Microbial inoculation to improve plant performance in mine-waste substrates: A test using pigeon pea (Cajanus cajan)

Mining activities alter soil physicochemical and biological properties that are critical for plant establishment. Revitalisation of soil biological properties via microbial inoculations can potentially be adopted to improve vegetation restoration. Here, we evaluate the feasibility of using beneficial microorganisms in the form of commercially available inoculants to enhance plant performance in a non-toxic and infertile mine-waste substrate, using pigeon pea [Cajanus cajan (L) Millsp.] as a test plant. Six treatments were established to investigate the effects of inoculants (Bradyrhizobium spp., microbial mix and uninoculated controls) and water availability (low and moderate) in a factorial design over 6 months. Plant performance was determined by physiological parameters (leaf gas exchange, leaf carbon, nitrogen and stable isotopes) and growth (height and biomass). Plant xylem sap phytohormones were measured to determine the plants' physiological status and effects of inoculation treatments. Results revealed that water had a greater effect on plant growth than inoculation treatments. Inoculation treatments, however, improved some physiological parameters. This study suggests that physical conditions such as soil moisture and nutrient availability may occlude more subtle (direct or interactive) effects of beneficial soil microbes on plant growth and plant condition. Prior knowledge on the biological and physicochemical properties of the soil to be amended, and on plant species-specific responses, would be needed to customise microbial inoculants for maximum benefits to ecological restoration, to support future adoption of this practice

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Single cell stable isotope probing with FISH-Raman spectroscopy for deciphering the ecophysiology of uncultured bacteria

Millions of microbial species thrive on Earth, but till today microbiologists have only succeeded in isolating a few thousand bacterial and archaeal species. Uncultured microbes are essential for the health of the planet and provide, due to their enormous genetic diversity, a potentially rich source for new biotechnologically relevant products. However, for most uncultured microbes no information on their physiological capabilities is available. Thus, there is an obvious need for the development of methods which allow to simultaneously identifying and investigating the physiology of microorganisms within their natural habitat. For this purpose, we have coupled fluorescence in situ hybridisation (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Cells which were 13C labelled through anabolic incorporation of the isotope exhibited key redshifted spectral peaks in the highly resolved Raman confocal spectra, the calculated ‘red shift ratio’ being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of probe-labeled microbial populations at the single cell level. In order to demonstrate the suitability of this technology for structure-function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms

Raman FISH

Linking both identity and function within natural microbial communities has long been seen as one of the ultimate goals for microbial ecologists. The phenotypic data obtained from single-cell Raman microspectroscopy can be combined with the phylogenetic resolution of fluorescence in situ hybridization (FISH) to provide a powerful tool for investigating the structure and function of natural microbial communities (Raman-FISH). This chapter provides information about the use of isotopically labeled substrates to track isotope incorporation and hence specific metabolic processes within bacterial functional groups, through the use of Raman-FISH. A general overview of the equipment and techniques necessary for conducting Raman-FISH analysis is followed by a case study highlighting the utility of this technique for discovering the bacterial communities responsible for naphthalene degradation in groundwater microcosms

Novel primer sets for next generation sequencing-based analyses of water quality

Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination

Advances in restoration ecology: rising to the challenges of the coming decades

Simultaneous environmental changes challenge biodiversity persistence and human wellbeing. The science and practice of restoration ecology, in collaboration with other disciplines, can contribute to overcoming these challenges. This endeavor requires a solid conceptual foundation based in empirical research which confronts, tests and influences theoretical developments. We review conceptual developments in restoration ecology over the last 30 years. We frame our review in the context of changing restoration goals which reflect increased societal awareness of the scale of environmental degradation and the recognition that inter-disciplinary approaches are needed to tackle environmental problems. Restoration ecology now encompasses facilitative interactions and network dynamics, trophic cascades, and above- and below ground linkages. It operates in a non-equilibrium, alternative states framework, at the landscape scale, and in response to changing environmental, economic and social conditions. Progress has been marked by conceptual advances in the fields of trait-environment relationships, community assembly, and understanding the links between biodiversity and ecosystem functioning. Conceptual and practical advances have been enhanced by applying evolving technologies, including treatments to increase seed germination and overcome recruitment bottlenecks, high throughput DNA sequencing to elucidate soil community structure and function, and advances in satellite technology and GPS tracking to monitor habitat use. The synthesis of these technologies with systematic reviews of context dependencies in restoration success, model based analyses and consideration of complex socio-ecological systems will allow generalizations to inform evidence based interventions. Ongoing challenges include setting realistic, socially acceptable goals for restoration under changing environmental conditions, and prioritizing actions in an increasingly space-competitive world. Ethical questions also surround the use of genetically modified material, translocations, taxon substitutions, and de-extinction, in restoration ecology. Addressing these issues, as the Ecological Society of America looks to its next century, will require current and future generations of researchers and practitioners, including economists, engineers, philosophers, landscape architects, social scientists and restoration ecologists, to work together with communities and governments to rise to the environmental challenges of the coming decades

Microbial inoculation to improve plant performance in mine‐waste substrates: A test using pigeon pea (Cajanus cajan)

Mining activities alter soil physicochemical and biological properties that are critical for plant establishment. Revitalisation of soil biological properties via microbial inoculations can potentially be adopted to improve vegetation restoration. Here, we evaluate the feasibility of using beneficial microorganisms in the form of commercially available inoculants to enhance plant performance in a non-toxic and infertile mine-waste substrate, using pigeon pea [Cajanus cajan (L) Millsp.] as a test plant. Six treatments were established to investigate the effects of inoculants (Bradyrhizobium spp., microbial mix and uninoculated controls) and water availability (low and moderate) in a factorial design over 6 months. Plant performance was determined by physiological parameters (leaf gas exchange, leaf carbon, nitrogen and stable isotopes) and growth (height and biomass). Plant xylem sap phytohormones were measured to determine the plants' physiological status and effects of inoculation treatments. Results revealed that water had a greater effect on plant growth than inoculation treatments. Inoculation treatments, however, improved some physiological parameters. This study suggests that physical conditions such as soil moisture and nutrient availability may occlude more subtle (direct or interactive) effects of beneficial soil microbes on plant growth and plant condition. Prior knowledge on the biological and physicochemical properties of the soil to be amended, and on plant species-specific responses, would be needed to customise microbial inoculants for maximum benefits to ecological restoration, to support future adoption of this practice

Microbial analysis of soil and groundwater from a gasworks site and comparison with a sequenced biological reactive barrier remediation process

Aims: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. Methods and Results: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. Conclusion: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. Significance and Impact of the Study: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process

Novel raman microspectroscopy and stable isotope probing to investigate a gas station site contaminated by naphthalene

A meeting abstract on Novel raman microspectroscopy and stable isotope probing to investigate a gas station site contaminated by naphthalene. The major concepts being: Methods and Techniques; Pollution Assessment Control and Management; Molecular Genetics (Biochemistry and Molecular Biophysics); Groundwater Ecology (Ecology, Environmental Sciences