Efficient and simple method for determination of suspendibility of bio-inoculant suspensions.

This study assessed the utilization of viscosity and zeta potential as a novel method to evaluate suspendibility of formulation of Sinorhizobium meliloti grown in starch industry wastewater for use as bio-inoculants. For this objective, sorbitol was used as a suspending agent at concentrations of 0 to 10% w/v. Model, based on multiple linear regression (with pH as dependant variable, and zeta potential, average particle size and sorbitol concentration as independent variables) demonstrated an important relation which was significant (p<0.001, R2=0.98). Sigmoid regression revealed a significant relation between zeta potential and suspendibility with p value=0.007 and R-squared=0.86, and between viscosity and suspendibility (p value<0.0001 and R squared=0.9823). Thus, these direct correlations established the lowering of measurement time from 12 h to 5 min.

Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency approximately 68 %) but was recovered infrequently ( approximately 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

Agro-industrial waste materials and wastewater sludge for rhizobial inoculant production: a review.

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the availability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g. plant compost, filtermud, fly-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in wastewater sludge are efficient for nodulation and nitrogen fixation with legumes as compared to standard inoculants. This new approach described in this review offers a safe environmental alternative for both waste treatment/disposal and inoculant production.

Evidence of a two-stage thermal denaturation process in lysozyme: a Raman scattering and differential scanning calorimetry investigation.

Raman spectroscopy (in the low-frequency range and the amide I band region) and modulated differential scanning calorimetry investigations have been used to analyze temperature-induced structural changes in lysozyme dissolved in 1H2O and 2H2O in the thermal denaturation process. Low-frequency Raman data reveal a change in tertiary structure without concomitant unfolding of the secondary structure. Calorimetric data show that this structural change is responsible for the configurational entropy change associated with the strong-to-fragile liquid transition and correspond to about 1/3 of the native-denaturated transition enthalpy. This is the first stage of the thermal denaturation which is a precursor of the secondary structure change and is determined to be strongly dependent on the stability of the hydrogen-bond network in water. Low-frequency Raman spectroscopy provides information on the flexibility of the tertiary structure (in the native state and the transient folding state) in relation to the fragility of the mixture. The unfolding of the secondary structure appears as a consequence of the change in the tertiary structure and independent of the solvent. Protein conformational stability is directly dependent on the stability of the native tertiary structure. The structural transformation of tertiary structure can be detected through the enhanced 1H/2H exchange inhibited in native proteins. Taking into account similar features reported in the literature observed for different proteins it can be considered that the two-stage transformation observed in lysozyme dissolved in water is a general mechanism for the thermal denaturation of proteins.

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.

Growth of alfalfa in sludge-amended soils and inoculated with rhizobia produced in sludge.

The efficiency of rhizobial inoculants produced in wastewater sludge used as a growth medium and as a carrier was compared with that of inoculants produced in yeast mannitol broth (YMB) medium and by using peat as a carrier. Alfalfa (Medicago sativa L.) plants were inoculated with solid and liquid Sinorhizobium meliloti inoculants and grown in pots containing two soil types (Kamouraska clay soil and Saint-André sandy soil). The effect of various levels of sludge amendment (60 and 120 kg N/ha) and nitrogen fertilizer (60 kg N/ ha) was also studied. The sludge-based inoculants showed the same symbiotic efficiency (nodulation and plant yield) as YMB-based inoculants. The inoculation increased the nodulation indexes from 4-6 to 8-12, and the rhizobial number from 10(3) (uninoculated soils) to 10(6)-10(7) cells/g in inoculated soils. However, the shoot dry weights and the nitrogen contents were not increased significantly by the inoculation. Applying sludge as an amendment enhanced the rhizobial number in soils from 10(3) to 10(4) cells/g and improved significantly the plant growth (shoot dry weights and nitrogen contents). This improvement increased with sludge rate and with the cut (three cuts). Compared with sludge, N fertilizer gave lower plant yields. The nodulation was not affected by sludge and N-fertilizer application. The texture and physico-chemical properties of soil were found to affect the yield and nitrogen content of the plants. In this study, macroelements and heavy metals were at acceptable levels and were not considered to be negative factors.

Production of S. meliloti using wastewater sludge as a raw material: effect of nutrient addition and pH control.

The utilization of wastewater sludge as a raw material for the production of legume inoculant is considered as a new viable alternative for recycling. The effect of addition of nutrient sources (yeast extract and glycerol) and the pH control on S. meliloti was investigated in a shake flask and at controlled pH in a 15 l fermentor. Different concentrations of yeast extract (0.5, 1, 2 and 4 g l(-1)) and glycerol (2.5, 5, 7.5 and 10 g l(-1)) were added to the secondary sludge. The cell yield as well as the generation time were affected by the addition of these nutrient sources. The maximum cell yield (8.85x10(9) cfu ml(-1)) was achieved in 32 hours of incubation with the addition of 4 g l(-1) of yeast extract. This value was 3.2 times higher than from the non-supplemented sludge. Moreover, at this yeast extract concentration, the cell concentration in the stationary phase did not decrease. The addition of glycerol to sludge samples containing 4 g l(-1) of yeast extract further improved the rhizobial growth but not significantly compared with the control. The highest yield (16.5x10(9) cfu ml(-1)) was obtained with 7.5 g l(-1) of glycerol and 4 g l(-1) of yeast extract. In fermentor experiments, pH did not seem to be a limiting factor and the increase of pH up to 8.85 in uncontrolled fermentor seems to have no effect on rhizobial growth and cell yield.

Wastewater sludge as a substrate for growth and carrier for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti.

The inoculation of legumes with rhizobia is used to maximise nitrogen fixation and enhance the plant yield without using N fertilisers. For this reason many inoculant types were developed and optimised. In our study, the effects of the growth medium, the carrier, the temperature and the storage period were determined on the survival of Sinorhizobium meloliti. Secondary sludge from Communauté Urbaine de Quebec wastewater treatment plant and standard medium (YMB) were used for rhizobial growth. Dehydrated sludge from Jonquière wastewater treatment plant, peat and a mixture of peat and sludge were used as carrier materials. Results showed that the wastewater sludge offered better protection for rhizobia survival during freezing and thawing at -20 degrees C than the standard medium. In general, results also showed the suitability of using sludge as a carrier because it had the same or a higher potential than peat to support survival of S. meliloti. In the case of YMB-grown rhizobia, peat- and sludge-based carriers appeared to be similar in terms of survival rate during the storage at 4 and 25 degrees C. For secondary sludge-grown rhizobia, the survival was better in sludge than in peat based carrier. Generally, the cell count remained higher than 10(8) cells/g for up to 80 days at 4 and 25 degrees C in both carriers (sludge and peat). However, for the secondary sludge-grown cells stored in peat-based carrier at 4 degrees C, the viable cells decreased under 10(8) cells/g at the 81st day of storage but remained acceptable compared to the standard (10(7) cells/g of carrier).

Acid and alkaline treatments for enhancing the growth of rhizobia in sludge.

Wastewater sludges have been proposed as an effective media for the production of rhizobia. The effect of total suspended solid (TSS) concentrations and pretreatments of sludge on the growth of Sinorhizobium meliloti were investigated. Acid (pH 2.0-6.0 obtained with H2SO4) and alkaline (50-200 mequiv.wt./L of NaOH) treatments were applied to enhance the biodegradability of primary (0.325%-3.2% TSS obtained by dilution of original sample) and secondary (0.2%-0.4% TSS obtained by concentration of original sample) sludges. In primary sludge without pretreatment, the highest cell count (11.10 x 10(9) cfu/mL) was obtained with 1.3% TSS. However, a maximum cell count of 13.00 x 10(9) cfu/mL was reached using an acid treatment of pH 2.0 and a 0.325% TSS concentration. Moreover, the alkaline treatment with 100 mequiv.wt./L of NaOH and 0.65% TSS increased the cell yield to 21.00 x 10(9) cfu/mL. For secondary sludge without pretreatment, no enhancement of growth was observed while increasing TSS concentration. This may be due to the increase of inhibitory substances, such as heavy metals, and of the Ca and Mg concentrations. As in primary sludge, some acid and alkaline treatments of secondary sludge tend to improve the cell count of S. meliloti. However, the highest value of 9.80 x 10(9) cfu/mL obtained with 0.4% TSS at pH 2.0 was lower than that obtained with primary sludge. It was also observed that S. meliloti grown in treated sludges maintained its capacity to nodulate alfalfa.

[Effects of inoculation with Rhizobium leguminosarum biovar trifolii on wheat cultivated in clover crop rotation agricultural soil in Morocco]. / Effets de l'inoculation avec des souches de Rhizobium leguminosarum biovar trifolii sur la croissance du blé dans deux sols du Maroc.

One hundred strains of Rhizobium leguminosarum bv. trifolii were isolated from roots of wheat cultivated in rotation with clover in two different regions of Morocco. The isolates were first screened for their effect on the growth of the cultivar Rihane of wheat cultivated in an agricultural soil under greenhouse conditions. After 5 weeks of growth, 14 strains stimulating the fresh or dry matter yield of shoots were selected and used in a second pot inoculation trial performed with two different agricultural soils. The results show that the strains behaved differently according to the soil used. In the loamy sand Rabat, strain IAT 168 behaved potentially like a plant growth promoting rhizobacteria (PGPR), as indicated by the 24% increases (P < 0.1) observed in wheat shoot dry matter and grain yields. In the silty clay Merchouch, no PGPR activity was observed, and 6 strains showed a significant deleterious effect on yields. These observations suggest that it is very important in a crop rotation system to choose a R. leguminosarum bv. trifolii strain that is effective with clover and shows PGPR activity with wheat to avoid deleterious effects on wheat yields.

Experimental and theoretical evidence for the existence of absolute acoustic band gaps in two-dimensional solid phononic crystals.

Experimental measurements of acoustic transmission through a solid-solid two-dimensional binary-composite medium constituted of a triangular array of parallel circular steel cylinders in an epoxy matrix are reported. Attention is restricted to propagation of elastic waves perpendicular to the cylinders. Measured transmitted spectra demonstrate the existence of absolute stop bands, i.e., band gaps independent of the direction of propagation in the plane perpendicular to the cylinders. Theoretical calculations of the band structure and transmission spectra using the plane wave expansion and the finite difference time domain methods support unambiguously the absolute nature of the observed band gaps.

Stochastic artificial retinas: algorithm, optoelectronic circuits, and implementation.

An analogy can be established between image processing and statistical mechanics. Many early- and intermediate-vision tasks such as restoration, image segmentation, and motion detection can be formulated as optimization problems that consist in finding the ground states of an energy function. This approach yields excellent results, but, once it is implemented in conventional sequential workstations, the computational loads are too extensive for practical purposes, and even fast suboptimal optimization approaches are not sufficient. We elaborate on dedicated massively-parallel integrated circuits, called stochastic artificial retinas, that minimize the energy function at a video rate. We consider several components of these artificial retinas: stochastic algorithms for restoration tasks in the presence of discontinuities, dedicated optoelectronic hardware to implement thermal motion by photodetection of speckles, and hybrid architectures that combine optoelectronic, asynchronous-analog, and clocked-digital circuits.

Physiological adaptation to low temperatures of strains of Rhizobium leguminosarum bv. viciae associated with Lathyrus spp.(1).

Strains of Rhizobium leguminosarum bv. viciae, isolated from the legume species Lathyrus japonicus and Lathyrus pratensis in northern Quebec (Canada), showed different capacities for growing at low temperature. In the present study, we investigated some mechanisms related to cold adaptation. Two cold-adapted strains (psychrotrophs) were compared to a poorly adapted strain and to a cold-sensitive strain (reference strain) for freezing survival, protein induction and fatty acid composition under low temperature. Following cold shocks (25 degrees C to 10, 5 and 0 degrees C), a common 6.1-kDa CSP (cold shock protein) was induced in all strains, but the total number of CSPs synthesized at 0 degrees C was higher in cold-adapted strains than in the cold-sensitive strain. The synthesis of CAPs (cold acclimation proteins) was observed under continuous growth at 5 degrees C in all three strains capable of growth at this temperature. Levels of survival after 24 h at -80 degrees C where higher in cold- (79%) and poorly adapted (64%) strains than in the cold-sensitive strain (33%), but a 2-h acclimation period at 5 degrees C before freezing doubled the survival of the cold-sensitive strain. Low temperature conditions affected similarly the fatty acid composition of all strains, regardless of their cold adaptation level. The proportion of unsaturated fatty acids increased significantly with the lowering of growth temperature from 25 to 5 degrees C, but showed a tendency to decrease after a cold shock from 25 to 5 degrees C. A specific unsaturated fatty acid, cis-12 octadecanoic acid, was produced during growth at 5 degrees C. The unsaturated cis-vaccenic acid was the principal component under all conditions. The cold adaptation trait was weakly reflected in symbiosis with the agronomic legume, Lathyrus sativus, with which one cold-adapted strain showed a slightly higher nitrogenase activity and shoot dry matter yield than a commercial strain under a sub-optimal temperature regime.

[Voluntary abortion: new perspectives in local anesthesia]. / Interruption volontaire de grossesse: nouvelles perspectives de l'anesthésie locale.

New perspectives have been envisioned for the TOP instrumental technique, through the utilization of pharmacologic products, whose property is to dilatate the cervix. Misoprostol or mifepristone, when administrated to a patient a few hours before a TOP by uterine suction curettage, enables a dilatation of the cervix in such a way that it allows the surgically induced abortion to be carried out with local anesthesia, under optimal comfort conditions for the patients as well as for the operator.

Genetic diversity of rhizobial symbionts isolated from legume species within the genera Astragalus, Oxytropis, and Onobrychis.

The genetic diversity of 44 rhizobial isolates from Astragalus, Oxytropis, and Onobrychis spp. originating from different geographic locations was evaluated by mapped restriction site polymorphism (MRSP) analysis of 16S rRNA genes and by PCR DNA fingerprinting with repetitive sequences (REP-PCR). A comparison of tree topologies of reference strains constructed with data obtained by MRSP and by 16S rRNA gene sequence analyses showed that the topologies were in good agreement, indicating that the MSRP approach results in reasonable estimates of rhizobial phylogeny. The isolates were distributed into 14 distinct 16S rRNA gene types clustering into three major groups which corresponded with three of the genera within the legume symbionts. Most of the isolates were within the genus Mesorhizobium. Five were identified with different genomic species nodulating Lotus spp. and Cicer arietinum. Three Astragalus isolates were classified as Bradyrhizobium, one being similar to Bradyrhizobium elkanii and another being similar to Bradyrhizobium japonicum. Six of the isolates were related to species within the genus Rhizobium. Two were similar to Rhizobium leguminosarum, and the remainder were identified as Rhizobium gallicum. DNA fingerprinting by REP-PCR revealed a high level of diversity within single 16S ribosomal DNA types. The 44 isolates were distributed into 34 REP groups. Rhizobial classification at the genus and probably also the species levels was independent of geographic origin and host plant affinity.

Classification of bacteria nodulating Lathyrus japonicus and Lathyrus pratensis in northern Quebec as strains of Rhizobium leguminosarum biovar viciae.

The diversity of two populations of rhizobia isolated from Lathyrus japonicus (30 strains) and Lathyrus pratensis (49 strains) growing in northern regions of Quebec, Canada, was determined on the basis of phenotypic characteristics, multilocus enzyme electrophoresis, DNA-DNA homology, and 16S ribosomal DNA sequencing. According to numerical analysis of phenotypic characteristics, strains were divided into four groups. Strains isolated from L. pratensis fell in groups I to III; the latter included reference strains of Rhizobium leguminosarum. All strains isolated from L. japonicus were included in group IV. All strains had nodulation characteristics similar to those of R. leguminosarum bv. viciae. Strains isolated from L. japonicus originating from an arctic region were usually able to grow at 5 degrees C and were more likely to be tolerant to copper (CuCl2.H2O, 100 micrograms/ml) and lead [Pb(CH3COO)2, 500 micrograms/ml] than strains isolated from L. pratensis from a boreal zone. However, both populations of Lathyrus strains were adapted to the cold in comparison to reference strains from temperate regions. Each population had similar genetic diversity (H = 0.45), determined by multilocus enzyme electrophoresis of the loci encoding eight enzymes, but the diversity obtained by analyzing all strains including the reference strains (H = 0.58) was higher. Representative strains of both populations showed high levels of DNA homology among themselves and with R. leguminosarum. Partial sequences of the 16S ribosomal RNA genes were similar to those reported for R. leguminosarum bv. viciae. We conclude that the strains isolated from L. japonicus and L. pratensis belong to R. leguminosarum bv. viciae but are distinguishable by growth at 5 degrees C, which is a characteristic related to their geographic origin.

Sequence and mutational analysis of the common nodBCIJ region of Rhizobium sp. (Oxytropis arctobia) strain N33, a nitrogen-fixing microsymbiont of both arctic and temperate legumes.

By heterologous hybridization, we have identified the common nodulation genes nodBCIJ of Rhizobium sp. strain N33 within a 8.2-kb PstI fragment. The nodBCIJ genes are located within a 4,620-bp region which also included a consensus nod box promoter. The four open reading frames coding for the nodBCIJ genes contain 657, 1,353, 915, and 789 nucleotides, respectively. We found that the nodA gene was not adjacent to the nodB gene, unlike the situation in many rhizobia. The DNA of the nodBCIJ genes of Rhizobium sp. strain N33 were found to be homologous to the corresponding genes of other rhizobia except for the 3'-coding region of the nodC gene. The deduced NodC protein was the longest of the rhizobia except Bradyrhizobium japonicum. Tn5 mutagenesis of the common nod region of strain N33 revealed that the nodBC genes were essential for nodulation on their temperate hosts Onobrychis viciifolia and Astragalus cicer. By contrast, mutations in the nodI and nodJ genes produced a Nod+ phenotype with a reduced number of nodules on the temperate hosts. Nodules formed on Onobrychis viciifolia by either nodI or nodJ mutants were approximately 10 times smaller than nodules formed by the wild type strain: this reduction in nodule size was not observed on Astragalus cicer.