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Actinobacteria are known by their ability to produce several antimicrobial compounds of biotechnological interest. Thus, in this study, we isolated and identified by partial 16S RNA sequencing ∼100 actinobacteria isolates from guarana (Paullinia cupana) bulk soil. Besides, we isolated from the actinobacteria Streptomyces morookaense AM25 a novel cyclic peptide, named gloeosporiocide, molecular formula C44H48N11O7S3 (calculated 938.2901), and characterized by the presence of cyclized cysteins to form three thiazols. The novel compound had activity against the plant pathogen Colletotrichum gloeosporioides, assayed by the paper disk diffusion method (42.7% inhibition, 0.1 mg disk-1) and by the microdilution assay (1.25 g L-1). Our results reveal the potential of the actinobacteria from the Amazon rhizospheric soils as biocontrol agents as well as producers of new compounds with antifungal activity. Thus, this work constitutes a step forward in the development of the biotechnology of actinobacteria in the production of compounds of agronomic interest., (© FEMS 2019.)

The wood protection industry has refined their products from chrome-, copper-, and arsenate-based wood preservatives toward solely copper-based preservatives in combination with organic biocides. One of these is Cu-HDO, containing the chelation product of copper and N-cyclohexyldiazenium dioxide (HDO). In this study, the fate of isotope-labeled ((13)C) and nonlabeled ((12)C) Cu-HDO incorporated in wood sawdust mixed with soil was investigated. HDO concentration was monitored by high-pressure liquid chromatography. The total carbon and the δ(13)C content of respired CO(2), as well as of the soil-wood-sawdust mixture, were determined with an elemental analyzer-isotopic ratio mass spectrometer. The concentration of HDO decreased significantly after 105 days of incubation, and after 24 days the (13)CO(2) concentration respired from soil increased steadily to a maximum after 64 days of incubation. Phospholipid fatty acid-stable isotope probing (PFA-SIP) analysis revealed that the dominant PFAs C(19:0)d8,9, C(18:0), C(18:1)ω7, C(18:2)ω6,9, C(17:1)d7,8, C(16:0), and C(16:1)ω7 were highly enriched in their δ(13)C content. Moreover, RNA-SIP identified members of the phylum Acidobacteria and the genera Phenylobacterium and Comamonas that were assimilating carbon from HDO exclusively. Cu-HDO as part of a wood preservative effectively decreased fungal wood decay and overall microbial respiration from soil. In turn, a defined bacterial community was stimulated that was able to metabolize HDO completely.

Methanotrophs in the rhizosphere play an important role in global climate change since they attenuate methane emission from rice field ecosystems into the atmosphere. Most of the CH(4) is emitted via transport through the plant gas vascular system. We used this transport for stable isotope probing (SIP) of the methanotrophs in the rhizosphere under field conditions and pulse-labelled rice plants in a Chinese rice field with CH(4) (99% (13)C) for 7 days. The rate of (13)CH(4) loss rate during (13)C application was comparable to the CH(4) oxidation rate measured by the difluoromethane inhibition technique. The methanotrophic communities on the roots and in the rhizospheric soil were analyzed by terminal-restriction fragment length polymorphism (T-RFLP), cloning and sequencing of the particulate methane monooxygenase (pmoA) gene. Populations of type I methanotrophs were larger than those of type II. Both methane oxidation rates and composition of methanotrophic communities suggested that there was little difference between urea-fertilized and unfertilized fields. SIP of phospholipid fatty acids (PLFA-SIP) and rRNA (RNA-SIP) were used to analyze the metabolically active methanotrophic community in rhizospheric soil. PLFA of type I compared with type II methanotrophs was labelled more strongly with (13)C, reaching a maximum of 6.8 atom-%. T-RFLP analysis and cloning/sequencing of 16S rRNA genes showed that methanotrophs, especially of type I, were slightly enriched in the 'heavy' fractions. Our results indicate that CH(4) oxidation in the rice rhizosphere under in situ conditions is mainly due to type I methanotrophs.

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH(4) into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH(4) flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using (13)C-labelled CH(4) directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to (13)CH(4) for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.

The purpose of this work was to determine the extent of microbial metabolic potential for polychlorinated biphenyls (PCBs) in soils that have had no previous exposure to this class of xenobiotic pollutants. Soil and sediment samples of distinct characteristics from six sites in Germany were used to inoculate PCB oil (Aroclor 1242) microdroplets. All samples yielded multispecies biofilms, as revealed by single-strand conformation polymorphism (SSCP) analyses of polymerase chain reaction (PCR) analysis of 16S rRNA genes, and sequence analysis of the main amplicons. Microbes representing 20 different operational taxonomic units (OTUs) were identified in the biofilms, but only a few were common to all biofilms, namely those closely related to Aquabacterium sp., Caulobacter sp., Imtechium assamiensis, Nevskia ramosa, Parvibaculum lavamentivorans and Burkholderia sp. The PCB biofilm communities were always distinct from control biofilms developing from the same samples in the absence of PCB. All PCB droplet-grown biofilms degraded multiple PCB congeners but differed in the congener spectra they degraded. These findings reveal that microbial potential to degrade PCBs is widespread in soils that have not been subjected to PCB contamination, and that this potential is characteristic of consortia of very diverse phylogenetic composition.

This report is part of a serial study applying stable isotope labelling to rice microcosms to track the utilization of recently photosynthesized carbon by active microbiota in the rhizosphere. The objective of the present study was to apply phospholipid fatty acid-based stable isotope probing (PLFA-SIP) to detect the spatial variation of active microorganisms associated with rhizosphere carbon flow. In total, 49 pulses of 13CO2 were applied to rice plants in a microcosm over a period of 7 days. Rhizosphere soil was separated from bulk soil by a root bag. Soil samples were taken from rhizosphere and bulk soil, and the bulk soil samples were further partitioned both vertically (up layer and down layer) and horizontally with increasing distance to the root bag. Incorporation of 13C into PLFAs sharply decreased with distance to the roots. The labelling of 16:1omega9, 18:1omega7, 18:1omega9, 18:2omega6,9 and i14:0 PLFAs was relatively stronger in the rhizosphere while that of i15:0 and i17:0 increased in the bulk soil. The microorganisms associated with 16:1omega9 were active in both up- and down-layer soils. The microorganisms represented by i14:0, 18:1omega7 and 18:2omega6,9 exhibited a relatively higher activity in up-layer soil, whereas those represented by i15:0 and i17:0 were more active in down-layer soil. These results suggest that in the rhizosphere Gram-negative and eukaryotic microorganisms were most actively assimilating root-derived C, whereas Gram-positive microorganisms became relatively more important in the bulk soil. The active populations apparently differed between up- and down-layer soil and in particular changed with distance to the roots, demonstrating systematic changes in the activity of the soil microbiota surrounding roots.

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.

A food chain consisting of toluene, toluene-degrading Pseudomonas sp. PS+ and a bacterivorous flagellated amoebae Vahlkampfia sp. was established in a batch culture. This culture was amended with [U-13C]toluene and served as a model system to elucidate the flux of carbon in the food chain by quantifying bacterial biovolumes and 13C enrichment of phospholipid fatty acid (PLFA) biomarkers of the bacteria and the heterotrophic protists. Major PLFA detected in the batch co-culture included those derived from Pseudomonas sp. PS+ (16:1omega7c and 18:1omega7c) and Vahlkampfia sp. (20:4omega6c and 20:3omega6c). A numerical model including consumption of toluene by the bacteria and predation of the bacteria by the heterotrophic protists was adjusted to the measured toluene carbon, bacterial carbon and delta13C values of bacterial and protist biomass. Using this model, we estimated that 28+/-7% of the consumed toluene carbon was transformed into bacterial biomass, and 12+/-4% of the predated bacterial carbon was incorporated into heterotrophic protist biomass. Our study showed that the 13C enrichment of PLFA biomarkers coupled to biomass determination via biovolume calculations is a suitable method to trace carbon fluxes in protist-inclusive microbial food chains because it does not require the separation of protist cells from bacterial cells and soil particles.

Recent advances in the degradation of polychlorinated biphenyls (PCBs) have focussed on the use of experimental enrichment cultures to obtain PCB-degrading communities, and the use of culture-independent approaches to characterize natural and experimental PCB-degrading communities and to identify the key members in this process. PCB-degrading communities can be surprisingly diverse. Novel types of composite bacteria-mineral biofilm communities have been described. Community metabolism of PCBs may lead to the formation of protoanemonin, a dead-end product in some instances but, in others, a seemingly productive intermediate. Analysis of isotope fractionation and preferred enantiomer degradation has provided new information on degradation of PCBs in anaerobic settings. The first defined community capable of dehalorespiration of PCBs has been described, and important community members identified. Here, we provide an overview of the current knowledge of aerobic and anaerobic degradation of PCBs in microbial consortia and in the environment, including novel approaches to determine in situ PCB degradation.

Abstract: Plant species exert strong effects on ecosystem functions and one of the emerging, and difficult to test hypotheses, is that plants alter soil functions through changing the community structure of soil microorganisms. We tested the hypothesis for atmospheric CH4 oxidation by using soil samples from a Siberian afforestation experiment and exposing them to 13C–CH4. We determined the activity of the soil methanotrophs under different tree species at three levels of initial CH4 concentration (30, 200 and 1000 ppm) thus distinguishing the activities of low- and high-affinity methanotrophs. Half of the samples were incubated with 13C-enriched CH4 (99.9%) and half with 12C–CH4. This allowed an estimation of the amount of 13C incorporated into individual PLFAs and determination of PLFAs of methanotrophs involved in CH4 oxidation at the different CH4 concentrations. Tree species strongly altered the activity of atmospheric CH4 oxidation without appearing to change the composition of high-affinity methanotrophs as evidenced by PLFA 13C labeling. The low diversity of atmospheric CH4 oxidizers, presumably belonging to the UCSα group, may explain the lack of tree species effects on the composition of soil methanotrophs. We submit that the observed tree species effects on atmospheric CH4 oxidation indicate an effect on biomass or cell-specific activities rather than by a community change and this may be related to the impact of the tree species on soil N cycling. [Copyright &y& Elsevier]