Burkholderia gladioli MB39 an Antarctic Strain as a Biocontrol Agent.

Bioprospecting sub-explored environments such as Antarctic locations leads to finding out diverse activities, reducing harmful chemical usage that affects both human health and the environment. In this study, ~ 7000 cold-adapted bacterial strains were isolated from samples around Melchior Antarctic Base at 5 °C and more than 13,000 at 15 °C. Out of them, 900 different colony morphotypes were evaluated for antimicrobial production, and 13 isolates demonstrated antibacterial and antifungal activities. One isolate, closely related to Burkholderia gladioli according to 16S rDNA (99.8%), gyrB (99.6%) and Cpn60 (99.4%) gene sequence analysis, showed a consistent, broad antimicrobial spectrum against both pathogenic and phytopathogenic bacteria. Its potent antifungal activity inhibits the growth of various plant pathogenic fungi, whereas it was mainly studied against Penicillium digitatum and Macrophomina phaseolina, the causal agents of blue mould in postharvest fruits and charcoal rot in soybean crops, respectively. The antibacterial compound exhibited low molecular weight (< 6000 Da), resistance to lytic enzymes and stability in a broad range of temperature and pHs. Observations of the B. gladioli MB39 antifungal effects over M. phaseolina mycelia by scanning electron microscopy showed alterations in hyphal structures, reduced hyphal extension, and severe cell morphology changes such as cytoplasmic leakage, flattened and empty mycelia. Here we report the isolation and identification of a cold-adapted B. gladioli strain. The results describe the effectiveness of the antarctic strain for bacterial and fungal phytopathogens biocontrol and its potential for crop protection plans.

Cost-effective optimized scleroglucan production by Sclerotium rolfsii ATCC 201126 at bioreactor scale. A quantity-quality assessment.

Exopolysaccharide (EPS) secretion by Sclerotium rolfsii ATCC 201126 in submerged cultures, already identified as high-osmolarity responsive, was assessed by reducing C-source without compromising EPS yields. A designed medium with 80 g sucrose L-1 (MOPT80) was tested at 3 L-bioreactor scale at different temperature, agitation, aeration and pH (uncontrolled vs. controlled) values. Optimal operative conditions (200 rpm, 28 °C, 0.5 vvm and initial pH -pHi- 4.5) were validated, as well as the possibility to work at pHi 5.5 to reduce biomass production. Purified EPSs produced in MOPT80 at optimal and other valid operative conditions exhibited refined grade (<1 % proteins and ash, 3-4 % reducing sugars, 87-99 % total sugars). EPS purity, MW and rheological parameters led to discourage pH controlled at 4.5. Relatively constant MW (6-8 × 106 Da) and outstanding viscosifying ability were found. Polyphasic EPS analysis (titre, purity, macromolecular features and rheological fitness) would support to properly select production conditions.

Prevalence, serotypes and virulence genes of Streptococcus agalactiae isolated from pregnant women with 35-37 weeks of gestation.

BACKGROUND: In pregnant women Streptococcus agalactiae (GBS) can be transmitted to newborn causing severe infections. It is classified into 10 serotypes (Ia, Ib, II-IX). The severity of neonatal disease is determined by the capsular serotype and virulence factors such as the polysaccharide capsule, encoded by the cps gene, protein C, which includes the Cα surface proteins (bca gene), Rib (rib gene) and Cß (bac gene); the proteins Lmb (lmb gene), FbsB (fbsB gene), FbsA (fbsA gene), the cyl operon encoding a ß-hemolysin (hylB gene), the CAMP factor (cfb gene) and the C5a peptidase (scpB gene). The aim of this work was to determine the degree of GBS colonization in pregnant women, the serotypes distribution and to investigate virulence-associated genes. METHODS: We worked with 3480 samples of vagino-rectal swabs of women with 35-37 weeks of gestation. The identification of the strains was carried out using conventional biochemical tests and group confirmatory serology using a commercial latex particle agglutination kit. Two hundred GBS strains were selected. Their serotype was determined by agglutination tests. The monoplex PCR technique was used to investigate nine virulence-associated genes (cps, bca, rib, bac, lmb, fbsB, fbsA, hylB and scpB). RESULTS: The maternal colonization was 9.09%. The serotypes found were: Ia (33.50%), III (19.00%), Ib (15.50%), II (14.00%), V (7.00%) and IX (5.50%). 5.50% of strains were found to be non-serotypeable (NT). The nine virulence genes investigated were detected simultaneously in 36.50% of the strains. The genes that were most frequently detected were scpB (100.00%), fbsA (100.00%), fbsB (100.00%), cylB (95.00%), lmb (94.00%) and bca (87.50%). We found associations between serotype and genes bac (p = 0.003), cylB (p = 0.02), rib (p = 0.01) and lmb (p < 0.001). CONCLUSIONS: The frequency of vaginal-rectal colonization, serotypes distribution and associated virulence genes, varies widely among geographical areas. Therefore, epidemiological surveillance is necessary to provide data to guide decision-making and planning of prevention and control strategies.

Use of glycerol for the production of actinobacteria with well-known bioremediation abilities.

In recent years, there has been an increasing interest in the remediation of contaminated environments, and a suitable solution is in situ bioremediation. To achieve this, large-scale bacterial biomass production should be sustainable, using economic culture media. The main aim of this study was to optimize the physicochemical conditions for the biomass production of an actinobacterium with well-known bioremediation ability using inexpensive substrates and to scale-up its production in a bioreactor. For this, the growth of four strains of actinobacteria were evaluated in minimal medium with glucose and glycerol as carbon and energy sources. In addition, l-asparagine and ammonium sulfate were assayed as alternative nitrogen sources. The strain Streptomyces sp. A5 showed the highest biomass production in shake-flasks culture using glycerol and ammonium sulfate as carbon and nitrogen sources, respectively. Factorial designs with five factors (glycerol concentration, inoculum size, pH, temperature, and agitation) were employed to optimize the biomass production of Streptomyces sp. A5. The maximum biomass production was obtained using 5 g L-1 of glycerol, 0.25 µL of inoculum, pH 7, 30 °C and 200 rpm. Finally, the production was successfully scaled to a 2 L stirred tank bioreactor. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02588-5.

Effects of thermal, alkaline and ultrasonic treatments on scleroglucan stability and flow behavior.

Aqueous solutions (0.2%, w/v) of scleroglucans from Sclerotium rolfsii ATCC 201126 from different cultivation time or purification protocol (EPS I, EPS II, EPSi) as well as a commercial scleroglucan (LSCL) exhibited different sensitivity against thermal (65, 95 and 150°C), ultrasonic (1, 5 and 10 min; 20% amplitude) or alkaline (0.01-0.2 N NaOH) treatments. Scleroglucan triple helix usually showed signs of denaturation at 150°C or with 0.2 NaOH with a pronounced decrease in apparent viscosity and loss of pseudoplastic behavior. Differences in sensitivity could be noted depending on the scleroglucan sample, which may be likely related to polysaccharide conformational features, and these latter to production and/or downstream processing conditions. Transmission electron microscopy showed scleroglucan topologies in accordance with thermal and alkaline denaturation. Size exclusion chromatography of control scleroglucans revealed elution profiles compatible with macromolecular aggregates which tended to diminish or disappear as thermal, alkali or sonication treatments progressed. Scleroglucan granule dissolution process took ∼8-14 s, according to DIC-light microscopy, and showed to be facilitated by addition of NaOH.

Unraveling the decolourizing ability of yeast isolates from dye-polluted and virgin environments: an ecological and taxonomical overview.

Microcosm assays with dye-amended culture media under a shot-feeding strategy allowed us to obtain 100 yeast isolates from the wastewater outfall channel of a dyeing textile factory in Tucumán (Argentina). Meanwhile, 63 yeast isolates were obtained from Phoebe porphyria (Laurel del monte) samples collected from Las Yungas rainforest (Tucumán), via a classical isolation scheme. Isolated yeasts, both from dye-polluted and virgin environments, were compared for their textile dye decolourization ability when cultured on solid and liquid media. Nine isolates from wastewater and 17 from Las Yungas showed the highest decolourization potential on agar plates containing six different reactive dyes, either alone or as a mixture. Five yeasts from each environment were further selected on the basis of their high dye removal rate in Vilmafix(®) Red 7B-HE- or Vilmafix(®) Blue RR-BB-amended liquid cultures. Yeasts from wastewater showed slightly higher decolourization percentages after 36 h of culture than yeasts from Las Yungas (98-100% vs. 91-95%, respectively). However, isolates from Las Yungas exhibited higher specific decolourization rates than isolates from effluents (1.8-3.0 vs. 0.9-1.3 mg g(-1)h(-1), respectively). All selected isolates were first grouped according to microsatellite-PCR analysis and representative isolates from each group were subsequently identified based on the 26S rRNA gene sequence analysis. Yeasts from wastewater were identified as the ascomycetous Pichia kudriavzevii (100%) and closely related to Candida sorbophila (99.8%), whilst yeasts from Las Yungas were identified as the basidiomycetous Trichosporon akiyoshidainum and Trichosporon multisporum. It is suggested that findings concerning yeast selection during screening programs for dye-decolourizing yeasts may be explained in the light of the copiotroph-oligotroph microorganisms rationale.

Cold-adapted microorganisms as a source of new antimicrobials.

Thirty out of 8,000 different colony morphotypes isolated from soil samples of Isla de los Estados were selected based on their ability to produce antimicrobials. The significant influence of culture media and incubation temperature on antimicrobial production was proved, being LB medium and 8 degrees C the conditions of choice. Most of the psychrotolerant isolates were phylogenetically related to Serratia proteamaculans (96.4-97.9%) while the psychrophilic isolated 8H1 was closely related to Pseudomonas sp. (90-94% similarity). Produced antimicrobials showed a promising wide spectrum of activity both against gram-positive and gram-negative pathogenic bacteria. They were suspected to be microcin-like compounds (Mw <2,000 Da) and showed a marked tolerance to heat (1 h in boiling water bath) and pH-treatments (1-12). Antimicrobial compounds also showed to partially keep their activity even after overnight freezing at -20 and -80 degrees C and displayed a negative net charge at pH 8.0, a common feature of class II microcins.

Clostridium lundense sp. nov., a novel anaerobic lipolytic bacterium isolated from bovine rumen.

A strictly anaerobic, mesophilic, endospore-forming, lipolytic bacterium, designated strain R1T, was isolated from bovine rumen fluid and characterized. Cells of this isolate were Gram-positive, non-motile rods that formed spherical terminal spores. The overall biochemical and physiological characteristics indicated that this strain should be placed in the genus Clostridium. The strain grew at temperatures between 25 and 47 degrees C (optimum, 37 degrees C), at pH between 5.0 and 8.5 (optimum pH 5.5-7.0) and in NaCl concentrations of 0-3% (w/v). The isolate was not able to utilize glucose or other carbohydrates as carbon sources. The DNA G + C content was 31.2 mol%. Sequence analysis of the 16S rRNA gene of R1T revealed that it has the closest match (98 % similarity) with Clostridium tetanomorphum DSM 4474T. The highest levels of DNA-DNA relatedness of the isolate were 61.9 and 54.3% with Clostridium pascui DSM 10365T and C. tetanomorphum DSM 4474T, respectively. Based on 16S rRNA gene sequence similarity, phylogenetic analysis, DNA G + C content, DNA-DNA hybridization data and distinct phenotypic characteristics, strain R1T (= DSM 17049T = CCUG 50446T) was classified in the genus Clostridium, as a member of a novel species, for which the name Clostridium lundense sp. nov. is proposed.

Sequence analysis, cloning and over-expression of an endoxylanase from the alkaliphilic Bacillus halodurans.

The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-beta-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg-1.

Bacillus bogoriensis sp. nov., a novel alkaliphilic, halotolerant bacterium isolated from a Kenyan soda lake.

Strain LBB3(T) isolated from Bogoria soda lake in Kenya is an alkaliphilic, Gram-positive, strictly aerobic, non-motile, spore-forming bacterium. It was identified as a member of the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The organism grows optimally at 37 degrees C and pH 10. The G+C content of the genomic DNA is 37.5 mol%. 16S rRNA gene sequence analysis showed 95 and 96 % sequence similarity with Bacillus pseudofirmus (DSM 8715(T)) and Bacillus alcalophilus (DSM 485(T)), respectively. Furthermore, DNA-DNA hybridization against these two Bacillus species showed 39.0 and 55.5 % similarity, respectively. Based on our observations, strain LBB3(T) is proposed to represent a novel species of the genus Bacillus, for which the name Bacillus bogoriensis sp. nov. is proposed. The type strain of B. bogoriensis is LBB3(T) (=ATCC BAA-922(T)=LMG 22234(T)).

Lipase-producing microorganisms from a Kenyan alkaline soda lake.

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.

Characterization of a new xylitol-producer Candida tropicalis strain.

A xylitol-producer yeast isolated from corn silage and designated as ASM III was selected based on its outstanding biotechnological potential. When cultivated in batch culture mode and keeping the dissolved oxygen at 40% saturation, xylitol production was as high as 130 g l(-1) with a yield of 0.93 g xylitol g(-1) xylose consumed. A preliminary identification of the yeast was performed according to conventional fermentation and assimilation physiological tests. These studies were complemented by using molecular approaches based on PCR amplification, restriction-fragment length polymorphism analysis and sequencing of the rDNA segments: intergenic transcribed spacer (ITS) 1-5.8S rDNA-ITS 2, and D1/D2 domain of the 26S rRNA gene. Results from both the conventional protocols and the molecular characterization, and proper comparisons with the reference strains Candida tropicalis ATCC 20311 and NRRL Y-1367, led to the identification of the isolate as a new strain of C. tropicalis.

Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans.

Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.