Phylogenetic Analysis Of The Microbial Community In Hypersaline Petroleum Produced Water From The Campos Basin.

In this work the archaea and eubacteria community of a hypersaline produced water from the Campos Basin that had been transported and discharged to an onshore storage facility was evaluated by 16S recombinant RNA (rRNA) gene sequence analysis. The produced water had a hypersaline salt content of 10 (w/v), had a carbon oxygen demand (COD) of 4,300 mg/l and contains phenol and other aromatic compounds. The high salt and COD content and the presence of toxic phenolic compounds present a problem for conventional discharge to open seawater. In previous studies, we demonstrated that the COD and phenolic content could be largely removed under aerobic conditions, without dilution, by either addition of phenol degrading Haloarchaea or the addition of nutrients alone. In this study our goal was to characterize the microbial community to gain further insight into the persistence of reservoir community members in the produced water and the potential for bioremediation of COD and toxic contaminants. Members of the archaea community were consistent with previously identified communities from mesothermic reservoirs. All identified archaea were located within the phylum Euryarchaeota, with 98 % being identified as methanogens while 2 % could not be affiliated with any known genus. Of the identified archaea, 37 % were identified as members of the strictly carbon-dioxide-reducing genus Methanoplanus and 59 % as members of the acetoclastic genus Methanosaeta. No Haloarchaea were detected, consistent with the need to add these organisms for COD and aromatic removal. Marinobacter and Halomonas dominated the eubacterial community. The presence of these genera is consistent with the ability to stimulate COD and aromatic removal with nutrient addition. In addition, anaerobic members of the phyla Thermotogae, Firmicutes, and unclassified eubacteria were identified and may represent reservoir organisms associated with the conversion hydrocarbons to methane.

Performance of a horizontal-flow anaerobic immobilized biomass (HAIB) reactor and dynamics of the microbial community during degradation of pentachlorophenol (PCP)

The anaerobic biological treatment of pentachlorophenol (PCP) and methanol as the main carbon source was investigated in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor at 30 +/- 1 degrees C, during a 220-day trial period. The reactor biomass was developed as an attached biofilm on polyurethane foam particles, with 24 h of hydraulic retention time. The PCP concentrations, which ranged from 2.0 to 13.0 mg/L, were controlled by adding synthetic substrate. The HAIB reactor reduced 97% of COD and removed 99% of PCP. The microbial biofilm communities of the HAIB reactor amended with PCP, without previous acclimatization, were characterized by polymerase chain reaction (PCR) and amplified ribosomal DNA restriction analysis (ARDRA) with specific Archaea oligonucleotide primers. The ARDRA technique provided an adequate analysis of the community, revealing the profile of the selected population along the reactor. The biomass activities in the HAIB reactor at the end of the experiments indicated the development of PCP degraders and the maintenance of the population of methanogenic Archaea, ensuring the high efficiency of the system treating PCP with added methanol as the cosubstrate. The use of the simplified ARDRA method enabled us to monitor the microbial population with the addition of high concentrations of toxic compounds and highlighting a selection of microorganisms in the biofilm. (C) 2008 Published by Elsevier Ltd.

The effects of copper on the microbial community of a coral reef sponge

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators far sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha -proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 mug l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 mug l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 mug l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction In the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 mug l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morphotype actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 mug l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 mug l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 mug l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 mug l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor

A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67+/-13.86 mg P l(-1) was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04+/-1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 mum) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 mum) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria , but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.

Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM 156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Enrichment of a PHA producing microbial community in a continuous bioreactor setup

Polyhydroxyalkanoates (PHAs) are biosynthetic polyesters, biodegradable and biocompatible making them of great interest for industrial purposes. The use of low value substrates with mixed microbial communities (MMC) is a strategy currently used to decrease the elevated PHA production costs. PHA production process requires an important step for selection and enrichment of PHA-storing microorganisms which is usually carried out in a Sequencing Batch Reactor (SBR). The aim of this study was to optimize the PHA accumulating culture selection stage using a 2-stage Continuous Stirrer Tank Reactor (CSTR) system. The system was composed by two separate feast and famine bioreactors operated continuously, mimicking the feast and famine phases in a SBR system. Acetate was used as carbon source and biomass seed was highly enriched in Plasticicumulans acidivorans obtained from activated sludge. The system was operated under two different sets of conditions (setup 1 and 2), maintaining a system total retention time of 12 hours and an OLR of 2.25 Cmmol/L.h-1. An average PHB-content of 3.3 % wt was obtained in setup 1 and 4.8% wt in setup 2. Several other experiments were performed in order to better understand the continuous system behaviour, using biomass from the continuous system. With the fed-batch experiment a maximum of 8.1% PHB was stored and the maximum substrate uptake and specific growth rates obtained in the growth experiment (1.15 Cmol Cmol-1.h-1 and 0.53 Cmol Cmol-1.h-1) were close to the ones from continuous system (1.12 Cmol Cmol-1.h-1 and 0.59 Cmol Cmol-1.h-1). The microbial community was characterized trough microscopic visualization, Denaturing Gradient Gel Electrophoresis (DGGE) analysis and Fluorescent in situ hybridization (FISH). The last studied performed mimicked the continuous system by building up a SBR system with all the same operational conditions while adding an extra acetate dosage during the 12 h cycle, simulating the substrate passing from the feast to the famine reactors under continuous operation. It was shown that possibly the continuous system was not able to efficiently select for PHB storing organisms under the operational conditions imposed, although the selected culture was capable of consuming the substrate and grow fast. This main conclusion might have resulted from two major factors affecting the system performance: the ammonium concentration in the Feast reactor and the amount of substrate leaching from the Feast to the Famine reactor.

Saprotrophic fungi Hypholoma fasciculare effect on the fungal community associated to Castanea sativa

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.

La biodiversidad microbiana del suelo y su rol en el manejo de enfermedades causadas por hongos de suelo en soja. Soil microbial diversity and its key role in the management of soybean diseases caused by fungi.

Las poblaciones microbianas juegan un rol fundamental en la estabilidad de los sistemas agrícolas e indican los niveles de salud de un suelo, tanto que pueden ser utilizadas como indicadores de sustentabilidad de un agroecosistema. Los microorganismos reflejan el efecto que tienen las prácticas agrícolas sobre el suelo a través de modificaciones en la abundancia (biodiversidad estructural) y actividades de sus poblaciones (como control biológico de los patógenos, entre otras). Al cuantificarse la biodiversidad microbiana nativa se puede conocer la riqueza de un agroecosistema y utilizarla para el manejo sustentable de hongos patógenos. En este trabajo se evaluará el efecto de la rotación de cultivo (soja-maíz y soja en monocultivo) y los sistemas de labranza (siembra directa y labranzas reducida) sobre la biodiversidad microbiana. Se cuantificarán a partir de suelo: poblaciones de hongos y bacterias totales; agentes potenciales de biocontrol de como Trichoderma spp., Gliocladium spp. y micorrizas vesículo arbusculares (mediante la cuantificación de glomalina), biomasa y respiracion microbiana, y la biodiversidad de comunidades de microorganismos que habitan en el suelo mediante el análisis de perfiles de ácidos grasos (PLFA). Al final del ciclo de cultivo de soja se cuantificará la incidencia de enfermedades causadas por hongos de suelo. Dada la gran abundancia y diversidad de los microorganismos del suelo, las metodologías que se emplearán permitirán obtener información global de la riqueza microbiana de un agro-ecosistema. Se relacionará la biodiversidad microbiana con la incidencia de enfermedades por hongos de suelo, en respuesta a diferentes prácticas de manejo. Esto permitirá aprender a combinar las tecnologías para mejorar los beneficios de la produccion y preservar el agroecosistema en el marco de una agricultura sustentable, y no de una agricultura sostenida por insumos.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA)

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed

Soil microbial biomass under different management and tillage systems of permanent intercropped cover species in an orange orchard

To mitigate soil erosion and enhance soil fertility in orange plantations, the permanent protection of the inter-rows by cover species has been suggested. The objective of this study was to evaluate alterations in the microbial biomass, due to different soil tillage systems and intercropped cover species between rows of orange trees. The soil of the experimental area previously used as pasture (Brachiaria humidicola) was an Ultisol (Typic Paleudult) originating from Caiuá sandstone in the northwestern part of the State of Paraná, Brazil. Two soil tillage systems were evaluated: conventional tillage (CT) in the entire area and strip tillage (ST) (strip width 2 m), in combination with different ground cover management systems. The citrus cultivar 'Pera' orange (Citrus sinensis) grafted onto 'Rangpur' lime rootstock was used. Soil samples were collected after five years of treatment from a depth of 0-15 cm, under the tree canopy and in the inter-row, in the following treatments: (1) CT and an annual cover crop with the leguminous species Calopogonium mucunoides; (2) CT and a perennial cover crop with the leguminous peanut Arachis pintoi; (3) CT and an evergreen cover crop with Bahiagrass Paspalum notatum; (4) CT and a cover crop with spontaneous Brachiaria humidicola grass vegetation; and (5) ST and maintenance of the remaining grass (pasture) of Brachiaria humidicola. Soil tillage and the different cover species influenced the microbial biomass, both under the tree canopy and in the inter-row. The cultivation of brachiaria increased C and N in the microbial biomass, while bahiagrass increased P in the microbial biomass. The soil microbial biomass was enriched in N and P by the presence of ground cover species and according to the soil P content. The grass species increased C, N and P in the soil microbial biomass from the inter-row more than leguminous species.

An improved method for measuring soil microbial activity by gas phase flow injection analysis

The rate of carbon dioxide production is commonly used as a measure of microbial activity in the soil. The traditional method of CO2 determination involves trapping CO2 in an alkali solution and then determining CO2 concentration indirectly by titration of the remaining alkali in the solution. This method is still commonly employed in laboratories throughout the world due to its relative simplicity and the fact that it does not require expensive, specific equipment. However, there are several drawbacks: the method is time-consuming, requires large amounts of chemicals and the consistency of results depends on the operator's skills. With this in mind, an improved method was developed to analyze CO2 captured in alkali traps, which is cheap and relatively simple, with a substantially shorter sample handling time and reproducibility equivalent to the traditional titration method. A comparison of the concentration values determined by gas phase flow injection analysis (GPFIA) and titration showed no significant difference (p > 0.05), but GPFIA has the advantage that only a tenth of the sample volume of the titration method is required. The GPFIA system does not require the purchase of new, costly equipment but the device was constructed from items commonly found in laboratories, with suggestions for alternative configurations for other detection units. Furthermore, GPFIA for CO2 analysis can be equally applied to samples obtained from either the headspace of microcosms or from a sampling chamber that allows CO2 to be released from alkali trapping solutions. The optimised GPFIA method was applied to analyse CO2 released from degrading hydrocarbons from a site contaminated by diesel spillage.

Fusarium oxysporum strains as biocontrol agents against Fusarium wilt: effects on soil microbial biomass and activity

Before planning the large-scale use of nonpathogenic strains of Fusarium oxysporum as biocontrol agents of Fusarium wilt, their behaviour and potential impact on soil ecosystems should be carefully studied as part of risk assessment. The aim of this work was to evaluate the effects of antagonistic F. oxysporum strains, genetically manipulated (T26/6) or not (233/1), on soil microbial biomass and activity. The effects were evaluated, in North-western Italy, in two soils from different sites at Albenga, one natural and the other previously solarized, and in a third soil obtained from a 10-year-old poplar stand (Popolus sp.), near Carignano. There were no detectable effects on ATP, fluorescein diacetate hydrolysis, and biomass P that could be attributed to the introduction of the antagonists. A transient increase of carbon dioxide evolution and biomass C was observed in response to the added inoculum. Although the results showed only some transient alterations, further studies are required to evaluate effects on specific microorganism populations.

Effects of soil management systems on soil microbial activity, bulk density and chemical properties

The objective of this experiment was to study the effects of soil management systems on the bulk density, chemical soil properties, and on the soil microbial activity on a Latossolo Vermelho distrófico (Oxisol). Soil samples were collected from plots under the following management conditions: a) natural dense "cerrado" vegetation (savanna); b) degraded Brachiaria decumbens pasture, 20 years old; c) no-tillage treatment with annual crop sequence (bean, corn, soybean and dark-oat in continuous rotation), 8 years old; d) conventional tillage treatment with crop residues added to the soil, and annual crop sequence, 10 years old. The continuous use of no-tillage system resulted in an increase in microbial biomass and decrease in soil basal respiration, therefore displaying evident long-term effects on the increase of soil C content. The no-tillage system also provided an improvement in bulk density and chemical properties of the soil. Hence, the no-tillage management system could be an alternative for the conservation and maintenance of physical and chemical conditions and the productive potential of "cerrado" soils.