffii in that the former utilized infusion media containing either glucose or 10% lactose, whereas the latter did not. Both species utilized the same carbohydrates in a chemically defined medium, although the latter acted more sluggishly.     

The crystal structure of pseudoazurin from Alcaligenes faecalis S-6 determined at 2.9 A resolution

The three-dimensional structure of pseudoazurin, a single copper-containing protein from Alcaligenes faecalis strain S-6, has been determined at 2.9 A resolution by X-ray crystallography. The sequences of two other pseudoazurins from Pseudomonas AM1 and Achromobacter cycloclastes may also be accommodated in this structure. The structure, an eight-stranded beta-barrel, resembles closely those of plastocyanin and azurin. It possesses two extra alpha-helices at the C-terminus, whereas azurins have an alpha-helical flap in the middle of their sequences.     

The Effect of Freezing on the Radiation Sensitivity of Vegetative Bacteria

S ummary : Five strains of bacteria were irradiated, suspended in heart infusion broth or in phosphate buffer, in aerated or anoxic conditions, at temperatures of 10–13° or -79°. Survivors under the different conditions were enumerated by plate counts on heart infusion agar.
Exponential survivor-dose curves were obtained with a Pseudomonas strain and with Escherichia coli B/r when irradiated at room temperature with aeration, whereas an Alcaligenes strain and 2 strains of Streptococcus faecium gave sigmoid curves. The decreased radiosensitivity in the frozen state was measured by comparing the D₁₀ values for exponential curves, or for the exponential portion of sigmoid curves, with that observed for irradiation at room temperature with aeration. This 'D₁₀ ratio' svaried between 2°5 and 8·5. For the Alcaligenes strain it was about 4, whether the frozen irradiation took place in the presence or absence of oxygen. With the Pseudomonas irradiated in the frozen state in the absence of oxygen the 'D₁₀ ratio' was usually about 1·5 times higher than when oxygen was present. The highest ratio (8·5) was obtained for anoxic irradiation of the Pseudomonas strain.
In general, the shapes of survival curves for frozen irradiation differed from those obtained at room temperature. The sigmoid curves for the Alcaligenes strain irradiated under aerobic conditions when frozen showed a marked decrease in extrapolation numbers. E. coli B/r when frozen in heart infusion broth gave a double exponential curve with a shallow slope initially, followed by a steeper slope. The most radiation resistant strain, Strep. faecium R53, gave sigmoid curves with a D₁₀ value of 300 Krads when frozen, and was then of similar resistance to Clostridium botulinum spores.     

Enrichment of linear alkylbenzenesulphonate (LAS) degrading bacteria in continuous culture

Enrichment experiments were carried out in continuous-flow units using a mineral medium with commercial linear alkylbenzenesulphonate (LAS) as the limiting carbon- and energy-source. The mixed bacterial culture originating from the waste water of a detergent plant consisted of five strains belonging to the genus Pseudomonas and two strains each of the genera Achromobacter and Acinetobacter. The cultivation conditions corresponding to dilution rates of 0.025-0.1 h^-1 and LAS concentrations of 20–50 mg/1 were examined. During the experiments the composition of mixed cultures and the kinetics of LAS biodegradation were followed. Continuous-flow enrichment experiments resulted in the selection of six bacterial cultures with different compositions of individual species and capability to utilize LAS. From the original seven strains at lower dilution rates (0.025 and 0.05 h^-1) six were selected, excluding Pseudomonas sp. 3, while at the highest dilution rate (0.1/h^-1) five strains were selected after eliminating Pseudomonas sp. 5 and Achromobacter sp. 1. All enriched mixed cultures were more efficient in primary than in ultimate LAS degradation. Two of the culture strains were able to achieve primary LAS degradation ( Pseudomonas sp. 1 in mineral medium with LAS as the sole carbon- and energy-source and Acinetobacter sp. 3 in medium supplemented by yeast extract and nutrient broth).
None of the strains could degrade LAS completely, which indicates that many types of interactions based on combined metabolic attack as well as those based on provision of specific nutrients, may exist between culture members during the complete LAS bio-oxidation.