Mauricio R. Dimitrov

Effects of the antibiotic enrofloxacin on the ecology of tropical eutrophic freshwater microcosms

The main objective of the present study was to assess the ecological impacts of the fluoroquinolone antibiotic enrofloxacin on the structure and functioning of tropical freshwater ecosystems. Enrofloxacin was applied at a concentration of 1, 10, 100 and 1,000 μg/L for 7 consecutive days in 600-L outdoor microcosms in Thailand. The ecosystem-level effects of enrofloxacin were monitored on five structural (macroinvertebrates, zooplankton, phytoplankton, periphyton and bacteria) and two functional (organic matter decomposition and nitrogen cycling) endpoint groups for 4 weeks after the last antibiotic application. Enrofloxacin was found to dissipate relatively fast from the water column (half-dissipation time: 11.7h), and about 11% of the applied dose was transformed into its main by-product ciprofloxacin after 24h. Consistent treatment-related effects on the invertebrate and primary producer communities and on organic matter decomposition could not be demonstrated. Enrofloxacin significantly affected the structure of leaf-associated bacterial communities at the highest treatment level, and reduced the abundance of ammonia-oxidizing bacteria and ammonia-oxidizing archaea in the sediments, with calculated NOECs of 10 and <1 μg/L, respectively. The ammonia concentration in the microcosm water significantly increased in the highest treatment level, and nitrate production was decreased, indicating a potential impairment of the nitrification function at concentrations above 100 μg/L. The results of this study suggest that environmentally relevant concentrations of enrofloxacin are not likely to result in direct or indirect toxic effects on the invertebrate and primary producer communities, nor on important microbially mediated functions such as nitrification.

Impact of a wastewater treatment plant on microbial community composition and function in a hyporheic zone of a eutrophic river

The impact of the installation of a technologically advanced wastewater treatment plant (WWTP) on the benthic microbial community of a vinyl chloride (VC) impacted eutrophic river was examined two years before, and three and four years after installation of the WWTP. Reduced dissolved organic carbon and increased dissolved oxygen concentrations in surface water and reduced total organic carbon and total nitrogen content in the sediment were recorded in the post-WWTP samples. Pyrosequencing of bacterial 16S rRNA gene fragments in sediment cores showed reduced relative abundance of heterotrophs and fermenters such as Chloroflexi and Firmicutes in more oxic and nutrient poor post-WWTP sediments. Similarly, quantitative PCR analysis showed 1–3 orders of magnitude reduction in phylogenetic and functional genes of sulphate reducers, denitrifiers, ammonium oxidizers, methanogens and VC-respiring Dehalococcoides mccartyi. In contrast, members of Proteobacteria adapted to nutrient-poor conditions were enriched in post-WWTP samples. This transition in the trophic state of the hyporheic sediments reduced but did not abolish the VC respiration potential in the post-WWTP sediments as an important hyporheic sediment function. Our results highlight effective nutrient load reduction and parallel microbial ecological state restoration of a human-stressed urban river as a result of installation of a WWTP.

Cultivation-Based and Molecular Assessment of Bacterial Diversity in the Rhizosheath of Wheat under Different Crop Rotations.

A field study was conducted to compare the formationand bacterial communities of rhizosheaths of wheat grown under wheat-cotton and wheat-rice rotation and to study the effects of bacterial inoculation on plant growth. Inoculation of Azospirillum sp. WS-1 and Bacillus sp. T-34 to wheat plants increased root length, root and shoot dry weight and dry weight of rhizosheathsoil when compared to non-inoculated control plants, and under both crop rotations. Comparing both crop rotations, root length, root and shoot dry weight and dry weight of soil attached with roots were higher under wheat-cotton rotation. Organic acids (citric acid, malic acid, acetic acid and oxalic acid) were detected in rhizosheaths from both rotations, with malic acid being most abundant with 24.8±2 and 21.3±1.5 μg g-1 dry soil in wheat-cotton and wheat-rice rotation, respectively. Two sugars (sucrose, glucose) were detected in wheat rhizosheath under both rotations, with highest concentrations of sucrose (4.08±0.5 μg g-1and 7.36±1.0 μg g-1) and glucose (3.12±0.5 μg g-1 and 3.01± μg g-1) being detected in rhizosheaths of non-inoculated control plants under both rotations. Diversity of rhizosheath-associated bacteria was evaluated by cultivation, as well as by 454-pyrosequencing of PCR-tagged 16S rRNA gene amplicons. A total of 14 and 12 bacterial isolates predominantly belonging to the genera Arthrobacter, Azospirillum, Bacillus, Enterobacter and Pseudomonaswere obtained from the rhizosheath of wheat grown under wheat-cotton and wheat-rice rotation, respectively. Analysis of pyrosequencing data revealed Proteobacteria, Bacteriodetes and Verrucomicrobia as the most abundant phyla in wheat-rice rotation, whereas Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria were predominant in wheat-cotton rotation. From a total of 46,971 sequences, 10.9% showed ≥97% similarity with 16S rRNA genes of 32 genera previously shown to include isolates with plant growth promoting activity (nitrogen fixation, phosphate-solubilization, IAA production). Among these, the most predominant genera were Arthrobacter, Azoarcus, Azospirillum, Bacillus, Cyanobacterium, Paenibacillus, Pseudomonas and Rhizobium.

Molecular Assessment of Bacterial Community Dynamics and Functional End Points during Sediment Bioaccumulation Tests.

Whole sediment toxicity tests play an important role in environmental risk assessment of organic chemicals. It is not clear, however, to what extent changing microbial community composition and associated functions affect sediment test results. We assessed the development of bacterial communities in artificial sediment during a 28 day bioaccumulation test with polychlorinated biphenyls, chlorpyrifos, and four marine benthic invertebrates. DGGE and 454-pyrosequencing of PCR-amplified 16S rRNA genes were used to characterize bacterial community composition. Abundance of total bacteria and selected genes encoding enzymes involved in important microbially mediated ecosystem functions were measured by qPCR. Community composition and diversity responded most to the time course of the experiment, whereas organic matter (OM) content showed a low but significant effect on community composition, biodiversity and two functional genes tested. Moreover, OM content had a higher influence on bacterial community composition than invertebrate species. Medium OM content led to the highest gene abundance and is preferred for standard testing. Our results also indicated that a pre-equilibration period is essential for growth and stabilization of the bacterial community. The observed changes in microbial community composition and functional gene abundance may imply actual changes in such functions during tests, with consequences for exposure and toxicity assessment.

Effect of Burkholderia tropica and Herbaspirillum frisingense strains on sorghum growth is plant genotype dependent

Sorghum is a multipurpose crop that is cultivated worldwide. Plant growth-promoting bacteria (PGPB) have important roles in enhancing sorghum biomass and nutrient uptake and suppressing plant pathogens. The aim of this research was to test the effects of the endophytic bacterial species Kosakonia radicincitans strain IAC/BECa 99, Enterobacter asburiae strain IAC/BECa 128, Pseudomonas fluorescens strain IAC/BECa 141, Burkholderia tropica strain IAC/BECa 135 and Herbaspirillum frisingense strain IAC/BECa 152 on the growth and root architecture of four sorghum cultivars (SRN-39, Shanqui-Red, BRS330, BRS509), with different uses and strigolactone profiles. We hypothesized that the different bacterial species would trigger different growth plant responses in different sorghum cultivars. Burkholderia tropica and H. frisingense significantly increased the plant biomass of cultivars SRN-39 and BRS330. Moreover, cultivar BRS330 inoculated with either strain displayed isolates significant decrease in average root diameter. This study shows that Burkholderia tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 are promising PGPB strains for use as inocula for sustainable sorghum cultivation.

Dominance of bacterial ammonium oxidizers and fungal denitrifiers in the complex nitrogen cycle pathways related to nitrous oxide emission

Organic compounds and mineral nitrogen (N) usually increase nitrous oxide (N2O) emissions. Vinasse, a by‐product of bio‐ethanol production that is rich in carbon, nitrogen, and potassium, is recycled in sugarcane fields as a bio‐fertilizer. Vinasse can contribute significantly to N2O emissions when applied with N in sugarcane plantations, a common practice. However, the biological processes involved in N2O emissions under this management practice are unknown. This study investigated the roles of nitrification and denitrification in N2O emissions from straw‐covered soils amended with different vinasses (CV: concentrated and V: nonconcentrated) before or at the same time as mineral fertilizers at different time points of the sugarcane cycle in two seasons. N2O emissions were evaluated for 90 days, the period that occurs most of the N2O emission from fertilizers; the microbial genes encoding enzymes involved in N2O production (archaeal and bacterial amoA, fungal and bacterial nirK, and bacterial nirS and nosZ), total bacteria, and total fungi were quantified by real‐time PCR. The application of CV and V in conjunction with mineral N resulted in higher N2O emissions than the application of N fertilizer alone. The strategy of vinasse application 30 days before mineral N reduced N2O emissions by 65% for CV, but not for V. Independent of rainy or dry season, the microbial processes were nitrification by ammonia‐oxidizing bacteria (AOB) and archaea and denitrification by bacteria and fungi. The contributions of each process differed and depended on soil moisture, soil pH, and N sources. We concluded that amoA‐AOB was the most important gene related to N2O emissions, which indicates that nitrification by AOB is the main microbial‐driven process linked to N2O emissions in tropical soil. Interestingly, fungal nirK was also significantly correlated with N2O emissions, suggesting that denitrification by fungi contributes to N2O emission in soils receiving straw and vinasse application.

Successive DNA extractions improve characterization of soil microbial communities

Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies describing and comparing soil microbial communities are based on a single DNA extraction, which may not lead to a representative recovery of DNA from all organisms present in the soil. The use of successive DNA extractions might improve soil microbial characterization, but the benefit of this approach has only been limitedly studied. To determine whether successive DNA extractions of the same soil sample would lead to different observations in terms of microbial abundance and community composition, we performed three successive extractions, with two widely used commercial kits, on a range of clay and sandy soils. Successive extractions increased DNA yield considerably (1–374%), as well as total bacterial and fungal abundances in most of the soil samples. Analysis of the 16S and 18S ribosomal RNA genes using 454-pyrosequencing, revealed that microbial community composition (taxonomic groups) observed in the successive DNA extractions were similar. However, successive DNA extractions did reveal several additional microbial groups. For some soil samples, shifts in microbial community composition were observed, mainly due to shifts in relative abundance of a number of microbial groups. Our results highlight that performing successive DNA extractions optimize DNA yield, and can lead to a better picture of overall community composition.

Bacterial glycerol oxidation coupled to sulfate reduction at neutral and acidic pH

Glycerol is a main co-product of biodiesel production. Crude glycerol may serve as a cheap and attractive substrate in biotechnological applications, e.g. for the production of valuable chemicals or as an electron donor for reduction processes. In this work, sulfate reduction with glycerol was studied at neutral and acidic pH using bioreactor sludge samples and Tinto River sediments as a source of inoculum, respectively. Communities of sulfate-reducing bacteria (SRB) and fermentative bacteria were co-enriched at both pH values. Molecular analyses revealed that sequences belonging to Desulfomicrobium genus were dominant in the cultures enriched at pH 7, while Desulfosporosinus sequences dominated in the culture enriched at pH 4. Glycerol conversion was coupled to sulfate reduction, but the substrate was incompletely oxidized to acetate in the neutrophilic enrichments, and acetate, lactate, and 1,3-propanediol under low pH conditions. Two strains belonging to Desulfomicrobium and Proteiniphilum genera were isolated from the neutrophilic enrichments, but the first isolate was not able to use glycerol, which suggests a syntrophic relationship between glycerol-degrading fermentative bacteria and SRB. A Clostridium strain able to grow with glycerol was isolated from the low pH enrichment. Our data indicate that glycerol promotes the growth of sulfate-reducing communities to form sulfide, which can be used to precipitate and recover heavy metals.