Sugarcane trash levels in soil affects the fungi but not bacteria in a short-term field experiment

Abstract The sugarcane in Brazil is passing through a management transition that is leading to the abolition of pre-harvest burning. Without burning, large amounts of sugarcane trash is generated, and there is a discussion regarding the utilization of this biomass in the industry versus keeping it in the field to improve soil quality. To study the effects of the trash removal on soil quality, we established an experimental sugarcane plantation with different levels of trash over the soil (0%, 50% and 100% of the original trash deposition) and analyzed the structure of the bacterial and fungal community as the bioindicators of impacts. The soil DNA was extracted, and the microbial community was screened by denaturing gradient gel electrophoresis in two different seasons. Our results suggest that there are no effects from the different levels of trash on the soil chemistry and soil bacterial community. However, the fungal community was significantly impacted, and after twelve months, the community presented different structures among the treatments.

Sugarcane trash levels in soil affects the fungi but not bacteria in a short-term field experiment.

The sugarcane in Brazil is passing through a management transition that is leading to the abolition of pre-harvest burning. Without burning, large amounts of sugarcane trash is generated, and there is a discussion regarding the utilization of this biomass in the industry versus keeping it in the field to improve soil quality. To study the effects of the trash removal on soil quality, we established an experimental sugarcane plantation with different levels of trash over the soil (0%, 50% and 100% of the original trash deposition) and analyzed the structure of the bacterial and fungal community as the bioindicators of impacts. The soil DNA was extracted, and the microbial community was screened by denaturing gradient gel electrophoresis in two different seasons. Our results suggest that there are no effects from the different levels of trash on the soil chemistry and soil bacterial community. However, the fungal community was significantly impacted, and after twelve months, the community presented different structures among the treatments.

Subgingival bacterial community profiles in HIV-infected Brazilian adults with chronic periodontitis.

BACKGROUND AND OBJECTIVE: To compare the subgingival microbial diversity between non-HIV-infected and HIV-infected individuals with chronic periodontitis using denaturing gradient gel electrophoresis (DGGE). MATERIAL AND METHODS: Thirty-two patients were selected: 11 were HIV-infected and 21 were non-HIV-infected, and all had chronic periodontitis. Periodontal measurements included probing depth, clinical attachment level, visible supragingival biofilm and bleeding on probing. Subgingival biofilm samples were collected from periodontal sites (50% with probing depth ≤ 4 mm and 50% with probing depth ≥ 5 mm) and whole-genomic-amplified DNA was obtained. The DNA samples were subjected to amplification of a 16S rRNA gene fragment using universal bacterial primers, followed by DGGE analysis of the amplified gene sequences. RESULTS: The non-HIV-infected group presented higher mean full-mouth visible supragingival biofilm (p = 0.004), bleeding on probing (p = 0.006), probing depth (p < 0.001) and clinical attachment level (p = 0.001) in comparison with the HIV-infected group. DGGE analysis revealed 81 distinct bands from all 33 individuals. Banding profiles revealed a higher diversity of the bacterial communities in the subgingival biofilm of HIV-infected patients with chronic periodontitis. Moreover, cluster and principal component analyses demonstrated that the bacterial community profiles differed between these two conditions. High interindividual and intra-individual variability in banding profiles were observed for both groups. CONCLUSION: HIV-infected patients with chronic periodontitis present greater subgingival microbial diversity. In addition, the bacterial communities associated with HIV-infected and non-HIV-infected individuals are different in structure.

Tracking the dynamics of heterotrophs and nitrifiers in moving-bed biofilm reactors operated at different COD/N ratios.

In this study, the impact of COD/N ratio and feeding regime on the dynamics of heterotrophs and nitrifiers in moving-bed biofilm reactors was addressed. Based on DGGE analysis of 16S rRNA genes, the influent COD was found to be the main factor determining the overall bacterial diversity. The amoA-gene-based analysis suggested that the dynamic behavior of the substrate in continuous and pulse-feeding reactors influenced the selection of specific ammonium-oxidizing bacteria (AOB) strains. Furthermore, AOB diversity was directly related to the applied COD/N ratio and ammonium-nitrogen load. Maximum specific ammonium oxidation rates observed under non-substrate-limiting conditions were observed to be proportional to the fraction of nitrifiers within the bacterial community. FISH analysis revealed that Nitrosomonas genus dominated the AOB community in all reactors. Moreover, Nitrospira was found to be the only nitrite-oxidizing bacteria (NOB) in the fully autotrophic system, whereas Nitrobacter represented the dominant NOB genus in the organic carbon-fed reactors.

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar.

Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.

Tank bromeliad water: similar or distinct environments for research of bacterial bioactives?

The Atlantic Rainforest does not have a uniform physiognomy, its relief determines different environmental conditions that define the composition of its flora and fauna. Within this ecosystem, bromeliads that form tanks with their leaves hold water reservoirs throughout the year, maintaining complex food chains, based mainly on autotrophic and heterotrophic bacteria. Some works concluded that the water held by tank bromeliads concentrate the microbial diversity of their ecosystem. To investigate the bacterial diversity and the potential biotechnology of these ecosystems, tank bromeliads of the Neoregelia cruenta species from the Atlantic Rainforest in Brazil were used as models for this research. Bacteria isolated from these models were tested for production of bioactive compounds. DGGE of the water held by tank bromeliads was performed in different seasons, locations and sun exposure to verify whether these environmental factors affect bacterial communities. The DGGE bands profile showed no grouping of bacterial community by the environmental factors tested. Most of the isolates demonstrated promising activities in the tests performed. Collectively, these results suggest that tank bromeliads of the N. cruenta species provide important habitats for a diverse microbial community, suggesting that each tank forms a distinct micro-habitat. These tanks can be considered excellent sources for the search for new enzymes and/or new bioactive composites of microbial origin.

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.

Tank bromeliad water: similar or distinct environments for research of bacterial bioactives?

The Atlantic Rainforest does not have a uniform physiognomy, its relief determines different environmental conditions that define the composition of its flora and fauna. Within this ecosystem, bromeliads that form tanks with their leaves hold water reservoirs throughout the year, maintaining complex food chains, based mainly on autotrophic and heterotrophic bacteria. Some works concluded that the water held by tank bromeliads concentrate the microbial diversity of their ecosystem. To investigate the bacterial diversity and the potential biotechnology of these ecosystems, tank bromeliads of the Neoregelia cruenta species from the Atlantic Rainforest in Brazil were used as models for this research. Bacteria isolated from these models were tested for production of bioactive compounds. DGGE of the water held by tank bromeliads was performed in different seasons, locations and sun exposure to verify whether these environmental factors affect bacterial communities. The DGGE bands profile showed no grouping of bacterial community by the environmental factors tested. Most of the isolates demonstrated promising activities in the tests performed. Collectively, these results suggest that tank bromeliads of the N. cruenta species provide important habitats for a diverse microbial community, suggesting that each tank forms a distinct micro-habitat. These tanks can be considered excellent sources for the search for new enzymes and/or new bioactive composites of microbial origin.

Diversity of the candidate phylum Poribacteria in the marine sponge Aplysina fulva.

Poribacterial clone libraries constructed for Aplysina fulva sponge specimens were analysed with respect to diversity and phylogeny. Results imply the coexistence of several, prevalently "intra-specific" poribacterial genotypes in a single sponge host, and suggest quantitative analysis as a desirable approach in studies of the diversity and distribution of poribacterial cohorts in marine sponges.

Clinical antimicrobial efficacy of NiTi rotary instrumentation with NaOCl irrigation, final rinse with chlorhexidine and interappointment medication: a molecular study.

AIM: To evaluate clinically the antibacterial effects of root canal treatment procedures using molecular microbiology analyses. METHODOLOGY: Samples were taken from 14 necrotic root canals of teeth with apical periodontitis before (S1) and after instrumentation with NaOCl irrigation (S2), a final rinse with chlorhexidine (CHX) (S3) and then one-week interappointment medication with calcium hydroxide/CHX paste (S4). The parameters examined included the following: incidence of positive broad-range PCR results for bacterial presence; impact on bacterial community structures evaluated by PCR-Denaturing Gradient Gel Electrophoresis (DGGE); quantitative bacterial reduction determined by real-time PCR; and identification of bacterial persisters by cloning and sequencing. Data from the different tests were subjected to statistical analyses and diversity indicator calculations. RESULTS: All S1 samples were positive for bacteria in all tests. Treatment procedures promoted a decrease in microbial diversity and significantly reduced the incidence of positive results and the bacterial counts (P < 0.05). In general, each subsequent treatment step improved disinfection. No specific taxon or community pattern was associated with post-treatment samples. CONCLUSION: Supplementary steps consisting of a final rinse with CHX followed by calcium hydroxide interappointment medication promoted further decrease in the bacterial bioburden to levels significantly below those achieved by the chemomechanical procedures alone. Because the long-term outcome of root canal treatment is dependent upon maximal bacterial reduction, the present results are of clinical relevance.

Diversity of the candidate phylum Poribacteria in the marine sponge Aplysina fulva

Poribacterial clone libraries constructed for Aplysina fulva sponge specimens were analysed with respect to diversity and phylogeny. Results imply the coexistence of several, prevalently "intraspecific" poribacterial genotypes in a single sponge host, and suggest quantitative analysis as a desirable approach in studies of the diversity and distribution of poribacterial cohorts in marine sponges

Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Effect of different operational conditions on biofilm development, nitrification, and nitrifying microbial population in moving-bed biofilm reactors.

In this study, the effect of different operational conditions on biofilm development and nitrification in three moving-bed biofilm reactors (MBBRs) was investigated: two reactors were operated in a continuously fed regime and one in sequencing-batch mode. The presence of organic carbon reduced the time required to form stable nitrifying biofilms. Subsequent stepwise reduction of influent COD caused a decrease in total polysaccharide and protein content, which was accompanied by a fragmentation of the biofilm, as shown by scanning electron microscopy, and by an enrichment of the biofilm for nitrifiers, as observed by fluorescent in situ hybridization (FISH) analysis. Polysaccharide and protein concentrations proved to be good indicators of biomass development and detachment in MBBR systems. Ammonium- and nitrite-oxidizing bacteria activities were affected when a pulse feeding of 4 g of NH(4)-N/(m(2)·day) was applied. Free nitrous acid and free ammonia were likely the inhibitors for ammonium- and nitrite-oxidizing bacteria.

Comparison of different protocols for the extraction of microbial DNA from reef corals.

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Petroleum-degrading enzymes: bioremediation and new prospects.

Anthropogenic forces, such as petroleum spills and the incomplete combustion of fossil fuels, have caused an accumulation of petroleum hydrocarbons in the environment. The accumulation of petroleum and its derivatives now constitutes an important environmental problem. Biocatalysis introduces new ways to improve the development of bioremediation strategies. The recent application of molecular tools to biocatalysis may improve bioprospecting research, enzyme yield recovery, and enzyme specificity, thus increasing cost-benefit ratios. Enzymatic remediation is a valuable alternative as it can be easier to work with than whole organisms, especially in extreme environments. Furthermore, the use of free enzymes avoids the release of exotic or genetically modified organisms (GMO) in the environment.

Bacterial communities reflect the spatial variation in pollutant levels in Brazilian mangrove sediment.

The majority of oil from oceanic oil spills converges on coastal ecosystems such as mangrove forests. A major challenge to mangrove bioremediation is defining the mangrove's pollution levels and measuring its recuperation from pollution. Bioindicators can provide a welcome tool for defining such recovery. To determine if the microbial profiles reflected variation in the pollutants, samples from different locations within a single mangrove with a history of exposure to oil were chemically characterised, and the microbial populations were evaluated by a comprehensive range of conventional and molecular methods. Multivariate ordination of denaturing gradient gel electrophoresis (DGGE) microbial community fingerprints revealed a pronounced separation between the sediment and rhizosphere samples for all analysed bacterial communities (Bacteria, Betaproteobacteria, Alphaproteobacteria, Actinobacteria and Pseudomonas). A Mantel test revealed significant relationships between the sediment chemical fertility and oil-derived pollutants, most of the bacterial community fingerprints from sediment samples, and the counts by different cultivation strategies. The level of total petroleum hydrocarbons was significantly associated with the Bacteria and Betaproteobacteria fingerprints, whereas anthracene and the total level of polycyclic aromatic hydrocarbons were associated with the Actinobacteria. These results show that microbial communities from the studied mangrove reflect the spatial variation of the chemicals in the sediment, demonstrating the specific influences of oil-derived pollutants.

A decade of land use contributes to changes in the chemistry, biochemistry and bacterial community structures of soils in the Cerrado.

The bacterial community structures (BCSs) of Cerrado soils cultivated under conventional tillage (CT), no-tillage (NT) and under native Cerrado (NC) vegetation were evaluated using PCR/DGGE of bacterial 16S rRNA (rrs) and rpoB genes and of Pseudomonas group genes. Soil chemical analysis, microbial biomass and the enzyme activities were also evaluated and correlated with the BCS measurements. The multivariate ordinations of DGGE profiles showed differences between the BCS of the NC area and those from cultivated areas. The BCSs of the CT and NT areas also differed in all DGGE fingerprints, including changes in the profile of Pseudomonas populations, indicating that agricultural systems can also be responsible for changes within specific microbial niches, although the clearest differences were found in the rpoB profiles. The MRPP analysis demonstrated significant differences between the BCSs from different soil layers of NT areas based on all gene fingerprints and those of NC areas based on bacterial 16S rRNA and rpoB genes fingerprints. No differences were observed in the microbial fingerprints of CT samples from different depths, indicating that ploughing affected the original BCS stratification. The BCS from NC areas, based on all gene fingerprints, could be related to higher levels of soil acidity and higher amounts of MBC and of phosphatase activity. In contrast, the BCSs from cultivated areas were related to higher levels of Ca + Mg, P and K, likely as a result of a history of chemical fertilisation in these areas. The relationships between rpoB and Pseudomonas BCSs and all chemical and biochemical properties of soils were significant, according to a Mantel test (P < 0.05), indicating that the different changes in soil properties induced by soil use or management may drive the formation of the soil BCS.

Keratinolytic activity of Bacillus subtilis AMR using human hair.

AIMS: To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides. METHODS AND RESULTS: The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0.01% yeast extract increased the keratinolytic activity 4.2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0.01% yeast extract (HMY) at pH 8.0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50 degrees C and pH 9.0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800-2600 Da. CONCLUSIONS: This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair. These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.

Relationships between bacterial diversity, microbial biomass, and litter quality in soils under different plant covers in northern Rio de Janeiro State, Brazil.

Microbial populations are primarily responsible for the decomposition of organic residues, the nutrients cycle, and the flow of energy inside of soil. The present study was undertaken to link soil microbiological and soil biochemical parameters with soil- and litter-quality conditions in the surface layer from 5 sites differing in plant cover, in stand age, and in land-use history. The aim was to see how strongly these differences affect the soil microbial attributes and to identify how microbiological processes and structures can be influenced by soil and litter quality. Soil and litter samples were collected from 5 sites according to different land use: preserved forest, nonpreserved forest, secondary forest, pasture, and eucalyptus plantation. Soil and litter microbial biomass and activity were analysed and DNA was extracted from soil. The DNA concentrations and soil microbial C and N correlated positively and significantly, suggesting that these are decisive nutrients for microbial growth and time required for microbial biomass renewal. The litter microbial biomass represented a source of C and N higher than soil microbial biomass and can be an important layer to contribute to tropical soil with low C and N availability. The litter quality influenced the litter and soil microbial biomass and activity and the soil bacterial diversity. The chemical and nutritional quality of the litter influenced the structure and microbial community composition in the eucalyptus plantation.