Sphingobacterium Multivorum Research Articles

Evaluation of tomato seedling root-associated bacterial endophytes towards organic seedling production

The study was taken up with the objective of evaluating the bacterial endophytes isolated from tomato towards seedling growth promotion and organic seedling production. Five isolates from tomato cv. Arka Vikas (Pseudomonas oleovorans, Stenotrophomonas maltophilia, Bacillus pumilus, Agrobacterium tumefaciens, and Microbacterium pumilum) and nine isolates from cv. Arka Abha (P. oleovorans, Pseudomonas plecoglossicida, Pantoea ananatis, Citrobacter freundii, Staphylococcus hominis, Sphingobacterium multivorum, Enterobacter cloacae, Arthrobacter globiformis, and A. tumefaciens) isolated from the root tissue of 4-week-old seedlings were tested for the plant growth promotion (PGP) characteristics and the effect on seedling growth. Most isolates showed ammonia and indole production, while five isolates (one from Arka Vikas and four from Arka Abha) displayed phosphate solubilization capability. Following seed fortification (soaking seeds in 0.1 OD600nm bacterial suspension for 1 h), several isolates displayed improved seed germination and/or higher seedling vigor in paper towel and glasshouse trials. Two isolates of P. oleovorans (Tm-Av01 and Tm-Ab01 from Arka Vikas and Arka Abha, respectively) and one A. tumefaciens isolate (Tm-Ab09) from Arka Abha showed higher seedling vigor index over control in both cultivars. The two isolates Tm-Av01 and Tm-Ab01 (P. oleovorans) which formed the predominant natural endophyte in the respective hosts with the additional feature of high antagonistic activity against the bacterial wilt pathogen, Ralstonia solanacearum, have been identified as promising endophytes towards seedling growth enhancement and organic seedling production across both the cultivars.

Variedad bacteriana en cultivos piscícolas y su resistencia a antibacterianos

Para determinar la variedad y resistencia frente a antibacterianos, durante un periodo productivo en el cual no se utilizaron antibióticos, en dos piscícolas del departamento del Meta, se obtuvieron muestras de agua y sedimento de donde se aislaron bacterias pertenecientes al grupo de heterótrofas Gram negativas. Las muestras se sembraron en medios selectivos Agar TCBS, McConkey, Citófaga, Cetrimide y Plate Count Agar para hacer recuentos bacterianos con las Técnicas de Análisis Microbiológico de la ICMSF y el Standart Methods. Las colonias diferentes de cada medio se repicaron para identificación bioquímica con kit comercial BBL-cristal®, y para el estudio de resistencia a seis antibacterianos se empleó el procedimiento descrito por Kirby-Bauer. La multi-resistencia, se estimó con el índice resistencia múltiple a antibióticos (MAR), y el índice de abundancia relativa (IAR) se calculó de acuerdo con Latorre (2007). Se identificaron 19 cepas bacterianas, seis correspondieron a Klebsiella pneumoniae, cinco a Enterobacter sp y cloacae, y las demás a Pseudomonas fluorescens y aerouginosa, Chryseobacterium sp. y gleum, Sphingobacterium multivorum, Serratia marcescens y Tetrathiobacter mimigardefordensis. Todas las cepas aisladas fueron resistentes a Eritromicina y el mayor índice de multi- resistencia lo presentó Enterobacter cloacae con 1.00 y 0.8. El mayor IAR correspondió a enterobacterias (≥800ufc/g). El encontrar seis cepas de Klebsiella pneumonidae y cinco de Enterobacter spcondiferentes índices de resistencia en los dos ambientes analizados, indica la gran variabilidad de estas especies. La resistencia de todos los microorganismos a Eritromicina, y el alto IMR de Enterobacter, demuestran que el uso de antibióticos en acuicultura, sumado a la alta concentración de enterobacterias puede tener efectos ambientales adversos que podrían repercutir en la salud pública.

Preparation of petroleum-degrading bacterial agent and its application in remediation of contaminated soil in Shengli Oil Field, China

Two petroleum-degrading strains were screened from oil fields and denoted as SWH-1 (Bacillus subtilis) and SWH-2 (Sphingobacterium multivorum), which were used to ferment and prepare bacterial agent to remediate petroleum-contaminated sites in Shengli Oil Field in China. The optimal liquid fermentation medium and conditions were MgSO₄·7H₂O (0.5%), NaCl (0.5%), soybean dregs (3%), pH 7.0, culturing at 30 °C, and 220 r/min for 16 h. Peat was chosen as the bacterial carrier due to its ability of keeping microbial activity. Mixed fermented liquid was added into peat (1:2) and air-dried, and the bacterial agent was obtained. It was applied to the petroleum-contaminated soil, which was irrigated, tilled, and fertilized. The removal rate reached 67.7% after 2 months of remediation. During remediation, the quantity of indigenous bacteria varied a lot, while the inoculated bacteria remained stable; the dehydrogenase activity was at high levels and then decreased. Indigenous microorganisms, inoculated bacterial agent, nutrients, water, and soil permeability all played important roles. The study prepared an environment-friendly bacterial agent and established a set of bioremediation technique, which provided further insights into integration of fermentation engineering and soil remediation engineering.

Bioflocculation behaviours of microbial communities in water treatment

We studied the flocculation behaviours of microbial communities in 21 soil, wastewater and activated sludge samples to clarify the effects of culture medium types on flocculation ability and screening efficiency, and to analyze diverse functions and microbial compositions. The bioflocculants produced by 33% of the microbial communities had flocculating efficiencies higher than 90%. Six out of the eight microbial communities with efficiencies over 94% were screened from the culture medium using dibutyl phthalate (DBP) as the carbon source. BF-BCT, which was derived from the Chinese cabbage soil sample, had the highest flocculating efficiency (99.6%), species diversity and uniformity. Nine highly efficient strains were separated and purified from seven different microbial communities, indicating that flocculating microorganisms are widely distributed in ecosystems. The 16S rRNA gene testing shows that the eight bacterial and the one fungal strains are common soil microorganisms. The flocculating abilities of BB11 (Sphingobacterium multivorum) and SE3 (Galactomyces geotrichum) have never been reported hitherto. Six strains, including the most flocculating-active TB13 and JB17, were screened from the culture medium using DBP as the sole carbon source. In particular, we compared the performance of culture media and analyzed analogous microbial communities with a Biolog automatic micro-analysis system for the first time.

Assessing the xylanolytic bacterial diversity during the malting process

The presence of microorganisms producing cell wall hydrolyzing enzymes such as xylanases during malting can improve mash filtration behavior and consequently have potential for more efficient wort production. In this study, the xylanolytic bacterial community during malting was assessed by isolation and cultivation on growth media containing arabinoxylan, and identification by 16S rRNA gene sequencing. A total of 33 species-level operational taxonomic units (OTUs) were found, taking into account a 3% sequence dissimilarity cut-off, belonging to four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and 25 genera. Predominant OTUs represented xylanolytic bacteria identified as Sphingobacterium multivorum, Stenotrophomonas maltophilia, Aeromonas hydrophila and Pseudomonas fulva. DNA fingerprinting of all xylanolytic isolates belonging to S. multivorum obtained in this study revealed shifts in S. multivorum populations during the process. Xylanase activity was determined for a selection of isolates, with Cellulomonas flavigena showing the highest activity. The xylanase of this species was isolated and purified 23.2-fold by ultrafiltration, 40% ammonium sulfate precipitation and DEAE-FF ion-exchange chromatography and appeared relatively thermostable. This study will enhance our understanding of the role of microorganisms in the barley germination process. In addition, this study may provide a basis for microflora management during malting.

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32nmol/mg/min. The productivity of this enzyme in whole cell form is 500mg/1l of cultured cell. Its optimum reaction condition is 10mM of calcium ions added to a phosphate buffer with a pH of 8.5.

Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE

A laboratory-scale biofiltration system, the rotatory-switching biofilter (RSB), was operated for 199 days using toluene as a model pollutant. The target gaseous pollutant for the biofiltration experiment was ∼300 ppmv of toluene. Toluene removal efficiency (RE, %) was initially approximately 20% with a 247-ppmv concentration (0.9 g m−3) of toluene during the first 10 days. Although the RE decreased several times whenever nitrogen was consumed, it again reached almost 100% when the nitrogen source was in sufficient supply. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to assess the transformation of microflora during operation of the biofilter. The results based on a 16S rRNA gene profile showed that the microbial community structure changed with operation time. Although the microflora changed during the initial period (before day 40), transformation of the bacterial component was hardly observed after day 51. Statistical analyses of the DGGE profiles indicated that the bacterial community was almost unaffected by the environmental factors, such as adding ozone, high-level nitrogen supply, increase of loading toluene, and the shutdown of the RSB. The DGGE profile using tmoA-like genes, which encode proteins belonging to the hydroxylase component mono-oxygenases involved in the initial attack of aerobic benzene, toluene, ethylbenzene, and xylene degradation, confirmed the existence of toluene-degrading bacteria. There were at least four kinds of toluene-degradable bacteria having tmoA-like genes up to day 36, which decreased to two species after day 40. Sequence analysis after DGGE profiling revealed that Burkholderia cepacia, Sphingobacterium multivorum, and Pseudomonas putida were present in the biofilter. Only Alicycliphilus denitrificans was present throughout the whole operation period. In the initial stage of operating the RSB, many types of bacteria may have tried to adapt to the conditions, and subsequently, only selected bacteria were able to grow and to degrade toluene. Implications: Biofiltration technology, which is more cost-effective and environmentally friendly than any other physicochemical method, has been applied to the treatment of volatile organic compounds (VOCs). It was difficult to maintain a high level of activity because the performance of the biofilter depends on microbial degradation of pollutants. However, there is little knowledge about the transformation of bacteria during long-term biofiltration operations. This study revealed the transformation of microflora in a biofilter using a molecular biological method, PCR-DGGE.

Determination of petroleum-degrading bacteria isolated from crude oil-contaminated soil in Turkey

In order to establish an experimental basis for bioremediation of soil contaminated with crude oil, 33 strains of bacteria with hydrocarbon-degrading ability were isolated from the contaminated soil in Adana, Batman and Adiyaman, Turkey. The strains were identified as Pseudomons spp., Paucimonas lemoignei, Stenotrophomonas maltophilia, Eschericha spp., Enterobacter spp. Citrobacter koseri, Acinetobacter spp., Aeromonas caviae, Sphingobacterium multivorum, Klebsiella pneumoniae. Pseudomons aeruginosa, Pseudomonas putida biotype A, Citrobacter amalonaticus GC subgroup A and Acinetobacter genomospecies, respectively. The ability to utilize crude oil as carbon source for their growth was ascertained. These bacterial isolates obtained in this study have catabolic capabilities for the biodegradation of petroleum hydrocarbons. Further study under soil conditions may be necessary to determine biodegradation potential of bacterial mixture tested in contaminated soil. Key words : Soil, crude oil, soil pollution, bacteria, biodegradation.

Used lubricating oil degradation and biosurfactant production by SC-9 consortia obtained from oil-contaminated soil

Consortia of used lubricating oil-degrading bacteria were isolated from oil-contaminated soil. The most active consortia in the assimilation of used lubricating oil were SC-9. This consortia contained four bacterial isolates based on culture-dependent technique. They were identified as Agrobacterium tumefaciens, Bacillus cereus, Chryseobacterium sp., and Sphingobacterium multivorum based on 16S rDNA sequence analysis. The SC-9 consortia showed 40.5% oil-degrading activity within 7 days. The addition of molasses to the liquid medium had a positive effect and there was an increase in the percentage of oil degradation and a greater in reduction surface tension. The ability of selected bacteria to degrade oil increased in a comparable trend as its biosurfactant production increased. Oil-degrading bacterial isolates that display substantial potential for production of biosurfactants can be applied in the bioremediation of soils contaminated with petroleum hydrocarbons.

Influence of tetracycline exposure on the growth of wheat seedlings and the rhizosphere microbial community structure in hydroponic culture

In this study, the effects of tetracycline exposure on wheat growth and the microbial community structure in the rhizosphere were investigated under hydroponic culture conditions. Exposure to various concentrations of tetracycline resulted in significant suppression of the growth of wheat roots and shoots, with minimum doses of 0.8 mg L−1 and 4 mg L−1 resulting in inhibition rates of 32% and 15.4%, respectively. Complete inhibition of the growth of these two parts of wheat plants was observed in response to treatment with tetracycline at 20 mg L−1 and 100 mg L−1, respectively. However, the germination of wheat seeds was not sensitive to exposure to tetracycline. The effects of tetracycline exposure on the microbial community in the wheat rhizosphere were evaluated through traditional cultivation and molecular biological analyses. The cultivation results indicated that bacteria were the dominant population, being present in concentrations of 1× 108–2.45× 109CFUs mL−1, although 39% to 87% inhibition occurred in response to tetracycline. The concentration of fungi increased in all tetracycline treated samples to 2.5 to 15.8 times that of the control. The highest concentration of fungi (4.27× 108 CFU mL−1) was observed in response to 60 mg L−1 tetracycline after 15 days of cultivation. In this stage, a large amount of fungal colonies was observed on the surface of the culture solution, the wheat roots became rotted and the plants became atrophic or even died. Molecular biological analysis indicated that the bacterial community structure was significantly different in samples that were exposed to high levels of tetracycline (over 20 mg L−1) than in samples that were exposed to lower concentrations. As the concentration of tetracycline increased, the diversity of the bacteria decreased. Additionally, several dominant sensitive species such as Sphingobacterium multivorum were suppressed by tetracycline, while some resistant species such as Acinetobacter sp. appeared or were conserved. The bacteria population tended to stabilize when the drug concentration exceeded 40 mg L−1.

Structural Insights into the Enzymatic Mechanism of Serine Palmitoyltransferase from Sphingobacterium multivorum

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyses the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The crystal structure of SPT from Sphingobacterium multivorum GTC97 complexed with l-serine was determined at 2.3 A resolution. The electron density map showed the Schiff base formation between l-serine and PLP in the crystal. Because of the hydrogen bond formation with His138, the orientation of the Calpha-H bond of the PLP-l-serine aldimine was not perpendicular to the PLP-Schiff base plane. This conformation is unfavourable for the alpha-proton abstraction by Lys244 and the reaction is expected to stop at the PLP-l-serine aldimine. Structural modelling of the following intermediates indicated that His138 changes its hydrogen bond partner from the carboxyl group of l-serine to the carbonyl group of palmitoyl-CoA upon the binding of palmitoyl-CoA, making the l-serine Calpha-H bond perpendicular to the PLP-Schiff base plane. These crystal and model structures well explained the observations on bacterial SPTs that the alpha-deprotonation of l-serine occurs only in the presence of palmitoyl-CoA. This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction.

Sphingobacterium siyangense sp. nov., isolated from farm soil

The taxonomic position of a novel Gram-negative strain, designated SY1(T), isolated from a farm-soil sample obtained from Jiangsu Province, PR China, was characterized by using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 degrees C and at pH 6.0-8.0. Based on 16S rRNA gene sequence analysis, strain SY1(T) is a member of the genus Sphingobacterium; Sphingobacterium multivorum JCM 21156(T) was the nearest relative (98.5 % sequence similarity). The predominant fatty acids of strain SY1(T) were iso-C(15 : 0) (32.9 %), C(16 : 0) (10.9 %) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c; 24.1 %). The DNA G+C content was 38.5 mol%. The low level of DNA-DNA relatedness (2.2 %) to S. multivorum JCM 21156(T) in combination with differential morphological and biochemical properties demonstrated that strain SY1(T) (=KCTC 22131(T)=CGMCC 1.6855(T)) should be classified as representing a novel species of the genus Sphingobacterium for which the name Sphingobacterium siyangense sp. nov. is proposed.

Conversion of Lesquerolic Acid to 14-Oxo-11(Z)-Eicosenoic Acid by Genetically Variable Sphingobacterium multivorum Strains

We investigated new microbial systems for their ability to convert lesquerolic acid (LQA; 14-hydroxy-11(Z)-eicosenoic acid) to value-added products. A strain of Sphingobacterium multivorum (NRRL B-23212) was found previously to convert LQA to 14-oxo-11(Z)-eicosenoic acid (14-OEA) as determined by gas chromatography-mass spectrometry and nuclear magnetic resonance analyses. Conversion of LQA was subsequently extended to examine S. multivorum and closely related species of Pedobacter, Spirosoma, Chryseobacterium, and Flavobacterium. Among 25 of such environmental isolates, a group of bacteria, whose identity was further confirmed by 16S rRNA gene sequence analysis as S. multivorum, was the only species found to conduct LQA conversion to produce 14-OEA. Among these strains, however, NRRL B-14797 was a variant strain devoid of the specific biologic activity. A new culture medium at pH 7.0 was defined to include Fe(2+) and Mn(2+) mineral ions, glycerol, and EDTA.2Na to improve the production of 14-OEA from the initial yields of 2% to 13% to approximately > or =75% for the reactive S. multivorum strains. These S. multivorum strains represent the first group of bacteria reported to carry out the functional modification of LQA.

Acceleration of the Substrate Cα Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.

Sphingobacterium canadense sp. nov., an isolate from corn roots

A free-living Gram-negative bacterial strain CR11 T was isolated from corn roots. Polyphasic taxonomy was performed, including API20 NE and API50 CH bacterial identification kits, Biolog analysis, lipids and fatty acid analysis, DNA–DNA hybridization, 16S rRNA and cpn60 gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain CR11 T belonged to the genus Sphingobacterium and was closely related to Sphingobacterium multivorum IFO 14947 T (98% similarity) and Sphingobacterium. thalpophilum ATCC 43320 T (97% similarity). DNA–DNA hybridization showed 11% and 13% DNA re-association with S. multivorum LMG 8342 T and S. thalpophilum LMG 11520 T, respectively. Major fatty acids (16:0, 15:0 iso and 17:0 iso 3-OH) and the G+C content of the DNA (40.5 mol%), were also similar to those of the genus Sphingobacterium. The predominant respiratory quinone was MK-7. In all analyses, including phenotypic characterization, this isolate was found to be different from the closely related species, S. multivorum and S. thalpophilum. On the basis of these results, this strain represents a new species within the genus Sphingobacterium. The name Sphingobacterium canadense sp. nov. is suggested and the type strain is CR11 T (=NCCB 100125 T=LMG 23727 T).

Molecular characterization of membrane-associated soluble serine palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii.

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.

Production of 14‐Oxo‐cis‐11‐eicosenoic Acid from Lesquerolic Acid by Sphingobacterium multivorum NRRL B‐23212

AbstractThe objective of this study was to explore the extent of microbial conversion of lesquerolic acid (14‐hydroxy‐cis‐11‐eicosenoic acid; LQA) by whole cell catalysis and to identify the newly converted products. Among compost isolates including NRRL strains B‐23212 (Sphingobacterium multivorum), B‐23213 (Acinetobacter sp.), B‐23257 (Enterobacter cloacae B), B‐23259 (Escherichia sp.) and B‐23260 (Pseudomonas aeruginosa) the S.multivorum strain was the only microorganism that converted LQA to produce a new product identified as 14‐oxo‐cis‐11‐eicosenoic acid by GC‐MS and NMR analyses. The conversion yield was 47.4% in 48 h at 200 rpm and 28°C in small shake flask experiments. In comparison, both Acinetobacter and Pseudomonas strains failed to convert LQA to major new products but used LQA apparently as an energy source during fermentation. For structural analysis, 6.88 g of 14‐oxo‐cis‐11‐eicosenoic acid was produced from converting 11 g LQA (a 62% yield) in 72 h at 200 rpm and 28 °C in Fernbach flasks using 18‐h‐old NRRL B‐23212 cultures and an improved medium that also contained EDTA and glycerol in lieu of glucose as carbon source. NRRL B‐23212 was further identified by 16S rRNA gene sequence analysis as a unique strain of S. multivorum. Therefore, S. multivorum NRRL B‐23212 possesses an enzymatic activity presumably a secondary alcohol dehydrogenase for converting LQA to produce 14‐oxo‐cis‐11‐eicosenoic acid, a first report that demonstrates the functional modification of LQA by whole cell catalysis.

Evaluation of pyrrolidonyl arylamidase for the identification of nonfermenting Gram-negative rods

To evaluate the activity of pyrrolidonyl arylamidase (PYR) for the differentiation and identification of nonfermenting gram negative rods (NFGNR), 293 isolates were tested. A 24 h culture of each test organism was prepared. From this a 108–109 cfu/mL suspension was added to 0.25 mL of sterile physiologic solution. A PYR disk was then added and the test was incubated for 30 minutes at 35–37°C, at environmental atmosphere. Reading was done by adding 1 drop of cinnamaldehyde reagent. Strains of Acinetobacter baumannii, Acinetobacter haemolyticus, Alcaligenes faecalis, Bergeyella zoohelcum, Bordetella bronchiseptica, Bordetella hinzii, Brevundimonas diminuta, Brevundimonas vesicularis, Brucella ovis, Brucella spp., Brucella suis, Burkholderia cepacia complex, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Oligella ureolytica, Pseudomonas alcaligenes, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas Vb3, Psychrobacter phenylpyruvicus, and Stenotrophomonas maltophilia were PYR negative. On the other hand Achromobacter piechaudii, Achromobacter denitrificans, Achromobacter xylosoxidans, Burkholderia gladioli, Chryseobacterium gleum-indologenes, Comamonas testosroni, Cupriavidus pauculus, Delftia acidovorans, Elizabethkingia meningoseptica, Myroides spp., Ochrobactrum anthropi, Pseudomonas oryzihabitans, Ralstonia pickettii, Rhizobium radiobacter, Shewanella spp., Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Weeksella virosa were PYR positive. Finally, Acinetobacter lwoffii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Roseomonas spp., and Sphingomonas paucimobilis-parapaucimobilis were PYR variable. PYR testing should be considered as a useful tool to facilitate the identification of NFGNR.

Role of Bacteria in Mediating the Oviposition Responses of <I>Aedes albopictus</I> (Diptera: Culicidae)

The responses of Aedes albopictus to sources of oviposition attractants and stimulants were evaluated with a behavioral bioassay in which females attracted to odorants emanating from water were trapped on screens coated with an adhesive. Gravid mosquitoes were attracted to volatiles from larval-rearing water and soil-contaminated cotton towels. Bacteria were isolated from these substrates and from an organic infusion made with oak leaves. Through fatty acid-methyl ester analyses, six bacterial isolates from larval-rearing water, two isolates from soil-contaminated cotton towels, and three isolates from oak leaf infusion were identified to species. The response of gravid mosquitoes to these isolates was also evaluated in behavioral bioassays. Water containing Psychrobacter immobilis (from larval-rearing water), Sphingobacterium multivorum (from soil-contaminated cotton towels), and an undetermined Bacillus species (from oak leaf infusion) elicited significantly higher oviposition than control water without bacteria. Only volatiles collected from larval rearing water elicited significant electroantennogram responses in females.

Identification of novel eubacteria from spent mushroom compost (SMC) waste by DNA sequence typing: ecological considerations of disposal on agricultural land

A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material ( n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp , Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log 10 7.01 colony forming units (cfu) per gram SMC material (range: log 10 6.53–7.52 cfu/g). Fungal counts gave a mean count of log 10 4.57 cfu per gram SMC material (range: log 10 3.93–4.98 cfu/g). From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe–microbe and plant–microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 10 18 cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.