Recovering Greater Fungal Diversity from Pristine and Diesel Fuel Contaminated Sub-Antarctic Soil Through Cultivation Using Both a High and a Low Nutrient Media Approach

Novel cultivation strategies for bacteria are widespread and well described for recovering greater diversity from the “hitherto” unculturable majority. While similar approaches have not yet been demonstrated for fungi it has been suggested that of the 1.5 million estimated species less than 5% have been recovered into pure culture. Fungi are known to be involved in many degradative processes, including the breakdown of petroleum hydrocarbons, and it has been speculated that in Polar Regions they contribute significantly to bioremediation of contaminated soils. Given the biotechnological potential of fungi there is a need to increase efforts for greater species recovery, particularly from extreme environments such as sub-Antarctic Macquarie Island. In this study, like the yet-to-be cultured bacteria, high concentrations of nutrients selected for predominantly different fungal species to that recovered using a low nutrient media. By combining both media approaches to the cultivation of fungi from contaminated and non-contaminated soils, 91 fungal species were recovered, including 63 unidentified species. A preliminary biodegradation activity assay on a selection of isolates found that a high proportion of novel and described fungal species from a range of soil samples were capable of hydrocarbon degradation and should be characterized further

Microfluidic qPCR Enables High Throughput Quantification of Microbial Functional Genes but Requires Strict Curation of Primers

Quantification of microbial functional genes enhances predictions of soil biogeochemical process rates, but reliance on low-throughput quantitative PCR (qPCR) limits the scope of ecological studies to a handful of targets. Here, we explore whether microfluidic qPCR (MFQPCR) is a viable high-throughput alternative for functional gene quantification, by evaluating the efficiency, specificity and sensitivity of 29 established and 12 newly designed primer pairs targeting taxonomic, nitrogen-cycling, and hydrocarbon degradation genes in genomic DNA soil extracts, under three different sets of MFQPCR assay conditions. Without curation, commonly-used qPCR primer pairs yielded an extreme range of reaction efficiencies (25.9–100.1%), but when conditions were optimized, MFQPCR produced copy-number estimates comparable to traditional qPCR. To guide microbial soil ecologists considering adoption of MFQPCR, we present suggestions for primer selection, including omission of inosines, degeneracy scores of < 9, amplicon sizes of ≤ 211 bp, and GC content of 32–61%. We conclude that, while the nanoliter reaction volumes, rapid thermocycling and one-size-fits-all reaction conditions of MFQPCR necessitates more stringent primer selection criteria than is commonly applied in soil microbial ecology, the ability to quantify up to 96 targets in 96 samples makes MFQPCR a valuable tool for monitoring shifts in functional community abundances. MFQPCR will particularly suit studies targeting multiple clade-specific functional genes, or when primer design is informed by previous knowledge of the environment

Salicylate or phthalate: the main intermediates in the bacterial degradation of naphthalene

Pathways of naphthalene degradation, the key enzymes, and genetic regulation are the main subjects of the present review, representing an attempt to summarize the current knowledge about the mechanism of the microbial degradation of PAH

Microfluidic qPCR for Microbial Ecotoxicology in Soil: A Pilot Study

From the late 1990’s there have been numerous calls to increase the biological relevance of methods used in ecotoxicology, by including environmental variation in experimental designs and replacing single-species tests with community-wide assessments. Quantitative PCR (qPCR) allows researchers to assess the impact of contamination on microbial communities involved in key processes such as nitrogen cycling, but is labor intensive, costly and requires a high degree of operator skill. Investigations are therefore usually restricted to quantifying 3 - 4 genes. Here we present the first application of microfluidic qPCR (MFQPCR) to microbial processes in soil. Utilising existing primer sets, we developed a MFQPCR assay for soil hydrocarbon ecotoxicology targeting the nitrogen cycle, hydrocarbon degradation and taxa, including bacteria and fungi. With as little as 6.7 nl reaction volumes, each chip has the capacity to quantify 14 genes across 30 samples in less than 5 hours, with costs per reaction less than half that of traditional qPCR. We developed the FuelTox pipeline, combining our MFQPCR assay with long-term in-situ mesocosms (114 weeks), fingerprinting (ARISA), factor-qPCR and multi-variate analysis, to assess the ecotoxicology of residual hydrocarbons on soil microbes on sub-Antarctic Macquarie Island. Principal response curves (PRC) of MFQPCR-derived gene abundances revealed significant inhibition of the endemic microbial community in response to fuel spiking; with bacterial laccase-like and denitrification (nosZ, nirK & narG) genes the most sensitive. Unlike previous Macquarie Island studies with fresh fuel, we observed similar sensitivities over our entire spiking range of 50 – 10 000 mg/kg, with no stimulation of nosZ, alkB or nah genes, commonly associated with hydrocarbon degradation observed. By 69 weeks post-spiking we observed significant reductions in spiking compounds (54-99%) and most significantly the recovery of the microbial community to that prior to fuel spiking. This study demonstrates that MFQPCR is not only a fast and cost-effective alternative to traditional qPCR, but it can be used for multi-variate analysis, thereby producing results that are directly comparable with more traditional ecotoxicology studies, such as single species tests using invertebrates or larger organisms. Due to the flexibility of MFQPCR, the FuelTox pipeline has great potential to be adapted to assess other contaminants and environmental stressors, by simply interchanging the primer sets used to target alternative genes of interest

Atmospheric trace gases support primary production in Antarctic desert surface soil

LetterCultivation-independent surveys have shown that the desert soils of Antarctica harbour surprisingly rich microbial communities¹⁻³. Given that phototroph abundance varies across these Antarctic soils²·⁴, an enduring question is what supports life in those communities with low photosynthetic capacity³·⁵. Here we provide evidence that atmospheric trace gases are the primary energy sources of two Antarctic surface soil communities. We reconstructed 23 draft genomes from metagenomic reads, including genomes from the candidate bacterial phyla WPS-2 and AD3. The dominant community members encoded and expressed high-affinity hydrogenases, carbon monoxide dehydrogenases, and a RuBisCO lineage known to support chemosynthetic carbon fixation⁶·⁷. Soil microcosms aerobically scavenged atmospheric H₂ and CO at rates sufficient to sustain their theoretical maintenance energy and mediated substantial levels of chemosynthetic but not photosynthetic CO₂ fixation. We propose that atmospheric H₂, CO₂ and CO provide dependable sources of energy and carbon to support these communities, which suggests that atmospheric energy sources can provide an alternative basis for ecosystem function to solar or geological energy sources⁸·⁹. Although more extensive sampling is required to verify whether this process is widespread in terrestrial Antarctica and other oligotrophic habitats, our results provide new understanding of the minimal nutritional requirements for life and open the possibility that atmospheric gases support life on other planets.Mukan Ji, Chris Greening, Inka Vanwonterghem, Carlo R. Carere, Sean K. Bay, Jason A. Steen, Kate Montgomery, Thomas Lines, John Beardall, Josie van Dorst, Ian Snape, Matthew B. Stott, Philip Hugenholtz & Belinda C. Ferrar

Microbial Community Dynamics within Frost Boils of Browning Peninsula, Antarctica

Browning Peninsula is a barren and ice free landscape in the Windmill Island, Eastern Antarctica which has experienced very little human activity. It is located in low valley consisting of large number of frost boils approximately 2-10 m in diameter, combined with an active layer of about 30 cm. Our aim was to investigate the microbial diversity of this unique, pristine environment. For this study, 18 soils were collected across 3 parallel transects using a spatially explicit sampling design. Soil genomic DNA was extracted and 454 pyrotag sequencing along with qPCR was performed targeting the bacterial 16S rRNA and fungal ITS/18S rRNA genes. Both the microbial and environmental data were analysed using Primer 6 and Permanova software and the community diversity and their similarity was investigated. Our results showed the bacterial community to consist predominantly of Actinobacteria (up to 55%), Chloroflexi (≥ 10%), Acidobacteria, Cyanobacteria, Proteobacteria and Gemmatimonadetes. While Ascomycota (≥ 96%), Basidiomycota (> 1.5%) and Fungi incertae sedis (< 1%) were the dominant fungi. A significant relationship between microbial community structures and environmental properties (extrinsic and intrinsic) was observed. For bacterial communities elevation, sand, conductivity, Cl-, SO4-- and total nitrogen were driving community pattern, while mud, total nitrogen, total carbon, SiO2 and sodium oxide were affecting fungal community distributions. Bacterial communities from samples of same polygon were more similar than the communities from different polygons and distance. While Fungal community distributions were random (soil sample specific) ie no trend was observed over the landscape or individual polygons. In this study, a culture clash was observed between the culture dependent and independent approaches to microbial characterisation. The artificial media recovered 4 (Pedobacter, Aminobacter, Dyella, Shingopyxix) bacterial genera and 3 (Engyodontium, Peniopora, Phoma) fungal genera which neither present in SSMS nor in soil based 454 pyrosequencing data. Comprehensive analysis of interboil variation may uncover what was happening in these boils. In the future it is required to have polygon specific sampling across different positions (edge, center), various depths of individual polygon and use of illumina pyrotaq sequencing platform to understand the ecosystem within these polygons with greater microbial dataset coverage

FOUNDRIES OF THE FUTURE:

Since the 1970s, cities world-wide have been witness to radical de-industrialisation. Manufacturing was considered incompatible with urban life and was actively pushed out.&nbsp;As economies have grown, public officials and developers have instinctively shifted their priorities to short-term, high-yielding land uses such as offices, retail space and housing. Inner-city growth&nbsp;from New York to London and even Seoul have generally come at the expense of land uses such as manufacturing or logistics. Despite the odds, manufacturing is not in terminal decay in western cities. On the contrary, it is at the opening of a new chapter. Urban manufacturing can help cities to be more innovative, circular, inclusive and resilient. Recently, with increasing interest in the circular economy, with cleaner and more compact technology, with more progressive building codes for mixed use, with increasing awareness of the impacts of social inequality and with a clearer understanding of the value chains between the trade&nbsp;of material and immaterial goods, cities across the world are realising that manufacturing has&nbsp;an important place in the 21st century urban economy. While both enthusiasm for making is increasing&nbsp;and the value of manufacturing is becoming increasingly evident&nbsp;in cities, the topic remains extremely complex and challenging&nbsp;to manage. This book attempts to shed light on the ways manufacturing can address urban challenges, it exposes constraints for the manufacturing sector and provides fifty patterns for working with urban manufacturing. This book has been written as a manual to help politicians, public authorities, planners,&nbsp;designers and community organisations to be able to plan, discuss and collaborate by developing more productive urban manufacturing. The book is split into two parts.&nbsp; We first cover an abridged history of the late nineteenth and early&nbsp;twentieth centuries, noting how European cities evolved rapidly by harnessing manufacturing, and then how the late twentieth century led to a radical shift in how cities work and think. We’re now at a crossroads between actors that do not see the need for manufacturing in&nbsp;cities and those that consider it vital for a prosperous urban future. Part of the tension comes from the fact that manufacturing is considered a ‘weak land use’ compared to activities such as real-estate development, which has been considered more financially attractive by&nbsp;many actors in the private and public sector. This real estate-oriented development narrative is increasingly regarded as short-sighted, but will not change without an alternative vision. We have therefore elaborated a narrative on how urban manufacturing responds to four&nbsp;specific challenges facing cities and how in turn manufacturing needs cities. In practice, planning and design for a topic like this is highly challenging. The second part of the book is intended as a handbook. By synthesising our research and fieldwork conducted&nbsp;in a number of cities, we have encountered many similarities in terms of problems, challenges and solutions for urban manufacturing. Inspired by the seminal 1977 book, ‘A Pattern Language’ we have translated our findings into fifty patterns which help render the diversity&nbsp;of issues concerning manufacturing more tangible. As both teamwork and negotiation are necessary, exercises and methods are provided to use the patterns. Finally, we have set out twelve key action areas as possible starting points for supporting urban manufacturing

Investigating the biosynthetic potential of an Antarctic soil through metagenomics, cultivation, and heterologous expression

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as sources of new antimicrobials. Metagenomic sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, and metagenomic library studies have yielded several novel natural products. However, accessing these compounds remains challenging. The constraints of short-read sequencing mean that the assembly of full-length BGC sequences from uncultured bacteria is nigh impossible, thus making assessment of BGC diversity difficult and downstream cloning infeasible. Conversely, metagenomic library approaches suffer from a bias towards known compounds as well as difficulties with expressing recovered BGCs. In the present work, a three-pronged approach was taken to access the biosynthetic diversity of bacteria from an Antarctic soil: A hybrid shotgun metagenome was sequenced and BGCs cloned and expressed, a novel regulatory gene-based screen for libraries was developed, and a number of isolates were obtained by culturing. Through metagenomic sequencing, many highly divergent BGCs were found in phyla such as Acidobacteriota and Verrucomicrobiota, but also the methanotrophic gammaproteobacterial order UBA7966. Sequencing of isolates obtained from the same soil indicated little overlap between the biosynthetic potential of readily cultured and uncultured bacteria. Several metagenomic BGCs were PCR-amplified, cloned and expressed in Pseudomonas and Streptomyces. While the sequencing of Streptomyces exconjugants showed that many inserts were truncated, a phenotype was observed in Pseudomonas. The library screening approach was validated in isolates, but the targets were absent in the metagenomic library used. In conclusion, the results uncover the rich diversity of BGCs from uncultured lineages present in the soil, show the potential of long-read sequencing to recover full-length BGCs from uncultured soil bacteria and demonstrate the feasibility of cloning them. However, they also indicate the necessity of refined molecular tools for successful heterologous expression of metagenomic BGCs