Kaolin waste dumps contain a huge volume of material that cannot be commercialized due to the presence of variable amounts of iron minerals, which impart a yellowish color to the kaolin. Elimination of iron from kaolin using either a chemical or a biological method was the aim of this study. The chemical leaching of iron from kaolin was carried out using response surface methodology to optimize the reaction conditions. Time was found to be the most influential variable, although oxalic acid must be present to leach 32% of the iron. Biological leaching was also assayed for 14 days using a Bacillus strain. The results of bacterial leaching of iron from kaolin showed <1% iron elimination.

We explored the seed-associated bacterial endophytic microbiome in seeds of the endemic holoparasitic species Cistanche armena from a saline and arid habitat in Armenia. A combination of culture-dependent and molecular techniques was employed for identifying the seed endomicrobiome (culturable and unculturable). From surface-sterilized seeds, 10 phyla, comprising 256 endophytic bacterial genera, were identified. Of the culturable strains, we also investigated the plant growth-promoting (PGP) traits. Most of the isolates were spore forming, halotolerant and alkaliphile Bacillus spp., indicating that the endophytic bacteria of C. armena seeds own traits related to the natural habitat of their host plant. Our results confirm that Bacillus species are common and dominated endophytes from plants growing on saline and arid soils. Pantoea spp. and Stenotrophomonas spp. are more favourable PGP endophytes in seeds of C. armena. The PGP traits of these bacteria, such as production of indole, a precursor of auxin, ACC-deaminase and organic acids have the potential to improve the tolerance of their host plants against the abiotic stresses present in their natural habitat. To the best of our knowledge, this is the first report concerning bacterial seed endophytes of the C. armena.

Bacillus pumilus SAFR-032, an endospore-forming bacterial strain, was investigated to determine its methylation pattern (methylome) change, compared to ground control, after direct exposure to space conditions onboard the International Space Station (ISS) for 1.5 years. The resulting ISS-flown and non-flown strains were sequenced using the Nanopore MinION and an in-house method and pipeline to identify methylated positions in the genome. Our analysis indicated genomic variants and m6A methylation increased in the ISS-flown SAFR-032. To complement the broader omics investigation and explore phenotypic changes, ISS-flown and non-flown strains were compared in a series of laboratory-based chamber experiments using an X-ray irradiation source (doses applied at 250, 500, 750, 1000 and 1250 Gy); results show a potentially higher survival fraction of ISS-flown DS2 at the two highest exposures. Taken together, results from this study document lasting changes to the genome by methylation, potentially triggered by conditions in spaceflight, with functional consequences for the resistance of bacteria to stressors expected on long-duration missions beyond low Earth orbit.

Endophytic microbiomes play a beneficial role in the development and protection of plants. However, seed-borne endophytic bacteria have not yet been fully explored. Investigation of parasitic plants, whose existence depends on yet poorly understood and complicated relationships with microorganisms and hosts, is particularly crucial. Endophytic bacteria promote seed conservation and facilitate seed germination in soil. Several root holoparasites from the Orobanchaceae family are the most aggressive broomrape species, often causing serious yield losses in important crops. Parasitic plants are characterized by the production of a large number of some of the smallest seeds in the world's flora, allowing them to stay viable in the soil for several dozen years. This study's aim was to isolate and characterize the seed endophyte and surface bacteria of the most aggressive and widespread broomrape weed, Phelipanche ramosa. We isolated two endophytic bacteria from within the seeds which are closely related to Brevibacterium frigoritolerans and Bacillus simplex described as soil bacteria, highly resistant to environmental conditions, and as plant growth-promoting rhizobacteria. Moreover, we isolated three strains from the surface of non-sterile seeds; all three isolates were related to the Bacillus cereus group.

A 26-day trial with 18 Pietrain×(Landrace×Duroc) pigs was conducted to investigate the effect of two dose levels of a specifically selected Bacillus spp. direct-fed microbial (DFM) product, on the emission of environmentally harmful gasses (methane, ammonia and hydrogen sulphide) from manure. Pigs were assigned to one of three treatments in a randomized complete block design according to their sex and initial BW. Each treatment contained three replications with two pigs per pen. The test treatments included a Bacillus spp. DFM containing 3×108 colony-forming unit/g, added at a low (250 mg/kg) and high (500 mg/kg) dose to an antibiotic free high fibre-based diet, and a non-supplemented control diet. Manure from pigs fed with the supplemented diets emitted lower amounts of atmospheric contaminants. The most significant reduction was observed with low DFM supplementation, in which methane and ammonia volatilization decreased (P<0.05) by >40% and 50%, respectively, on fresh weight basis in relation to the control. Microbiome analysis of manure by high through put sequencing techniques on eubacterial and archaeal 16S rRNA genes highlighted the complex interactions between indigenous gut microflora and inoculated Bacillus spp. The tested Bacillus DFM could be considered as a best available technique in reducing the environmental impacts of growing pigs fed with high fibre-based diets.

The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

Following observations by a service engineer (mid-2007), 12 of the hundreds of installed packaged plant (PP) sewage systems were producing relatively large numbers of Culex quinquefasciatus (Say) and a variety of filth flies in George Town, Grand Cayman. Due to the vector potential of C. quinquefasciatus in the Caribbean, we explored the efficacy of industry-standard insecticides against C. quinquefasciatus. Since PP systems contain an organic biofilm on the water surface, we devised a modified technique for collecting immatures and for the application of insecticides. We treated functioning and non-functioning PP systems with Bacillus sphaericus (VectoLex® WDG), Bacillus thuringiensis var. israelensis (Bti; VectoBac® CG), temephos (Abate® 4-E), monomolecular film or ethyoxylated alcohol surfactant (Agnique® MMF) and (S)-methoprene (Altosid® Pro-G). B. thuringiensis var. israelensis gave control for 35 and 48 days for functioning and non-functioning systems, respectively, with less control from B. sphaericus (high label rate) (c. 25 and 35 days) and temephos (suggested label rate) (c. 27 and 33 days). B. sphaericus (low label rate), monomolecular film and (S)-methoprene gave limited control of immatures (i.e. no mortality detected after 24 h). Treatment options for PP systems are discussed with regard to economics, operational feasibility, and within the context of integrated mosquito management. Functioning systems were recolonized at a faster rate than those that were not functioning, but the level of infestation in the non-functioning systems was considerably greater.

Introduction. Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) can be considered important rhizospheric beneficial microorganisms. Their use as biocontrol strategies against soilborne pathogens such as nematodes should be taken into account. However, optimal management of soil microbiota communities is not easy because of the high specificity involved in these types of interactions. The aim of our study was to determine whether the combined inoculation of two AMF species and a Bacillus consortium based on three strains previously described as PGPR in other crops were able to reduce nematode infection and damage on papaya. Materials and methods. Papaya seedlings were inoculated with two AMF isolates (Glomus mosseae or G. manihotis) at the beginning of the nursery phase. Once the mycorrhizal symbiosis was established, a Bacillus consortium was applied. Nematode inoculum was applied 20 d after transplanting to individual pots. Plants were harvested 160 d after nematode inoculation. Results. In terms of plant development and nutrition, benefits due to AMF inoculation persisted in the presence of PGPR. However, the effect of dual inoculation was different, depending on the Glomus species. This positive effect was also evident in plants with nematode. Meloidogyne infection was significantly reduced in mycorrhizal plants. However, the addition of PGPR does not seem to improve the results of AMF single treatments in terms of nematode infection. Conclusion. Dual application of AMF and PGPR must be considered for papaya threatened by the root-knot nematode, although a previous screening should be done in order to select the best microbe combination to optimise results.

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5′ end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

Six genera of bacteria: Serratia, Providencia, Enterobacter, Aeromonas, Cedecea and Bacillus were isolated from field-collected G. pallidipes. Four genera: Serratia, Providencia, Enterobacter and Aeromonas were isolated from insectary-reared Glossina morsitans morsitans. All bacteria except, Bacillus spp. and Providencia alcalifaciens were pathogenic for teneral G. m. morsitans. Bacteria isolated from insectary-reared tsetse were more pathogenic than those isolated from the field. Fungi of three genera were also isolated from field-collected adult G. pallidipes, Penicillium, Aspergillus and Fusarium. Five genera, Aspergillus, Penicillium, Rhizopus, Mucor and Trichoderma were isolated from field-collected G. pallidipes pupae. The fungi, however, were only mildly pathogenic for adult tsetse and were not pathogenic for pupae.