Seasonal effects in a lake sediment archaeal community of the brazilian savanna

The Cerrado is a biome that corresponds to 24% of Brazil’s territory. Only recently microbial communities of this biome have been investigated. Here we describe for the first time the diversity of archaeal communities from freshwater lake sediments of the Cerrado in the dry season and in the transition period between the dry and rainy seasons, when the first rains occur. Gene libraries were constructed, using Archaea-specific primers for the 16S rRNA and amoA genes. Analysis revealed marked differences between the archaeal communities found in the two seasons. I.1a and I.1c Thaumarchaeota were found in greater numbers in the transition period, while MCG Archaea was dominant on the dry season. Methanogens were only found in the dry season. Analysis of 16S rRNA sequences revealed lower diversity on the transition period. We detected archaeal amoA sequences in both seasons, but there were more OTUs during the dry season. These sequences were within the same cluster as Nitrosotalea devanaterra’s amoA gene. The principal coordinate analysis (PCoA) test revealed significant differences between samples from different seasons. These results provide information on archaeal diversity in freshwater lake sediments of the Cerrado and indicates that rain is likely a factor that impacts these communities

Soil acidobacterial 16S rRNA gene sequences reveal subgroup level differences between savanna-like cerrado and atlantic forest brazilian biomes

16S rRNA sequences from the phylum Acidobacteria have been commonly reported from soil microbial communities, including those from the Brazilian Savanna (Cerrado) and the Atlantic Forest biomes, two biomes that present contrasting characteristics of soil and vegetation. Using 16S rRNA sequences, the present work aimed to study acidobacterial diversity and distribution in soils of Cerrado savanna and two Atlantic forest sites. PCA and phylogenetic reconstruction showed that the acidobacterial communities found in “Mata de galeria” forest soil samples from the Cerrado biome have a tendency to separate from the other Cerrado vegetation microbial communities in the direction of those found in the Atlantic Forest, which is correlated with a high abundance of Acidobacteria subgroup 2 (GP2). Environmental conditions seem to promote a negative correlation between GP2 and subgroup 1 (GP1) abundance. Also GP2 is negatively correlated to pH, but positively correlated to high Al3+ concentrations. The Cerrado soil showed the lowest Acidobacteria richness and diversity indexes of OTUs at the species and subgroups levels when compared to Atlantic Forest soils. These results suggest specificity of acidobacterial subgroups to soils of different biomes and are a starting point to understand their ecological roles, a topic that needs to be further explored

Microbial community profile and water quality in a protected area of the caatinga biome

The Caatinga is a semi-arid biome in northeast Brazil. The Paraguaçú River is located in the Caatinga biome, and part of its course is protected by the National Park of Chapada Diamantina (PNCD). In this study we evaluated the effect of PNCD protection on the water quality and microbial community diversity of this river by analyzing water samples obtained from points located inside and outside the PNCD in both wet and dry seasons. Results of water quality analysis showed higher levels of silicate, ammonia, particulate organic carbon, and nitrite in samples from the unprotected area compared with those from protected areas. Pyrosequencing of the 16S rRNA genes revealed that Burkholderiales was abundant in samples from all three sites during both seasons and was represented primarily by the genus Polynucleobacter and members of the Comamonadaceae family (e.g., genus Limnohabitans). During the dry season, the unprotected area showed a higher abundance of Flavobacterium sp. and Arthrobacter sp., which are frequently associated with the presence and/or degradation of arsenic and pesticide compounds. In addition, genes that appear to be related to agricultural impacts on the environment, as well as those involved in arsenic and cadmium resistance, copper homeostasis, and propanediol utilization, were detected in the unprotected areas by metagenomic sequencing. Although PNCD protection improves water quality, agricultural activities around the park may affect water quality within the park and may account for the presence of bacteria capable of pesticide degradation and assimilation, evidencing possible anthropogenic impacts on the Caatinga

Riqueza do domínio Archaea no solo do bioma Cerrado

Dissertação (mestrado)—Universidade de Brasília, Instituto de Biologia, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2012.O Domínio Archaea vem sendo descrito principalmente através de técnicas moleculares, independentes de cultivo e, atualmente, é dividido em dois filos formalmente aceitos, Crenarchaeota e Euryarchaeota, além de quatro outros propostos – Korarchaeota, Nanoarchaeota, Thaumarchaeota e Aigarchaeota. Uma vez que sequências do gene de rRNA 16S de Archaea foram descritas a partir de amostras de diferentes ambientes, as archaeas passaram a ser consideradas organismos ubíquos na natureza. Este trabalho tem como objetivo caracterizar a riqueza de archaeas em amostras de solo de Cerrado - a principal vegetação do Brasil Central, sendo a segunda maior do país. O DNA metagenômico de amostras em duplicata de solos provenientes de duas fitofisionomias distintas, cerrado denso e mata de galeria, foi extraído empregando-se o PowerSoil DNA Isolation Kit (MOBio Laboratories Inc.). A construção de bibliotecas genômicas foi feita a partir de experimentos de PCR, com iniciadores Archaea-específicos, visando amplificar regiões do gene de rRNA16S a partir do DNA extraído das diferentes amostras. Os resultados obtidos a partir dos fragmentos sequenciados revelaram identidade com o domínio Archaea em 96% das sequências, todas pertencentes ao filo Crenarchaeota (possivelmente Thaumarchaeota), principalmente aos grupos I.1b e I.1c. Somente nas amostras de Mata de Galeria foram encontradas sequências do grupo I.1a. A riqueza de Archaea foi maior em cerrado denso do que em mata de galeria. Os cálculos não-paramétricos tais como as curvas de rarefação indicam que a amostragem do ambiente foi adequada. As amostras das duas fitofisionomias apresentam diferenças significativas pelas técnicas estatísticas de β-diversidade ∫-Libshuff e pelo gráfico de Análise de Principais Coordenadas (PCA). ______________________________________________________________________________ ABSTRACTThe domain Archaea has been described mostly by culture-independent methods. Currently it is divided into two well accepted phyla, namely the Euryarchaeota and Crenarchaeota, and an additional four proposed phyla: Korarchaeota, Nanoarchaeota, Thaumarchaeota and Aigarchaeota. Since their 16S rDNA sequences have been described from diverse ecosystems not classified as extreme, Archaea is now considered ubiquitous and not a domain of only extreme organisms. This work describes the richness of the Archaea domain retrieved from soil samples of the Cerrado biome – the main vegetation of Central Brazil and second largest biome in the country. The metagenomic DNA from two replicas of each two distinct phytophysiognomies, cerrado denso and mata de galeria, was extracted using the PowerSoil DNA Isolation Kit (MOBio Laboratories Inc.). The four DNA libraries were constructed through PCR amplification of the 16S rDNA using Archaea-specific primers. Our results reveal that 96% of the sequenced fragments have identity with the domain Archaea. All of these sequences are from the phylum Crenarchaeota (possibly Thaumarchaeota), predominantly affiliated to group I.1b and I.1c. Sequences afilliated to the group I.1a were only found on the soil from mata de galeria. The soils from cerrado denso have a greater richness of Archaea than those from mata de galeria. The non-parametric estimatives showed that both the environments have been well sampled. There is a significant difference between the soil samples of the two phytophysiognomies shown by the statistical test of ∫-Libshuff and by the Principal Coordenates Analysis graphic (PCA)

Microbiologia do ciclo do nitrogênio em solos do cerrado

A interação entre as variáveis do solo e a microbiota influencia os processos que ocorrem no solo, tanto que, em ambientes terrestres o N é reciclado primariamente pela microbiota. No ciclo do N, a nitrificação é a etapa em que nitrato se torna disponível no solo para as plantas, mas também N é perdido por lixiviação de nitrato ou pela emissão de gases nitrogenados. Entretanto, as mudanças climáticas, a modificação do uso da terra e a aplicação de fertilizantes nitrogenados veem alterando a dinâmica de N. Um especial interesse é direcionado à maior savana na América do Sul, o bioma tropical sazonal seco que é o Cerrado, cuja paisagem vem sendo alterada pela agricultura. Fazendo uso da técnica de metagenômica, os atributos funcionais da microbiota do solo do Cerrado quanto ao ciclo do N foram comparados entre dois parques de conservação do bioma, distantes 500 km entre si, com variação na textura e no conteúdo de água do solo. Os tipos de vegetação amostradas dentro de cada parque mascararam os efeitos de altitude e distância entre os parques, e todas as amostras apresentaram uma maior abundância de genes para assimilação de amônia e amonificação. Isso corrobora a literatura encontrada sobre o metabolismo de amônia como forma principal de N no Cerrado. Em particular, o Campo limpo alagado, presente somente em um dos parques, apresentou a maior abundância de genes fixadores de nitrogênio. Ainda, foram detectados genes para denitrificação, mas somente dois hits foram observados para nitrificação. Sucessivamente, foi acessado o impacto do manejo do solo sobre a abundância de Archaea e Bacteria oxidantes de amônia por quantificação do gene marcador amoA ao longo do cultivo da soja no bioma Cerrado. A análise molecular, tal como as técnicas clássicas e de isótopos mostraram um maior conteúdo de C orgânico e de NH4+-N no pousio em comparação à área nativa de reserva legal adjacente ao plantio da soja. De mesma forma, observou-se um aumento na abundância de oxidantes de amônia e da taxa de nitrificação no solo agrícola em comparação à área nativa, com a menor razão amônia/nitrato observada no solo após revolvimento. A abundância de AOB apresentou correlação com o aumento de pH ao longo do cultivo da soja. Experimentos seguintes testaram o efeito de água e de pH em microcosmos contendo solo do Cerrado, tal como a possível inibição de nitrificação em slurries contendo uma mistura de solo do Cerrado com um solo agrícola (Craibstone) com reconhecida atividade de oxidação de amônia. No entanto, o acúmulo de NO3- estava abaixo do nível de detecção na maior parte das amostras, tanto naquelas com aumento no teor gravimétrico de água ou com aumento de pH, independente da alta concentração de amônia. A nitrificação não foi inibida nas misturas de slurries incubadas, e, ainda, após 21 dias de incubação foi possível detectar transcritos de amoA de AOA no slurry de solo de Cerrado. Os perfis de DGGE mostraram um maior número de bandas de AOA amoA nos slurries de Craibstone e das misturas dos dois solos, do que o perfil observado nos slurries incubados somente com solo do Cerrado. Considerando o exposto acima, este foi o primeiro trabalho apresentado sobre o metabolismo de N e mais especificamente sobre a oxidação de amônia, utilizando dados de metagenomas e de PCR em tempo real. A baixa detecção de nitrato nas amostras de campo e de incubações em laboratório sugerem que algum outro mecanismo ocorre nos solos do bioma Cerrado no sentido de preservação de N inorgânico preferencialmente na forma de amônia. Sugerimos que a nitrificação depende da presença de oxidantes de amônia, mas também da composição da comunidade microbiana, sendo que a sua diversidade afeta a dinâmica de N no solo. Provavelmente condições abióticas e bióticas influenciam na limitação de crescimento da comunidade de oxidantes de amônia autotróficos no Cerrado. Por exemplo a competição por amônia entre esses oxidantes autotróficos e plantas ou com microorganismos heterotróficos. Ainda a redução dissimilatória de nitrato a amônia ou a imobilização abiótica de nitrato podem influenciar o desenvolvimento daquela comunidade. ______________________________________________________________________________________________ ABSTRACTInteractions between soil characteristics and microbiota influence the processes in soil ecosystem, as the terrestrial N is primarily cycled by the microbiota. In the N cycle, nitrification enables plants’ access to nitrate, although N can be lost through nitrate leaching, or N trace gas emission. These N dynamics are being disturbed by climate change, land use modification and the employment of nitrogenous fertilizers. A special interest goes to the largest savanna in South America, the seasonally dry Cerrado biome, where agriculture is changing the biome landscape. Shotgun metagenomics was used to compare the functional attributes of N cycling from the soil microbiota present in two conservation parks of the Cerrado biome, 500 km distant from each other, with varying soil texture and water content. Types of vegetation sampled within each park masked the altitude and distance effects, but all samples showed higher abundance of genes for assimilation of ammonia and ammonification. This corroborates Cerrado literature of ammonia as the main soil N form. In addition, a flooded grassland presented the highest abundance of N fixation genes. Despite the detection of denitrification genes, only two hits for the nitrification process were described. Subsequently, we assessed the impact of soil management on the abundance of Archaea (AOA) and Bacteria (AOB) ammonia oxidizers by quantification of the marker gene (amoA)during different stages of soybean cultivation within the Cerrado. Molecular analysis and classic and isotope techniques exhibited higher content of organic C and NH4+-N during fallow than in the adjacent undisturbed field, and an increase in ammonia oxidizers abundance and nitrification rates in the agricultural soil than in the undisturbed site, with the lowest ammonium/nitrate ratio in tilled soil. AOB abundance was correlated with the increase in pH during soybean cultivation. Further experiments tested the effect of moisture and pH in microcosms containing Cerrado soil, and the possible nitrification inhibition in slurries assembled with a mixture of Cerrado and agricultural soil known for actively oxidizing ammonia (Craibstone soil). Nevertheless, very little NO3- accumulation was observed in Cerrado microcosms with either increasing moisture or pH, despite high ammonia concentration. Nitrification was not inhibited in the mixed soilslurries, and after 21 days it was possible to detect the activity of AOA with the quantification of amoA transcripts. Moreover, DGGE profiles showed a higher number of AOA amoA gene in the Craibstone-only slurries and similar to the mixed slurries, but lower in the Cerrado-only slurries. This was the first assessment of the N metabolism with metagenomic data and qPCR for ammonia oxidation in the Cerrado. However, the little accumulation of NO3- in the field soils or in the treated microcosms or slurries advocates that some other mechanism occurs in this ecosystem to preserve inorganic N preferentially in the NH3 form. Taken these findings together, it is likely that not only the presence of ammonia oxidizers is fundamental for nitrification to occur, but that the microbial community composition and diversity affects the direction in which N process occur in soil. Most possibly there is a correlation between abiotic and biotic conditions that limits the abundance of autotrophic ammonia oxidizers, as for example the competition for NH4+ by plants or heterotrophic microbes or through dissimilatory reduction of NO3- to NH4+

Riqueza do domínio Archaea no solo do bioma Cerrado

Dissertação (mestrado)—Universidade de Brasília, Instituto de Biologia, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2012.O Domínio Archaea vem sendo descrito principalmente através de técnicas moleculares, independentes de cultivo e, atualmente, é dividido em dois filos formalmente aceitos, Crenarchaeota e Euryarchaeota, além de quatro outros propostos – Korarchaeota, Nanoarchaeota, Thaumarchaeota e Aigarchaeota. Uma vez que sequências do gene de rRNA 16S de Archaea foram descritas a partir de amostras de diferentes ambientes, as archaeas passaram a ser consideradas organismos ubíquos na natureza. Este trabalho tem como objetivo caracterizar a riqueza de archaeas em amostras de solo de Cerrado - a principal vegetação do Brasil Central, sendo a segunda maior do país. O DNA metagenômico de amostras em duplicata de solos provenientes de duas fitofisionomias distintas, cerrado denso e mata de galeria, foi extraído empregando-se o PowerSoil DNA Isolation Kit (MOBio Laboratories Inc.). A construção de bibliotecas genômicas foi feita a partir de experimentos de PCR, com iniciadores Archaea-específicos, visando amplificar regiões do gene de rRNA16S a partir do DNA extraído das diferentes amostras. Os resultados obtidos a partir dos fragmentos sequenciados revelaram identidade com o domínio Archaea em 96% das sequências, todas pertencentes ao filo Crenarchaeota (possivelmente Thaumarchaeota), principalmente aos grupos I.1b e I.1c. Somente nas amostras de Mata de Galeria foram encontradas sequências do grupo I.1a. A riqueza de Archaea foi maior em cerrado denso do que em mata de galeria. Os cálculos não-paramétricos tais como as curvas de rarefação indicam que a amostragem do ambiente foi adequada. As amostras das duas fitofisionomias apresentam diferenças significativas pelas técnicas estatísticas de β-diversidade ∫-Libshuff e pelo gráfico de Análise de Principais Coordenadas (PCA). ______________________________________________________________________________ ABSTRACTThe domain Archaea has been described mostly by culture-independent methods. Currently it is divided into two well accepted phyla, namely the Euryarchaeota and Crenarchaeota, and an additional four proposed phyla: Korarchaeota, Nanoarchaeota, Thaumarchaeota and Aigarchaeota. Since their 16S rDNA sequences have been described from diverse ecosystems not classified as extreme, Archaea is now considered ubiquitous and not a domain of only extreme organisms. This work describes the richness of the Archaea domain retrieved from soil samples of the Cerrado biome – the main vegetation of Central Brazil and second largest biome in the country. The metagenomic DNA from two replicas of each two distinct phytophysiognomies, cerrado denso and mata de galeria, was extracted using the PowerSoil DNA Isolation Kit (MOBio Laboratories Inc.). The four DNA libraries were constructed through PCR amplification of the 16S rDNA using Archaea-specific primers. Our results reveal that 96% of the sequenced fragments have identity with the domain Archaea. All of these sequences are from the phylum Crenarchaeota (possibly Thaumarchaeota), predominantly affiliated to group I.1b and I.1c. Sequences afilliated to the group I.1a were only found on the soil from mata de galeria. The soils from cerrado denso have a greater richness of Archaea than those from mata de galeria. The non-parametric estimatives showed that both the environments have been well sampled. There is a significant difference between the soil samples of the two phytophysiognomies shown by the statistical test of ∫-Libshuff and by the Principal Coordenates Analysis graphic (PCA)

Comparative analysis of pesticide-related genes in Paraguaçú River water samples obtained in the wet season.

<p>Samples were compared by ANOVA, followed by the Tukey–Kramer post hoc test (p < 0.05) without correction for multiple comparisons. Taxa with small effect sizes were removed by filtering (effect size = 8.00) using STAMP software.</p

Comparative analysis of pesticide-related genes in Paraguaçú River water samples obtained in the dry season.

<p>Samples were compared by ANOVA, followed by the Tukey–Kramer post hoc test (p < 0.05) without correction for multiple comparisons. Taxa with small effect sizes were removed by filtering (effect size = 8.00) using STAMP software.</p

Principal component analysis (PCA) of the physicochemical parameters and bacterial community of Paraguaçú River water samples obtained from three sites (P1, unprotected area; P2 and P3, protected areas) during both wet and dry seasons.

<p>The ordination analysis was performed with ammonia, nitrite, nitrate, total nitrogen, orthophosphate, total phosphorous, silicate, dissolved organic carbon (DOC), particulate organic carbon (POC), and bacterial counts based on the correlation matrix.</p