Arnaldo Chaer Borges

Characterizing the microbiota across the gastrointestinal tract of a Brazilian Nelore steer.

The gastrointestinal tracts (GIT) of herbivores harbor dense and diverse microbiota that has beneficial interactions with the host, particularly for agriculturally relevant animals like ruminants such as cattle. When assessing ruminant health, microbiological indicators are often derived from the rumen or feces. However, it is probable that ruminal and fecal microbiota do not reflect the microbial communities within the GIT of ruminants. To test this, we investigated the compartments of the GIT from a Brazilian Nelore steer and performed a 16S rRNA pyrosequencing analysis on the collected samples. Our results showed high intra-individual variation, with samples clustering according to their location in the GIT including the forestomach, small intestine, and large intestine. Although sequences related to the phyla Firmicutes and Bacteroidetes predominated all samples, there was a remarkable variation at the family level. Comparisons between the microbiota in the rumen, feces, and other GIT components showed distinct differences in microbial community. This work is the first intensive non-culture based GIT microbiota analysis for any ruminant and provides a framework for understanding how host microbiota impact the health of bovines.

Evaluation of bacterial surfactant toxicity towards petroleum degrading microorganisms

The acute toxicity of bacterial surfactants LBBMA111A, LBBMA155, LBBMA168, LBBMA191 and LBBMA201 and the synthetic surfactant sodium dodecyl sulfate (SDS) on the bioluminescent bacterium Vibrio fischeri was evaluated by measuring the reduction of light emission (EC(20)) by this microorganism when exposed to different surfactant concentrations. Moreover, the toxic effects of different concentrations of biological and synthetic surfactants on the growth of pure cultures of isolates Acinetobacter baumannii LBBMA04, Acinetobacter junni LBBMA36, Pseudomonas sp. LBBMA101B and Acinetobacter baumanni LBBMAES11 were evaluated in mineral medium supplemented with glucose. The EC(20) values obtained confirmed that the biosurfactants have a significantly lower toxicity to V. fischeri than the SDS. After 30 min of exposure, bacterial luminescence was almost completely inhibited by SDS at a concentration of 4710 mg L(-1). Growth reduction of pure bacterial cultures caused by the addition of biosurfactants to the growth medium was lower than that caused by SDS.

Biodegradability of bacterial surfactants.

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO2 evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO2 emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.

Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

Inter- and intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp), were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp.), and Laccaria (L. bicolor and L. amethystea). Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.

Epulorhiza epiphytica sp. nov. isolated from mycorrhizal roots of epiphytic orchids in Brazil

Abstract Epulorhiza epiphytica sp. nov. isolated from the roots of two Brazilian native epiphytic orchid species is described. In culture, it differs from the known species of Epulorhiza in the minute size of monilioid cells with foveate surfaces. This is the first report of an orchid mycorrhizal fungus from Brazil.

Vesicular-arbuscular-/ecto-mycorrhiza succession in seedlings of. Eucalyptus spp.

The occurrence of vesicular-arbuscular mycorrhizae (AM) and ectomycorrhizae (ECM) in the same root system was observed when species of Eucalyptus urophylla S.T. Blake, E. citriodora Hook f., E. grandis W. Hill ex Maiden, E. cloeziana F. Muell. and E. camaldulensis Dehnh were simultaneously inoculated with Glomus etunicatum Becker & Gederman and Pisolithus tinctorius (Per.) Cocker & Couch, isolate Pt 90A. The succession between the two fungi was observed. In general ectomycorrhizal colonization increased followed by a decrease in AM. Pisolithus tinctorius was favored in simultaneous inoculation with G. etunicatum, and the positive effect of the simultaneous inoculation of both fungi in the percent colonization by the AM fungus occurred up to 60 days after inoculation. After 120 days, colonization of roots by G. etunicatum decreased in the presence of P. tinctorius. When inoculated simultaneously, the proportion of AM and ECM varied with evaluation time, while the combined percentage of mycorrhizal roots approached the maximum and remained more or less constant after 60 days, suggesting that there could be competition between the fungi for limiting substrate. The maximum percent mycorrhizal colonization varied with Eucalyptus species and the highest value was observed for E. camaldulensis, followed in order by E. citriodora, E. urophylla, E. grandis and E. cloeziana.

ITS sequences and mitochondrial DNA polymorphism in Pisolithus isolates.

Genetic variability among isolates of the ectomycorrhizal fungus genus Pisolithus collected in Brazil, USA and France was examined, based on nucleotide sequence of ITS from rDNA genes and restriction polymorphism of the mitochondrial DNA (mtDNA). Alignment of the ITS sequences showed 97.0% homology among isolates from Brazil, 97.8% homology among isolates from USA and France and 75.7-78.5% among the isolates from Brazil and USA/France. These sequences were aligned with ITS sequences of Australian, Brazilian and Kenyan Pisolithus isolates described in the literature. A consensus tree generated by sequence data of all Pisolithus isolates showed the presence of a number of species among the isolates. The mtDNA analysis was carried out by total DNA restriction analysis with different enzymes (Dra I, EcoR I, EcoR V, Hha I-Xba I, Hind III and Rsa I) and probing with purified mtDNA labelled with fluorescein d-UTP. The size of the mitochondrial genome of isolates Pt 90A, Pt 185R and PF cleaved with EcoR I was estimated to be, approx. 40.5, 47.2 and 47.6 kb, respectively. Cluster analysis based on the mtDNA restriction fragments grouped the isolates into two distinct groups, which coincide with their host specificity and geographical origins. PCR-RFLP analysis of mitochondrial rDNA (mt rDNA) from 12 Pisolithus isolates did not show any polymorphism. These data corroborate previous results obtained in our laboratory with RAPD-PCR and rDNA PCR-RFLP markers and support recent taxonomic studies where a number of Pisolithus species were described.

Oil Recovery from Fuel Oil Storage Tank Sludge Using Biosurfactants

The petroleum industry generates large amounts of solid and semisolid wastes known as oily sludges. The composition of oily sludge varies due to the large diversity in the quality of crude oils, differences in the processes used for oil-water separation, leakages during industrial processes, and also mixing with the existing oily sludge. Usually, the oily sludge contains water, sand, oils, grease, organic compounds, chemical elements, and metals. Those sludges can be generated in several steps of the petroleum production and refining, such as in oil/water separation steps and in the bottom of tanks. The accumulation of oily residues in petroleum industry poses a serious environmental problem. The purpose of this work was evaluate an alternative process to removal of oily sludges through the use of biosurfactants to reduce the viscosity and promote formation of oil/water emulsions making sludge pumping easier and permitting crude oil recovery after breaking the emulsion. Five bacterial isolates were selected for their biosurfactant production potential after screening microorganisms recovered from oil-contaminated sites. Supernatants obtained from autoclave cell suspensions (hereby referred to as autoclaved-supernatant) were mixed with oily sludge collected from fuel oil storage tanks to a final concentration of 0.01%, in order to separate the oil from the inert material. The process proved to be highly efficient for oil recovery, and resulted in up to 95% reduction in sludge volume. The use of cell-free supernatant medium obtained from biosurfactant-producing bacterial strains to treat oily sludges may be an economically and environmentally viable technology, considering the small volume of microbial culture required for the treatment.

Mobilisation of bacteria in a fine-grained residual soil by electrophoresis.

An investigation of electrokinetic bacterial mobilisation in a residual soil from gneiss is presented here. The experimental program aimed at assessing the efficacy of electrophoresis against the electro-osmotic flow to transport endospores of Bacillus subtilis LBBMA 155 and nitrogen-starved cells of Pseudomonas sp. LBBMA 81. Electrokinesis was performed on a low hydraulic reconstituted clayey soil column submitted to a 5mA electrical current for 24h. Cells were coccoid-shaped and characterised as possessing low surface hydrophobicity and less than 1microm in diameter. Distribution coefficient for B. subtilis in the soil was between 16.8 and 19.9 times higher than that for Pseudomonas sp. Distribution coefficient for B. subtilis between eluate and anionic exchange column was 11.8 times higher than that for Pseudomonas sp. After the electrokinesis, it was shown that cells and endospores were distributed hyperbolically through the soil probe and moved against the electro-osmotic flow; however, endospores were transported throughout all soil core and starved cells only till half of its length. The higher transport efficiency of B. subtilis endospores was attributed to their higher negative charge on cell surface. These results demonstrate that electrokinesis can be used for bacteria transport in soils with low hydraulic conductivity, even against the electro-osmotic flow.

Efeito da monensina e extrato de própolis sobre a produção de amônia e degradabilidade in vitro da proteína bruta de diferentes fontes de nitrogênio

This experiment had as objective to evaluate protein fermentation of three nitrogen sources (trypticase, soybean meal and fish meal), with or without monensin or propolis extract addition. Incubations were done by using 7.2 mL of McDougall buffer, 2.0 mL of inocula, 0.2 mL of ethanolic solution with or without monensin or propolis and 84.4, 150 or 112.5 mg/10 mL of trypticase, soybean meal and fish meal, respectively, in a 3x3 factorial arranjement. The flasks were incubated anaerobically at 39oC in water bath during 120 hours, and samples were collected from the media over time for ammonia, microbial protein, soluble protein and protein degradability determinations. Fish meal caused lesser ammonia production than trypticase and soybean meal in control treatment, due to its lower degradability. Monensin and propolis decreased ammonia production in the trypticase and soybean meal treatments, leading to accumulation of soluble protein in the media. The microbial protein synthesis was similar among the three feed sources and with presence or absence of inhibitors, except in the case of propolis that estimulated synthesis in the fish meal treatment. There was greater protein degradability of the control treatment for soybean meal (73%) than fish meal (42%). Monensin reduced degradation of trypticase and soybean meal, by inhibiting ammonia production, and propolis increased degradation of fish meal, by increasing soluble protein concentration in the media. Due to the in vitro effect of propolis on fermentation activity, it is necessary to carry out researches in order to verify its effect on ruminal fermentation and animal performance.