Cryopreservation of a soil microbiome using a Stirling 1 cycle approach - a genomic assessment

Soil microbiomes are dynamic systems that respond to biotic and abiotic environmental factors such as those presented at seasonal scales or due to long-term anthropogenic regime shifts. These can affect the composition and function of microbiomes. Investigation of microbiomes can uncover hidden microbial roles in health and disease and discover microbiome-based interventions. Collections of soil samples are kept by various institutions in either a refrigerated or occasionally frozen state, but conditions are not optimised to ensure the integrity of soil microbiome. In this manuscript, we describe cryopreservation with a controlled rate cooler and estimate the genomic content of an exemplar soil sample before and after cryopreservation. The first hypothesis was to test the genomic integrity of the microbiome. We also enriched the soil sample with a liquid medium to estimate the growth of bacteria and compared their growth before and after cryopreservation. Sequence-based rRNA metabarcoding was used to demonstrate that the controlled rate cooler maintains intact the DNA content of the microbiome. Two methods of cryopreservation were applied and compared with control aliquots of soil. An optimised cryopreservation of soil samples is essential for the development of microbiome research in order to retain stable, functionally intact microbiomes. Our results showed that metabarcoding of 16S and ITS rRNA were useful methods to estimate successful cryopreservation. The soil microbiome after enrichment with liquid medium exhibited a similar response of cryopreserved soil and this was estimated with the comparison of the ten most abundant bacterial taxa. These findings support a successful process of cryopreservation and are promising for future use of this technology. To the best of our knowledge, this study is the first report of cryopreservation of soil using a Stirling cycle cooling approach

The UK Crop Microbiome Cryobank: a utility and model for supporting Phytobiomes research

Plant microbiomes are the microbial communities essential to the functioning of the phytobiome—the system that consist of plants, their environment, and their associated communities of organisms. A healthy, functional phytobiome is critical to crop health, improved yields and quality food. However, crop microbiomes are relatively under-researched, and this is associated with a fundamental need to underpin phytobiome research through the provision of a supporting infrastructure. The UK Crop Microbiome Cryobank (UKCMC) project is developing a unique, integrated and open-access resource to enable the development of solutions to improve soil and crop health. Six economically important crops (Barley, Fava Bean, Oats, Oil Seed Rape, Sugar Beet and Wheat) are targeted, and the methods as well as data outputs will underpin research activity both in the UK and internationally. This manuscript describes the approaches being taken, from characterisation, cryopreservation and analysis of the crop microbiome through to potential applications. We believe that the model research framework proposed is transferable to different crop and soil systems, acting not only as a mechanism to conserve biodiversity, but as a potential facilitator of sustainable agriculture systems

Draft genome sequence of <i>Bacillus thuringiensis</i> strain BrMgv02-JM63, a chitinolytic bacterium isolated from oil-contaminated mangrove soil in Brazil

Here, we report the draft genome sequence and the automatic annotation of Bacillus thuringiensis strain BrMgv02-JM63. This genome comprises a set of genes involved in the metabolism of chitin and N-acetylglucosamine utilization, thus suggesting the possible role of this strain in the cycling of organic matter in mangrove soils

Interspecific variation of the bacterial community structure in the phyllosphere of the three major plant components of mangrove forests

Mangrove forests encompass a group of trees species that inhabit the intertidal zones, where soil is characterized by the high salinity and low availability of oxygen. The phyllosphere of these trees represent the habitat provided on the aboveground parts of plants, supporting in a global scale, a large and complex microbial community. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonizers, which is influenced by numerous environmental factors in addition to leaf physical and chemical properties. Here, a combination of culture-base methods with PCR-DGGE was applied to test whether local or plant specific factors shape the bacterial community of the phyllosphere from three plant species (Avicenia shaueriana, Laguncularia racemosa and Rhizophora mangle), found in two mangroves. The number of bacteria in the phyllosphere of these plants varied between 3.62 x 10(4) in A. schaeriana and 6.26 x 10³ in R. mangle. The results obtained by PCR-DGGE and isolation approaches were congruent and demonstrated that each plant species harbor specific bacterial communities in their leaves surfaces. Moreover, the ordination of environmental factors (mangrove and plant species), by redundancy analysis (RDA), also indicated that the selection exerted by plant species is higher than mangrove location on bacterial communities at phyllosphere

A comprehensive study on diesel oil bioremediation under microcosm conditions using a combined microbiological, enzymatic, mass spectrometry, and metabarcoding approach

This study aims at the application of a marine fungal consortium (Aspergillus sclerotiorum CRM 348 and Cryptococcus laurentii CRM 707) for the bioremediation of diesel oil-contaminated soil under microcosm conditions. The impact of biostimulation (BS) and/or bioaugmentation (BA) treatments on diesel-oil biodegradation, soil quality, and the structure of the microbial community were studied. The use of the fungal consortium together with nutrients (BA/BS) resulted in a TPH (Total Petroleum Hydrocarbon) degradation 42% higher than that obtained by natural attenuation (NA) within 120 days. For the same period, a 72 to 92% removal of short-chain alkanes (C12 to C19) was obtained by BA/BS, while only 3 to 65% removal was achieved by NA. BA/BS also showed high degradation efficiency of long-chain alkanes (C20 to C24) at 120 days, reaching 90 and 92% of degradation of icosane and heneicosane, respectively. In contrast, an increase in the levels of cyclosiloxanes (characterized as bacterial bioemulsifiers and biosurfactants) was observed in the soil treated by the consortium. Conversely, the NA presented a maximum of 37% of degradation of these alkane fractions. The 5-ringed PAH benzo(a)pyrene, was removed significantly better with the BA/BS treatment than with the NA (48 vs. 38 % of biodegradation, respectively). Metabarcoding analysis revealed that BA/BS caused a decrease in the soil microbial diversity with a concomitant increase in the abundance of specific microbial groups, including hydrocarbon-degrading (bacteria and fungi) and also an enhancement in soil microbial activity. Our results highlight the great potential of this consortium for soil treatment after diesel spills, as well as the relevance of the massive sequencing, enzymatic, microbiological and GC-HRMS analyses for a better understanding of diesel bioremediation.This research was financially supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant #2016/07957-7) and by Conselho Nacional de Desenvolvimento Tecnológico (CNPq) (grant #407986/2018-6).Peer reviewe

The Microbiome of Brazilian Mangrove Sediments as Revealed by Metagenomics

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H2S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

The Influence of Different Land Uses on the Structure of Archaeal Communities in Amazonian Anthrosols Based on 16S rRNA and amoA Genes

Soil from the Amazonian region is usually regarded as unsuitable for agriculture because of its low organic matter content and low pH; however, this region also contains extremely rich soil, the Terra Preta Anthrosol. A diverse archaeal community usually inhabits acidic soils, such as those found in the Amazon. Therefore, we hypothesized that this community should be sensitive to changes in the environment. Here, the archaeal community composition of Terra Preta and adjacent soil was examined in four different sites in the Brazilian Amazon under different anthropic activities. The canonical correspondence analysis of terminal restriction fragment length polymorphisms has shown that the archaeal community structure was mostly influenced by soil attributes that differentiate the Terra Preta from the adjacent soil (i.e., pH, sulfur, and organic matter). Archaeal 16S rRNA gene clone libraries indicated that the two most abundant genera in both soils were Candidatus nitrosphaera and Canditatus nitrosocaldus. An ammonia monoxygenase gene (amoA) clone library analysis indicated that, within each site, there was no significant difference between the clone libraries of Terra Preta and adjacent soils. However, these clone libraries indicated there were significant differences between sites. Quantitative PCR has shown that Terra Preta soils subjected to agriculture displayed a higher number of amoA gene copy numbers than in adjacent soils. On the other hand, soils that were not subjected to agriculture did not display significant differences on amoA gene copy numbers between Terra Preta and adjacent soils. Taken together, our findings indicate that the overall archaeal community structure in these Amazonian soils is determined by the soil type and the current land use.CNPq[Proc. 474455/2007-6]CNPq[Proc. 152405/2007-0]FAPESP[Proc. 2006/06700-0

Microbial community response to a simulated hydrocarbon spill in mangrove sediments

In this study, we examined the hypothesis that the microbial communities in mangrove sediments with different chemical and historical characteristics respond differently to the disturbance of a hydrocarbon spill. Two different mangrove sediments were sampled, one close to an oil refinery that had suffered a recent oil spill and another that had not been in contact with oil. Based on the sampled sediment, two sets of mesocosms were built, and oil was added to one of them. They were subjected to mimicked mangrove conditions and monitored for 75 days. Archaeal and bacterial communities were evaluated through PCR-DGGE. Both communities showed the emergence of small numbers of novel bands in response to oil pollution. 16S rRNA gene clone libraries were constructed from both mesocosms before the addition of oil and at day 75 after oil addition. LIBSHUFF analysis showed that both mangrove-based mesocosms contained similar communities at the start of the experiment and that they were different from the initial one, as well as from each other, after 75 days. These results hint at a role of environmental history that is not obvious from community diversity indicators, but is apparent from the response to the applied stress