Insights into metal tolerance and resistance mechanisms in Trichoderma asperellum unveiled by de novo transcriptome analysis during bioleaching.

This study investigates the genetic responses of the fungus Trichoderma asperellum (T. asperellum) during bioleaching of ore and tailing samples, comparing one-step, two-step, and spent media bioleaching processes. HPLC analysis quantified oxalic acid, citric acid, and propionic acids, with oxalic acid identified as the primary organic acid involved in metal bioleaching. Metal analysis revealed differences in recovery between ore and tailing samples and among bioleaching processes. The two-step bioleaching process yielded the highest zinc (>54%) and nickel (>60%) recovery in tailings and ore, respectively. Nickel's efficient recovery in ore bioleaching was attributed to the presence of manganese, while its precipitation as nickel oxalate in tailings hindered recovery. Additional metals such as Co, Mn, Mg, Cu, and As were also successfully recovered. Transcriptomic analyses showed significant upregulation of genes associated with biological processes and cellular components, particularly those related to cell membrane structure and function, indicating T. asperellum's adaptation to environmental stresses during metal bioleaching. These findings enhance our understanding of the diverse mechanisms influencing metal recovery rates in bioleaching processes.

Impact of current anthropogenic activities on Blesbokspruit wetland microbiome and functions.

Till present, natural wetlands have been continuously subjected to intensive pollution stress in recent years, mainly because of the rapidly growing industrialization and urbanization that are associated with a myriad of anthropogenic activities and land use practices. These man-made sources of pollution change the chemical properties of the natural wetlands, which in turn alter their microbial ecological biodiversity and functions. For the first time, the impact of the current anthropogenic activities and land use practices on the Blesbokspruit wetland chemical status and their consequential effect on the microbial structure and functions were investigated. Sites of high pollution intensity were identified using geographic information systems mapping (GISMapping) and the wetland microbiome and functional profile were studied through the use of high throughput shotgun metagenomics sequencing analysis. The predominant phyla that stemmed along the Blesbokspruit wetland were found to be Proteobacteria which was more dominant in water (93 %) than in the sediments (89 %), followed by firmicutes which was more abundant in sediments (9 %) than in water (6 %), and Bacteroidetes were relatively low in abundance within both the sediments (2 %) and the overlying water (1 %). The genera Klebsiella (70.4 %-28.2 %), Citrobacter (52.0 %-30.6 %), Escherichia (51.0 %-8.4 %), and Lynsinibacillus (9.3 %-1.5 %) were observed in most water and sediment samples. Within the six polluted sites, Site 2 was found to be the most highly polluted site in the Blesbokspruit wetland with very high COD (900 mg/L), TOC (11.60 mg/L), NO3- (39.74 mg/L), NO2- (12.64 mg/L), PO43 (4.14 mg/L), Fl- (143.88 mg/L), Cl- (145.95 mg/L) concentrations recorded in the water and high levels of TOC (0.37 mg/L), TC (6.92 %), TN (1.82 %), TS (0.53 %) in sediments. The microbial community structure and functions were found to be strongly influenced by the high organic content from the intense agricultural activities and sewage spillages and heavy metals from the mining activities nearby.

Phytoextraction of anthropogenic heavy metal contamination of the Blesbokspruit wetland: Potential of wetland macrophytes.

Blesbokspruit wetland is one of the least conserved ecosystems in the Southern Africa region with active and latent threats of anthropogenic contamination stretching over decades of mining wastewater discharge, agricultural run-off, and a consistent influx of untreated sewage. This study provides an insight into the present-day spatial distribution of heavy metal contamination and the role of localised macrophytes in their phytoremediation. With exception of the first sampling point, the concentration of heavy metals in water samples throughout the wetland was within limits however findings from sediment samples were the inverse. The concentrations of Chromium and Nickel significantly exceeded both effect range low (ERL) and effect range medium (ERM) limits (250-430 mg/Kg and 73-151 mg/Kg respectively) as set out by international sediment quality guidelines. Emergent- Phragmites australis, Typha capensis, and free-flowing-Eichhornia crassipes macrophytes, which are naturally localised to the wetland were found to have varying bioaccumulation potential for different heavy metals; Bioconcentration of heavy metals in emergent macrophytes was relatively low especially for Nickel and Chromium compared to free-flowing macrophytes. E. crassipes accumulated significant amounts of the heavy metals with root concentrations of up to 17.23, 116.6, 330.5, and 342.9 mg/Kg for Arsenic, Lead, Nickel, and Chromium respectively. The emergent macrophytes were however found to bioconcentrate Arsenic up to 1.15 L/Kg (T. capensis) and 9.9 L/Kg (P. Australis) at sites 4 and 5 respectively.   Findings with regards to bioconcentration especially of the E. crassipes, validate recommendations for the utilization of hyperaccumulating macrophytes for the natural recovery of these heavy metals towards alleviating the anthropogenic stress on this valuable ecosystem.

Applying EDTA in Chelating Excess Metal Ions to Improve Downstream DNA Recovery from Mine Tailings for Long-Read Amplicon Sequencing of Acidophilic Fungi Communities.

The hostile environment of mine tailings contains unique microbial life capable of bioleaching. The metagenomic analysis of such an environment provides an in-depth understanding of the microbial life and its potential, especially in biomining operations. However, DNA recovery from samples collected in those environments is challenging due to the presence of metal ions that interfere with the DNA analysis. A varied concentration of EDTA (4-13 µg/µL) to chelate the metal ions of enriched tailing samples prior to DNA extraction was performed. The results show that 9 µg/µL of EDTA was effective in most samples. However, the increasing concentration of EDTA negatively affected the DNA recovery. The sequencing of the successfully extracted DNA revealed a diverse range of fungal genera, some of which have not been previously reported in tailing or bioleaching applications. The dominant genera include Fodinomyces, Penicillium, Recurvomuces, Trichoderma, and Xenoacremonium; their traits were determined using the FungalTraits database. This study demonstrates the need to include a preliminary metal-chelating step using EDTA before DNA extractions for samples collected from metal-rich environments. It further showed the need for optimization but provided a benchmark range, particularly for tailings. However, we caution that a further EDTA removal step from the extracted DNA should be included to avoid its interferences in downstream applications.

Insufficiency in functional genomics studies, data, and applications: A case study of bio-prospecting research in ruminant microbiome.

Over the last two decades, biotechnology has advanced at a rapid pace, propelled by the incorporation of bio-products into various aspects of pharmaceuticals, industry, and the environment. These developments have sparked interest in the bioprospecting of microorganisms and their products in a variety of niche environments. Furthermore, the use of omics technologies has greatly aided our analyses of environmental samples by elucidating the microbial ecological framework, biochemical pathways, and bio-products. However, the more often overemphasis on taxonomic identification in most research publications, as well as the data associated with such studies, is detrimental to immediate industrial and commercial applications. This review identifies several factors that contribute to the complexity of sequence data analysis as potential barriers to the pragmatic application of functional genomics, utilizing recent research on ruminants to demonstrate these limitations in the hopes of broadening our horizons and drawing attention to this gap in bioprospecting studies for other niche environments as well. The review also aims to emphasize the importance of routinely incorporating functional genomics into environmental metagenomics analyses in order to improve solutions that drive rapid industrial biocatalysis developments from derived outputs with the aim of achieving potential benefits in energy-use reduction and environmental considerations for current and future applications.

Addressing the water-energy nexus: A focus on the barriers and potentials of harnessing wastewater treatment processes for biogas production in Sub Saharan Africa.

Several anthropogenic activities reduce the supply of freshwater to living organisms in all ecological systems, particularly the human population. Organic matter in derived wastewater can be converted into potential energy, such as biogas (methane), through microbial transformation during anaerobic digestion (AD). To address the current lack of data and values for wastewater generation in Sub-Saharan Africa, this review analyzes and estimates (at 50% and 90% conversion rates) the potential amount of wastewater-related sludge that can be generated from domestic freshwater withdrawals using the most recent update in 2017 from the World Bank repository and database on freshwater status in Sub-Saharan Africa. The Democratic Republic of the Congo (DRC) could potentially produce the highest estimate of biogas in Sub-Saharan Africa from domestic wastewater sludge of approximately 90 billion m3, which could be converted to 178 million MWh of electricity annually, based on this extrapolation at 50% conversion rates. Using same conversion rates estimates, at least nine other countries, including Guinea, Liberia, Nigeria, Sierra Leone, Angola, Cameroon, Central African Republic, Gabon, and Congo Republic, could potentially produce biogas in the range of 1-20 billion m3. These estimates show how much energy could be extracted from wastewater treatment plants in Sub-Saharan Africa. AD process to produce biogas and energy harvesting are essential supplementary operations for Sub-Saharan African wastewater treatment plants. This approach could potentially solve the problem of data scarcity because these values for Freshwater withdrawals are readily available in the database could be used for estimation and projections towards infrastructure development and energy production planning. The review also highlights the possibilities for energy generation from wastewater treatment facilities towards wastewater management, clean energy, water, and sanitation sustainability, demonstrating the interconnections and actualization of the various related UN Sustainable Development Goals.

Physico-Chemical and Metagenomic Profile Analyses of Animal Manures Routinely Used as Inocula in Anaerobic Digestion for Biogas Production.

Anaerobic digestion (AD) of organic waste is considered a sustainable solution to energy shortage and waste management challenges. The process is facilitated by complex communities of micro-organisms, yet most wastes do not have these and thus need microbial inoculation using animal manures to initiate the process. However, the degradation efficiency and methane yield achieved in using different inocula vary due to their different microbial diversities. This study used metagenomics tools to compare the autochthonous microbial composition of cow, pig, chicken, and horse manures commonly used for biogas production. Cows exhibited the highest carbon utilisation (>30%) and showed a carbon to nitrogen ratio (C/N) favourable for microbial growth. Pigs showed the least nitrogen utilisation (<3%) which explains their low C/N whilst horses showed the highest nitrogen utilisation (>40%), which explains its high C/N above the optimal range of 20−30 for efficient AD. Manures from animals with similar gastrointestinal tract (GIT) physiologies were observed to largely harbour similar microbial communities. Conversely, some samples from animals with different GITs also shared common microbial communities plausibly because of similar diets and rearing conditions. Insights from this study will lay a foundation upon which in-depth studies of AD metabolic pathways and strategies to boost methane production through efficient catalysis can be derived.

Trends and Applications of Omics Technologies to Functional Characterisation of Enzymes and Protein Metabolites Produced by Fungi.

Identifying and adopting industrial applications for proteins and enzymes derived from fungi strains have been at the focal point of several studies in recent times. To facilitate such studies, it is necessary that advancements and innovation in mycological and molecular characterisation are concomitant. This review aims to provide a detailed overview of the necessary steps employed in both qualitative and quantitative research using the omics technologies that are pertinent to fungi characterisation. This stems from the understanding that data provided from the functional characterisation of fungi and their metabolites is important towards the techno-economic feasibility of large-scale production of biological products. The review further describes how the functional gaps left by genomics, internal transcribe spacer (ITS) regions are addressed by transcriptomics and the various techniques and platforms utilised, including quantitive reverse transcription polymerase chain reaction (RT-qPCR), hybridisation techniques, and RNA-seq, and the insights such data provide on the effect of environmental changes on fungal enzyme production from an expressional standpoint. The review also offers information on the many available bioinformatics tools of analysis necessary for the analysis of the overwhelming data synonymous with the omics approach to fungal characterisation.

Applying PICRUSt and 16S rRNA functional characterisation to predicting co-digestion strategies of various animal manures for biogas production.

An estimated 25 million tons of animal manure is produced globally every year, causing considerable impact to the environment. These impacts can be managed through the use of anaerobic digestion (AD) This process achieves waste degradation through enzymatic activity, the efficiency of the AD process is directly related to microorganisms that produce these enzymes. Biomethane potential (BMP) assays remain the standard theoretical framework to pre-determine biogas yield and have been used to determine the feasibility of substrates or their combination for biogas production. However, an integrated approach that combines substrate choice and co-digestion would provide an improvement to the current predictive models. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) addresses the limitations of assays in this regard. In this paper, the biochemical functions of horse, cow, and pig manures are predicted. A total of 135 predicted KEGG Orthologies (KOs) showed amino acids, carbohydrate, energy, lipid, and xenobiotic metabolisms in all the samples. Linear discriminant analysis (LDA) combined with the effect size measurements (LEfSe), showed that fructose, mannose, amino acid and nucleotide sugar, phosphotransferase (PST) as well as starch and sucrose metabolisms were significantly higher in horse manure samples. 36 of the KOs were related to the acidogenesis and/or acetogenesis AD stages. Extended bar plots showed that 11 significant predictions were observed for horse-cow, while 5 were predicted for horse-pig and for cow-pig manures. Based on these predictions, the AD process can be enhanced through co-digestion strategies that takes into account the predicted metabolic contributions of the manure samples. The results supported the BMP calculations for the samples in this study. Biogas yields can be improved if this combined approach is employed in routine analysis before co-digesting different substrates.

Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70.

Plasmodium falciparum heat shock protein (PfHsp70) has been proposed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. However, the biochemical and chaperone properties of PfHsp70 have not been elucidated. The heterologous overproduction of P. falciparum proteins in Escherichia coli is problematic because of its AT-rich genome and the usage of codons that are rarely used in E. coli. In this paper, we describe the successful overproduction of (His)(6)-PfHsp70 in E. coli using the pQE30 expression vector system. Initial experiments with E. coli [pQE30/PfHsp70] resulted in the overproduction of the full-length protein and truncated derivatives. The RIG plasmid, which encodes tRNAs for rare codons, was engineered into the E. coli [pQE30/PfHsp70] strain, resulting in significant reduction of the truncated (His)(6)-PfHsp70 derivatives and improved yields of the full-length protein. (His)(6)-PfHsp70 was successfully purified using nickel-chelating Sepharose affinity chromatography and its biochemical properties were determined. The V(max), K(m), and k(cat) for the basal ATPase activity of (His)(6)-PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 microM, and 1.03 min(-1), respectively. Gel filtration studies indicated that (His)(6)-PfHsp70 existed largely as a monomer in solution. This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.