Copper nanoparticles embedded fungal chitosan as a rational and sustainable bionanozyme with robust laccase activity for catalytic oxidation of phenolic pollutants.

Despite their potential for oxidation of persistent environmental pollutants, the development of rational and sustainable laccase nanozymes with efficient catalytic performance remains a challenge. Herein, fungal-produced chitosan-copper (CsCu) is proposed as a rational and sustainable bionanozyme with intrinsic laccase activity. The CsCu nanozyme was prepared by in situ reduction of copper on chitosan extracted from Irpex sp. isolate AWK2 a native fungus, from traditional fermented foods, yielding a low molecular weight chitosan with a 70% degree of deacetylation. Characterizations of the nanozyme using SEM-EDX, XRD, and XPS confirmed the presence of a multi-oxidation state copper on the chitosan matrix which is consistent with the composition of natural laccase. The laccase memetic activity was investigated using 2,4-DP as a substrate which oxidized to form a reddish-pink color with 4-AP (λmax = 510 nm). The CsCu nanozyme showed 38% higher laccase activity than the pristine Cu NPs at pH 9, indicating enhanced activity in the presence of chitosan structure. Further, CsCu showed significant stability in harsh conditions and exhibited a lower Km (0.26 mM) which is competitive with that reported for natural laccase. Notably, the nanozyme converted 92% of different phenolic substrates in 5 h, signifying a robust performance for environmental remediation purposes.

Bacterial Nanocellulose/Copper as a Robust Laccase-Mimicking Bionanozyme for Catalytic Oxidation of Phenolic Pollutants.

Industrial effluents containing phenolic compounds are a major public health concern and thus require effective and robust remediation technologies. Although laccase-like nanozymes are generally recognized as being catalytically efficient in oxidizing phenols, their support materials often lack resilience in harsh environments. Herein, bacterial nanocellulose (BNC) was introduced as a sustainable, strong, biocompatible, and environmentally friendly biopolymer for the synthesis of a laccase-like nanozyme (BNC/Cu). A native bacterial strain that produces nanocellulose was isolated from black tea broth fermented for 1 month. The isolate that produced BNC was identified as Bacillus sp. strain T15, and it can metabolize hexoses, sucrose, and less expensive substrates, such as molasses. Further, BNC/Cu nanozyme was synthesized using the in situ reduction of copper on the BNC. Characterization of the nanozyme by scanning electron microscopy (SEM) and X-ray diffraction (XRD) confirmed the presence of the copper nanoparticles dispersed in the layered sheets of BNC. The laccase-mimetic activity was assessed using the chromogenic redox reaction between 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) with characteristic absorption at 510 nm. Remarkably, BNC/Cu has 50.69% higher catalytic activity than the pristine Cu NPs, indicating that BNC served as an effective biomatrix to disperse Cu NPs. Also, the bionanozyme showed the highest specificity toward 2,4-DP with a Km of 0.187 mM, which was lower than that of natural laccase. The bionanozyme retained catalytic activity across a wider temperature range with optimum activity at 85 °C, maintaining 38% laccase activity after 11 days and 46.77% activity after the fourth cycle. The BNC/Cu bionanozyme could efficiently oxidize more than 70% of 1,4-dichlorophenol and phenol in 5 h. Thereby, the BNC/Cu bionanozyme is described here as having an efficient ability to mimic laccase in the oxidation of phenolic compounds that are commonly released into the environment by industry.

Kerosene Biodegradation by Highly Efficient Indigenous Bacteria Isolated From Hydrocarbon-Contaminated Sites.

Kerosene is widely used in Ethiopia as a household fuel (for lighting and heating), as a solvent in paint and grease, and as a lubricant in glass cutting. It causes environmental pollution and escorts to loss of ecological functioning and health problems. Therefore, this research was designed to isolate, identify, and characterize indigenous kerosene-degrading bacteria that are effective in cleaning ecological units that have been contaminated by kerosene. Soil samples were collected from hydrocarbon-contaminated sites (flower farms, garages, and old-aged asphalt roads) and spread-plated on mineral salt medium (Bushnell Hass Mineral Salts Agar Medium: BHMS), which consists of kerosene as the only carbon source. Seven kerosene-degrading bacterial species were isolated, 2 from flower farms, 3 from garage areas, and 2 from asphalt areas. Three genera from hydrocarbon-contaminated sites were identified, including Pseudomonas, Bacillus, and Acinetobacter using biochemical characterization and the Biolog database. Growth studies in the presence of various concentrations of kerosene (1% and 3% v/v) showed that the bacterial isolates could metabolize kerosene as energy and biomass. Thereby, a gravimetric study was performed on bacterial strains that proliferated well on a BHMS medium with kerosene. Remarkably, bacterial isolates were able to degrade 5% kerosene from 57.2% to 91% in 15 days. Moreover, 2 of the most potent isolates, AUG2 and AUG1, resulted in 85% and 91% kerosene degradation, respectively, when allowed to grow on a medium containing kerosene. In addition, 16S rRNA gene analysis indicated that strain AAUG1 belonged to Bacillus tequilensis, whereas isolate AAUG showed the highest similarity to Bacillus subtilis. Therefore, these indigenous bacterial isolates have the potential to be applied for kerosene removal from hydrocarbon-contaminated sites and the development of remediation approaches.

Multi-faceted CRISPR-Cas9 strategy to reduce plant based food loss and waste for sustainable bio-economy - A review.

Currently, international development requires innovative solutions to address imminent challenges like climate change, unsustainable food system, food waste, energy crisis, and environmental degradation. All the same, addressing these concerns with conventional technologies is time-consuming, causes harmful environmental impacts, and is not cost-effective. Thus, biotechnological tools become imperative for enhancing food and energy resilience through eco-friendly bio-based products by valorisation of plant and food waste to meet the goals of circular bioeconomy in conjunction with Sustainable Developmental Goals (SDGs). Genome editing can be accomplished using a revolutionary DNA modification tool, CRISPR-Cas9, through its uncomplicated guided mechanism, with great efficiency in various organisms targeting different traits. This review's main objective is to examine how the CRISPR-Cas system, which has positive features, could improve the bioeconomy by reducing food loss and waste with all-inclusive food supply chain both at on-farm and off-farm level; utilising food loss and waste by genome edited microorganisms through food valorisation; efficient microbial conversion of low-cost substrates as biofuel; valorisation of agro-industrial wastes; mitigating greenhouse gas emissions through forestry plantation crops; and protecting the ecosystem and environment. Finally, the ethical implications and regulatory issues that are related to CRISPR-Cas edited products in the international markets have also been taken into consideration.

Nanocellulose/Fe3O4/Ag Nanozyme with Robust Peroxidase Activity for Enhanced Antibacterial and Wound Healing Applications.

Peroxidase memetic nanozymes with their free radical-mediated catalytic actions proved as efficacious antibacterial agents for combating bacterial resistance. Herein, nanocellulose (NC) extracted from Eragrostis teff straw was used to prepare NC/Fe3O4/Ag peroxidase nanozyme as an antibacterial and wound healing agent. Characterization of the nanozyme with XRD, FTIR, SEM-EDX, and XPS confirmed the presence of silver NPs and the magnetite phase of iron oxide dispersed on nanocellulose. The peroxidase activity of the prepared nanozyme was examined using TMB and H2O2 as substrates which turned blue in acidic pH (λmax = 652 nm). With a lower Km (0.387 mM), the nanozyme showed a comparable affinity for TMB with that reported for the HRP enzyme. Furthermore, the nanozyme remained efficient over a broader temperature range while maintaining 61.53% of its activity after the fourth cycle. In vitro, antibacterial tests against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacterial strains showed that NC/Fe3O4/Ag exhibits concentration-dependent and enhanced antibacterial effect for Escherichia coli compared to NC and NC-Fe3O4 and negative control. Furthermore, the wound-healing performance of the NC-Fe3O4-Ag nanozyme was investigated in vivo using an animal model (mice). The nanozyme showed 30% higher wound healing performance compared to the control base ointment and is comparable with the commercial nitrofurazone ointment. The results show the potential of the prepared nanozyme for wound-healing purposes.

Isolation and Characterization of Diesel-Degrading Bacteria from Hydrocarbon-Contaminated Sites, Flower Farms, and Soda Lakes.

Hydrocarbon-derived pollutants are becoming one of the most concerning ecological issues. Thus, there is a need to investigate and develop innovative, low-cost, eco-friendly, and fast techniques to reduce and/or eliminate pollutants using biological agents. The study was conducted to isolate, characterize, and identify potential diesel-degrading bacteria. Samples were collected from flower farms, lakeshores, old aged garages, asphalt, and bitumen soils and spread on selective medium (Bushnell Haas mineral salt agar) containing diesel as the growth substrate. The isolates were characterized based on their growth patterns using optical density measurement, biochemical tests, and gravimetric analysis and identified using the Biolog database and 16S rRNA gene sequencing techniques. Subsequently, six diesel degraders were identified and belong to Pseudomonas, Providencia, Roseomonas, Stenotrophomonas, Achromobacter, and Bacillus. Among these, based on gravimetric analysis, the three potent isolates AAUW23, AAUG11, and AAUG36 achieved 84%, 83.4%, and 83% diesel degradation efficiency, respectively, in 15 days. Consequently, the partial 16S rRNA gene sequences revealed that the two most potent bacterial strains (AAUW23 and AAUG11) were Pseudomonas aeruginosa, while AAUG36 was Bacillus subtilis. This study demonstrated that bacterial species isolated from hydrocarbon-contaminated and/or uncontaminated environments could be optimized to be used as potential bioremediation agents for diesel removal.

Elevated Level of Imipenem-Resistant Gram-Negative Bacteria Isolated from Patients Attending Health Centers in North Gondar, Ethiopia.

BACKGROUND: The frequent identification of resistant bacteria in hospitals constantly presents antimicrobial therapy with a challenge. Imipenem, once considered an extremely powerful antibiotic against multidrug-resistant bacterial infections, is losing its effectiveness. Its use in empirical therapy with inadequate or nonexistent antimicrobial stewardship programs has further triggered bacterial resistance in low-income countries. Therefore, this study aimed at identifying imipenem-resistant Gram-negative bacteria from patients who were referred to health centers in North Gondar, Ethiopia. METHODS: A total of 153 sputum samples were used to isolate Gram-negative bacteria. The isolates, which were resistant to imipenem, were identified by standard biochemical tests and 16S rRNA sequencing. The Kirby-Bauer disk diffusion method was used to determine the sensitivity or resistance of the isolate to diverse antimicrobial agents. RESULTS: The study identified 79 imipenem-resistant bacterial isolates from eight genera with clinically relevant microorganisms, including Acinetobacter baumannii (20.77%), Klebsiella pneumoniae (19.48%), Pseudomonas aeruginosa (16.88%), and Serratia marcescens (14.28%). Overall, imipenem-resistant bacterial isolates were detected in 31 samples (20.26%). Additionally, a remarkably high level of resistance to most antibiotics was observed among isolates of Klebsiella pneumoniae and Acinetobacter baumannii. Gentamycin is the most active antibiotic against many of the isolates, while ß-lactams appear to be less effective. CONCLUSION: The study indicated that many Gram-negative bacteria were resistant to imipenem with parallel resistances to other antimicrobials. Hence, the prescription of imipenem within the region should be according to the antibiotic resistance profiles of the multi-drug resistant bacteria.

Analysis of Phylogenetic Variation of Stenotrophomonas maltophilia Reveals Human-Specific Branches.

Stenotrophomonas maltophilia is a non-fermenting Gram-negative bacterium that is ubiquitous in the environment. In humans, this opportunistic multi-drug-resistant pathogen is responsible for a plethora of healthcare-associated infections. Here, we utilized a whole genome sequencing (WGS)-based phylogenomic core single nucleotide polymorphism (SNP) approach to characterize S. maltophilia subgroups, their potential association with human infection, and to detect any possible transmission events. In total, 89 isolates (67 clinical and 22 environmental) from Germany were sequenced. Fully finished genomes of five strains were included in the dataset for the core SNP phylogenomic analysis. WGS data were compared with conventional genotyping results as well as with underlying disease, biofilm formation, protease activity, lipopolysaccharide (LPS) SDS-PAGE profiles, and serological specificity of an antibody raised against the surface-exposed O-antigen of strain S. maltophilia K279a. The WGS-based phylogenies grouped the strains into 12 clades, out of which 6 contained exclusively human and 3 exclusively environmental isolates. Biofilm formation and proteolytic activity did correlate neither with the phylogenetic tree, nor with the origin of isolates. In contrast, the genomic classification correlated well with the reactivity of the strains against the K279a O-specific antibody, as well as in part with the LPS profiles. Three clusters of clinical strains had a maximum distance of 25 distinct SNP positions, pointing to possible transmission events or acquisition from the same source. In conclusion, these findings indicate the presence of specific subgroups of S. maltophilia strains adapted to the human host.

Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and ß-Lactamase Expression.

Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas maltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the L1 and L2 ß-lactamases in response to ß-lactam treatment. Here we report that the patient isolate S. maltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bla L1 and bla L2 were transcriptionally the most strongly upregulated genes. Promoter fusions of bla L1 and bla L2 genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla L2 expressing cells as identified by RNA-seq analysis. Overexpression of comE in S. maltophilia K279a reduced the level of cells that were in a bla L2-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including bla L1, bla L2, and comE.