Changes in the microbial community during bioremediation of gasoline-contaminated soil

Abstract We aimed to verify the changes in the microbial community during bioremediation of gasoline-contaminated soil. Microbial inoculants were produced from successive additions of gasoline to municipal solid waste compost (MSWC) previously fertilized with nitrogen-phosphorous. To obtain Inoculant A, fertilized MSWC was amended with gasoline every 3 days during 18 days. Inoculant B received the same application, but at every 6 days. Inoculant C included MSWC fertilized with N–P, but no gasoline. The inoculants were applied to gasoline-contaminated soil at 10, 30, or 50 g/kg. Mineralization of gasoline hydrocarbons in soil was evaluated by respirometric analysis. The viability of the inoculants was evaluated after 103 days of storage under refrigeration or room temperature. The relative proportions of microbial groups in the inoculants and soil were evaluated by FAME. The dose of 50 g/kg of inoculants A and B led to the largest CO2 emission from soil. CO2 emissions in treatments with inoculant C were inversely proportional to the dose of inoculant. Heterotrophic bacterial counts were greater in soil treated with inoculants A and B. The application of inoculants decreased the proportion of actinobacteria and increased of Gram-negative bacteria. Decline in the density of heterotrophic bacteria in inoculants occurred after storage. This reduction was bigger in inoculants stored at room temperature. The application of stored inoculants in gasoline-contaminated soil resulted in a CO2 emission twice bigger than that observed in uninoculated soil. We concluded that MSWC is an effective material for the production of microbial inoculants for the bioremediation of gasoline-contaminated soil.

Changes in the microbial community during bioremediation of gasoline-contaminated soil.

We aimed to verify the changes in the microbial community during bioremediation of gasoline-contaminated soil. Microbial inoculants were produced from successive additions of gasoline to municipal solid waste compost (MSWC) previously fertilized with nitrogen-phosphorous. To obtain Inoculant A, fertilized MSWC was amended with gasoline every 3 days during 18 days. Inoculant B received the same application, but at every 6 days. Inoculant C included MSWC fertilized with N-P, but no gasoline. The inoculants were applied to gasoline-contaminated soil at 10, 30, or 50g/kg. Mineralization of gasoline hydrocarbons in soil was evaluated by respirometric analysis. The viability of the inoculants was evaluated after 103 days of storage under refrigeration or room temperature. The relative proportions of microbial groups in the inoculants and soil were evaluated by FAME. The dose of 50g/kg of inoculants A and B led to the largest CO2 emission from soil. CO2 emissions in treatments with inoculant C were inversely proportional to the dose of inoculant. Heterotrophic bacterial counts were greater in soil treated with inoculants A and B. The application of inoculants decreased the proportion of actinobacteria and increased of Gram-negative bacteria. Decline in the density of heterotrophic bacteria in inoculants occurred after storage. This reduction was bigger in inoculants stored at room temperature. The application of stored inoculants in gasoline-contaminated soil resulted in a CO2 emission twice bigger than that observed in uninoculated soil. We concluded that MSWC is an effective material for the production of microbial inoculants for the bioremediation of gasoline-contaminated soil.

Diversity and distribution of the endophytic bacterial community at different stages of Eucalyptus growth.

The relationships between plants and endophytic bacteria significantly contribute to plant health and yield. However, the microbial diversity in leaves of Eucalyptus spp. is still poorly characterized. Here, we investigated the endophytic diversity in leaves of hybrid Eucalyptus grandis x E. urophylla (Eucalyptus "urograndis") by using culture-independent and culture-dependent approaches, to better understand their ecology in leaves at different stages of Eucalyptus development, including bacteria with N2 fixation potential. Firmicutes, Proteobacteria (classes alpha-, beta- and gamma-) and Actinobacteria were identified in the Eucalyptus "urograndis" endophytic bacterial community. Within this community, the species Novosphingobium barchaimii, Rhizobium grahamii, Stenotrophomonas panacihumi, Paenibacillus terrigena, P. darwinianus and Terrabacter lapilli represent the first report these bacteria as endophytes. The diversity of the total endophytic bacteria was higher in the leaves from the 'field' (the Shannon-Wiener index, 2.99), followed by the indices obtained in the 'clonal garden' (2.78), the 'recently out from under shade (2.68), 'under shade' (2.63) and 'plants for dispatch' (2.51). In contrast, for diazotrophic bacteria, the highest means of these indices were obtained from the leaves of plants in the 'under shade' (2.56), 'recently out from under shade (2.52)' and 'field' stages (2.54). The distribution of the endophytic bacterial species in Eucalyptus was distinct and specific to the development stages under study, and many of the species had the potential for nitrogen fixation, raising the question of whether these bacteria could contribute to overall nitrogen metabolism of Eucalyptus.

Calcium oxalate crystals in eucalypt ectomycorrhizae: morphochemical characterization.

Ectomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants.

Isolation and characterization of endophytic bacteria isolated from the leaves of the common bean (Phaseolus vulgaris)

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris. leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10² to 2.8 x 10³ CFU g-1 of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.

Isolation and characterization of endophytic bacteria isolated from the leaves of the common bean (Phaseolus vulgaris).

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris) leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10(2) to 2.8 x 10(3) CFU g(-1) of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.

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.

Fotossíntese em alfafa (Medicago sativa L. ) sob supressäo e ressuprimento de fosfato / Photosynthesis in alfalfa (Medicago sativa L. ) under phosphate suppression and ressuply

Neste estudo, foram avaliados os efeitos da supressäo e do ressuprimento de fosfato (Pi) sobre a fotossíntese e eficiência fotoquímica de plantas de Medicago sativa cv. Flórida 77, em diferentes estádios do desenvolvimento vegetativo (V3, V4) e reprodutivo (R6, R8). O ensaio foi conduzido em casa de vegetaçäo e as plantas cultivadas na soluçäo nutritiva de HOAGLAND & ARNON (1950), contendo 0,14mmol L-1 de Pi. A supressäo de Pi por dez dias reduziu os teores de fósforo nas folhas amostradas, em todos os estádios do desenvolvimento. Entretanto, com o ressuprimento, somente nos estádios vegetativos, os valores foram semelhantes ao tratamento controle. A fotossíntese por área foliar, em todos os estádios do desenvolvimento diminuiu com a supressäo de Pi. De modo geral, o ressuprimento de Pi à soluçäo nutritiva resultou em recuperaçäo na fotossíntese, excetuando-se as plantas no estádio V3, uma indicaçäo de que o período de supressäo näo causou danos permanentes no aparato fotossintético. Os teores dos pigmentos fotossintéticos e a eficiência fotoquímica do fotossistema II (FS II), avaliada pela relaçäo Fv/Fm, näo foram alterados quando as plantas foram submetidas à supressäo de Pi. Esse resultado demonstra que o transporte de elétrons através do FS II näo limitou a fotossíntese nas folhas amostradas, sob supressäo de Pi, sugerindo que a supressäo causou efeito mais pronunciado na etapa bioquímica da fotossíntese

Detecting structural and functional differences in activated sludge bacterial communities originating from laboratory treatment of elementally and totally chlorine-free bleaching effluents.

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF-and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20-22%) and gamma (11-12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.

Fixação do nitrogênio em alfafa nodulada sob supressão e ressuprimento de fósforo / Nitrogen fixation in alfalfa nodulated under phosphorus supression and resupply

Estudaram-se os efeitos da supressäo e do ressuprimento de fósforo (Pi) sobre a fixaçäo biológica do nitrogênio atmosférico (N2) em plantas de alfafa cv. Flórida 77 inoculadas com Sinorhizobium meliloti, em diferentes estádios do desenvolvimento vegetativo (V3 e V4) e reprodutivo (R6 e R8). O ensaio foi conduzido em casa de vegetaçäo e as plantas cultivadas em soluçäo nutritiva. O período de supressäo de Pi por dez dias reduziu os teores de Pi nas folhas e nos nódulos em todos os estádios do desenvolvimento, enquanto nas raízes essa reduçäo somente foi observada nos estádios vegetativos. Após o ressuprimento de Pi à soluçäo nutritiva por igual período, dez dias, foi observada a recuperaçäo nos teores de Pi nos estádios R6 e R8 para folhas, V3 e V4 para raízes e V3 para os nódulos. A supressäo de Pi alterou o processo de fixaçäo do N2, estimado pela concentraçäo de aminoácidos totais nas folhas e nas raízes. Os teores de aminoácidos nas folhas e nas raízes foram significativamente menores nas plantas sob supressäo de Pi, em relaçäo aos das plantas do tratamento controle. Após o ressuprimento os teores de aminoácidos totais nas raízes, em todos os estádios do desenvolvimento, alcançaram valores similares àqueles das plantas do tratamento controle, enquanto nas folhas isso só ocorreu nos estádios vegetativos. A supressäo de Pi näo influenciou a proporçäo de aminoácidos livres na seiva do xilema

Nodulação, fixação de nitrogênio e produção de matéria seca de alfafa em resposta a doses de calcário, com diferentes relações cálcio: magnésio / Nodulation, nitrogen fixation and alfalfa dry matter production as affected by rates of limestone with different calcium: magnesium ratios applyed

Altas concentraçöes de alumínio reduzem a eficência da nodulaçäo e da fixaçäo biológica do nitrogênio atmosférico em leguminosas. O presente ensaio foi realizado em casa de vegetaçäo com o objetivo de avaliar a necessidade de calagem para o cultivo da alfafa em LATOSSOLO VERMELHO-AMARELO álico, textura argilosa. Também foi avaliado o efeito da relaçäo Ca:Mg do calcário aplicado ao solo, sobre a nodulaçäo, os teores de nitrogênio e a produçäo de matéria seca da parte aérea. Os tratamentos consistiram de duas cultivares de alfafa (Flórida 77 e Crioula), quatro doses de calcário (1,2; 2,4; 4,8; 7,2 t ha-1) e cinco relaçöes percentuais estequiométricas entre Ca e Mg (100:0; 75:25; 50:50; 25:75; 0:100), em delineamento fatorial inteiramente casualizado, com quatro repetiçöes. A nodulaçäo e os teores de nitrogênio na parte aérea aumentaram com a adiçäo de doses crescentes de calcário ao solo. A produçäo de matéria seca da parte aérea das duas cultivares aumentou em funçäo do aumento das doses de calcário, sendo que somente o cultivar Flórida 77 apresentou diferenças significativas entre as relaçöes Ca:Mg. A relaçäo 100:0 foi a que rendeu maior produçäo de matéria seca da parte aérea

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

A ocorrência de micorrizas arbusculares (AM) e ectomicorrizas (ECM) no mesmo sistema radicular foi observada quando Eucalyptus urophylla S.T. Blake, E. citriodora Hook F., E. grandis W. Hill ex Maiden, E. cloeziana F. Muell e E. camaldulensis Dehnh foram inoculadas simultaneamente com Glomus etunicatum Becker & Gederman and Pisolithus tinctorius (Per.) Cocker & Couch. A sucessão entre os dois fungos foi observada. De modo geral, o aumento da colonização ECM foi acompanhado de um decréscimo em AM. A inoculação simultânea resultou em percentagens de colonização diferenciadas das obtidas com a inoculação isolada. Pisolithus tinctorius beneficiou-se da inoculação simultânea com G. etunicatum. Para o fungo AM, o efeito positivo da inoculação simultânea ocorreu até aos 60 dias após a inoculação. A partir de 120 dias, a colonização das raízes por G. etunicatum diminuiu na presença de P. tinctorius. A proporção de AM e ECM variou com o tempo de avaliação e o total da percentagem de raízes colonizadas aproximou-se do valor máximo aos 60 dias da inoculação, mantendo-se constante. Os maiores valores foram observados para E. camaldulensis, seguido por E. citriodora, E. urophylla, E. grandis e E. cloeziana.

Growth and nutritional status of Brazilian wood species Cedrella fissilis and Anadenanthera peregrina in bauxite spoil in response to arbuscular mycorrhizal inoculation and substrate amendment

The growth of Cedrella fissilis Vell. (Cedro Rosa) and of Anadenanthera peregrina Benth (Angico Vermelho) in bauxite spoil was studied to evaluate their response to substrate amendment or to inoculation with arbuscular mycorrhizal fungi (AMF). The plants were grown in bauxite spoil, topsoil or spoil amended with either topsoil or compost, and inoculated with the AMF Acaulospora scrobiculata, Gigaspora margarita or Glomus etunicatum. Root colonization was highly dependent on the interaction plant-fungus-substrate. In C. fissilis, root colonization by Gigaspora margarita dropped from 75(per cent) in bauxite spoil to only 4(per cent) in topsoil. Contrarily, root colonization of A. peregrina by the same fungus increased from 48(per cent) in spoil to 60(per cent) in topsoil. Root colonization of C. fissilis in topsoil was lower than in the three other substrates. The opposite was observed for A. peregrina. Inoculation of the plants with Acaulospora scrobiculata or Glomus etunicatum was very effective in promoting plant growth. Plants of both C. fissilis and A. peregrina did not respond to amendments of bauxite spoil unless they were mycorrhizal. Also, a preferential partitioning of photosynthates to the shoots of A. peregrina inoculated with G. etunicatum or A. scrobiculata, and of C. fissilis inoculated with any of the three species of AMF was observed. C. fissilis showed a greater response to mycorrhizal inoculation than A. peregrina. The mean mycorrhizal efficiency (ME) for dry matter production by C. fissilis was 1,847(per cent) for A. scrobiculata, 1,922(per cent) for G. etunicatum, and 119(per cent) for G. margarita. In A. peregrina, the ME was 249(per cent) for A. scrobiculata, 540(per cent) for G. etunicatum, and 50(per cent) for G. margarita. The effect of mycorrhizal inoculation on plant growth seems to be related in part to an enhanced phosphorus absorption by inoculated plants. Moreover, the efficiency with which the absorbed nutrients were used to produce plant biomass was much greater in plants inoculated with A. scrobiculata or G. etunicatum.

Estabilidade de Plasmídio Recombinante em Zymomonas mobilis Ag11 / Stability of a recombinant plasmid in zymomonas mobilis Ag11

Resumo: Foram obtidos 19 plasmídios recombinantes contendo fragmentos de DNA plamidial de Zymomonas mobilis Ag11, clonados no sítio de EcoRI do pBR325. Os fragmentos clonados variaram de <0,5 a 8,6 Kb. O plasmídio recombinante pZMB12 teve seu peso aumentado, por processos de recombinaçäo, durante sua permanência em Z. mobilis Ag11. Este plasmídio denominado pZMBA12, ao ser clivado com seu tamanho original. O plasmídio recombinantee pZMB12 apresentou maior estabilidade em Z. mobilis do que em E. coli (au)

Isolamento e caracterizaçäo de bacteriófagos de Lactococcus lactis e Lactococcus cremosis / Isolation and characterization of lactococcal bacteriophages from cheese whey

Amostras de soro, coletadas em indústrias de queijo de Minas Gerais, Brasil, foram usada para isolar e caracterizar bacteriófagos de bactérias lácticas. Dez bacteriófagos foram observados em microscópio eletrônico e seus DNAs foram analisados em gel de agarose após a clivagem com várias enzimas de restriçäo. Sete bacteriófagos foram específicos para Lactococcus lactis subsp. cremosis e três para L. lactis subsp. lactis. As micrografias eletrônicas mostraram que todos os bacteriófagos apresentavam morfologia similar, contendo cabeça isométrica e cauda curta näo contrátil. Baseando-se no padräo de restriçäo do DNA e no tamanho do genoma, os bacteriófagos foram classificados em quatro grupos distintos. Todos os três bacteriófagos específicos para L. lactis subsp. lactis se situaram no mesmo grupo.