Relative abundance and the fate of human rotavirus in wastewater during treatment processes: identification of potential infectious rotavirus in the final effluents and receiving aquatic milieu in Durban area, South Africa.

The occurrence and persistence of rotaviruses in raw and treated wastewater and their discharge into rivers represent a significant health risk for humans and animals, worldwide. In this study, samples were collected monthly from each of the four Durban wastewater treatment plants (DWWTPs) and receiving rivers for a period of 3 months. Rotavirus was quantified by real-time quantitative PCR (RT-qPCR), and viability was assessed using integrated cell culture (ICC)-qPCR. Rotavirus was detected consistently in 100% of influent wastewaters (mean concentration range, 4.36-4.46 log10 genome equivalent (GE) copies/L) and final effluent samples of three DWWTPs (range, 3.35-3.61 log10 GE copies/L). Overall, 94% (45/48) of the wastewater analyzed and 95% (20/21) of the associated river water samples were positive for rotavirus (range, 2.04-6.77 log10 GE copies/L). The activated sludge process with 0.10-0.43 log10 reduction values (LRV) only moderately reduced the viral loads. Similarly, one of the DWWTPs that operated the biofilter modality produced 0.20 LRV. Though the additional treatment with chlorine produced higher LRV (range, 0.31-0.53) than the corresponding activated sludge or biofilter process, the difference in viral removals was not significant (p > 0.05). The equivalent treatment efficiencies of the four DWWTPs varied from 19 to 43% decay in the population of rotavirus. Further, infectious rotavirus ranging from 66.67 to 100%, 50 to 100%, and 66.67 to 100% were detected in the post-activated sludge, final effluents, and river water samples, respectively. In conclusion, the findings of infectious rotavirus in both the final effluents and associated rivers represent an infection risk for humans or animals during contact. Thus, close monitoring for rotavirus and risk assessment studies under distinct exposure scenarios may further shed light on the health-related risks associated with water recovery and reuse in urban settings.

Low-cost and reliable substrate-based phenotyping platform for screening salt tolerance of cutting propagation-dependent grass, paspalum vaginatum.

BACKGROUND: Salt tolerance in plants is defined as their ability to grow and complete their life cycle under saline conditions. Staple crops have limited salt tolerance, but forage grass can survive in large unexploited saline areas of costal or desert land. However, due to the restriction of self-incompatible fertilization in many grass species, vegetative propagation via stem cuttings is the dominant practice; this is incompatible with current methodologies of salt-tolerance phenotyping, which have been developed for germination-based seedling growth. Therefore, the performance of seedlings from cuttings under salt stress is still fuzzy. Moreover, the morphological traits involved in salt tolerance are still mostly unknown, especially under experimental conditions with varying levels of stress. RESULTS: To estimate the salt tolerance of cutting propagation-dependent grasses, a reliable and low-cost workflow was established with multiple saline treatments, using Paspalum vaginatum as the material and substrate as medium, where cold stratification and selection of stem segments were the two variables used to control for experimental errors. Average leaf number (ALN) was designated as the best criterion for evaluating ion-accumulated salt tolerance. The reliability of ALN was revealed by the consistent results among four P. vaginatum genotypes, and three warm-season (pearl millet, sweet sorghum, and wild maize) and four cold-season (barley, oat, rye, and ryegrass) forage cultivars. Dynamic curves simulated by sigmoidal mathematical models were well-depicted for the calculation of the key parameter, Salt50. The reliability of the integrated platform was further validated by screening 48 additional recombinants, which were previously generated from a self-fertile mutant of P. vaginatum. The genotypes displaying extreme ALN-based Salt50 also exhibited variations in biomass and ion content, which not only confirmed the reliability of our phenotyping platform but also the representativeness of the aerial ALN trait for salt tolerance. CONCLUSIONS: Our phenotyping platform is proved to be compatible with estimations in both germination-based and cutting propagation-dependent seedling tolerance under salt stresses. ALN and its derived parameters are prone to overcome the species barriers when comparing salt tolerance of different species together. The accuracy and reliability of the developed phenotyping platform is expected to benefit breeding programs in saline agriculture.

Molecular Characterization and Phylogenetic analyses of Rotaviruses Circulating in Municipal Sewage and Sewage-Polluted River Waters in Durban Area, South Africa.

Globally, rotavirus continues to be the leading etiology of severe pediatric gastroenteritis, and transmission of the disease via environmental reservoirs has become an emerging concern in developing countries. From August to October 2021, a total of 69 samples comprising 48 of raw and treated sewage, and 21 surface waters, were collected from four Durban wastewater treatment plants (DWWTP), and effluent receiving rivers, respectively. Rotaviruses recovered and identified from the samples were subjected to sequencing, genotyping, and phylogenetic analysis. Of the 65 (94.2%) rotavirus-positive samples, 33.3% were from raw sewage, 16% from activated sludge, 15.9% from final effluents, and 29.0% were from the receiving river samples. A total of 49 G and 41 P genotypes were detected in sewage while 15 G and 22 P genotypes were detected in river samples. G1 genotype predominated in sewage (24.5%) followed by G3 (22.4%), G2 (14.3%), G4 (12.2%), G12 (10.2%), G9 (8.2%), and G8 (6.1%). Similarly, G1 predominated in river water samples (33.3%) and was followed by G2, G4 (20.0% each), G3, and G12 (13.3% each). Rotavirus VP4 genotypes P[4], P[6], and P[8] accounted for 36.6%, 29.3%, and 9.8%, respectively, in sewage. Correspondingly, 45.5%, 31.8%, and 13.6% were detected in river samples. The G and P genotypes not identified by the methods used were 2.1% versus 24.3% and 0.1% versus 9.1% for sewage and river water samples, respectively. Sequence comparison studies indicated a high level of nucleotide identity in the G1, G2, G3, G4, G8 VP7, and P[4], P[6], and P[8] VP4 gene sequences between strains from the environment and those from patients in the region. This is the first environmental-based study on the G and P genotypes diversity of rotavirus in municipal wastewater and their receiving rivers in this geographical region. The high similarity between environmental and clinical rotavirus strains suggests both local circulation of the virus and potential exposure risks. In addition, it highlights the usefulness of sewage surveillance as an additional tool for an epidemiological investigation, especially in populations that include individuals with subclinical or asymptomatic infections that are precluded in case-based studies.

A multifunctional enolase mediates cytoadhesion and interaction with host plasminogen and fibronectin in Mycoplasma hyorhinis.

Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights as an adhesin in Mycoplasma hyorhinis adhesion to epithelial cells as well as a plasminogen receptor mediating extracellular matrix degradation.

Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Production strategies and biotechnological relevance of microbial lipases: a review.

Lipases are enzymes that catalyze the breakdown of lipids into long-chain fatty acids and glycerol in oil-water interface. In addition, they catalyze broad spectrum of bioconversion reactions including esterification, inter-esterification, among others in non-aqueous and micro-aqueous milieu. Lipases are universally produced from plants, animals, and microorganisms. However, lipases from microbial origin are mostly preferred owing to their lower production costs, ease of genetic manipulation etc. The secretion of these biocatalysts by microorganisms is influenced by nutritional and physicochemical parameters. Optimization of the bioprocess parameters enhanced lipase production. In addition, microbial lipases have gained intensified attention for a wide range of applications in food, detergent, and cosmetics industries as well as in environmental bioremediation. This review provides insights into strategies for production of microbial lipases for potential biotechnological applications.

Production and potential biotechnological applications of microbial surfactants: An overview.

Microbial surfactants are amphipathic molecules that consist of hydrophilic and hydrophobic domains, which allow partition of two fluid phases of varying degree of polarity. They are classified into two main groups: bioemulsifier and biosurfactant, depending on their molecular weight. Microbial surfactants occur in various categories according to their chemical nature and producing organisms. These biomolecules are produced by diverse groups of microorganisms including fungi, bacteria, and yeasts. Their production is significantly influenced by substrate type, fermentation technology and microbial strains. Owing to inherent multifunctional properties and assorted synthetic aptitude of the microbes, microbial surfactants are mostly preferred than their chemical counterparts for various industrial and biomedical applications including bioremediation, oil recovery; as supplements in laundry formulations and as emulsion-stabilizers in food and cosmetic industries as well as therapeutic agents in medicine. The present review discusses on production of microbial surfactants as promising and alternative broad-functional biomolecules for various biotechnological applications.

Production and characterization of bioemulsifiers from Acinetobacter strains isolated from lipid-rich wastewater.

In this study, two indigenous bacterial strains (Ab9-ES and Ab33-ES) isolated from lipid-rich wastewater showed potential to produce bioemulsifier in the presence of 2% (v/v) olive oil as a carbon source. These bacterial strains were identified as Acinetobacter sp. Ab9-ES and Acinetobacter sp. Ab33-ES by polymerase chain reaction and analysis of 16S rRNA gene sequences. Bioemulsifier production by these strains was found to be growth-linked. Maximum emulsifying activities (83.8% and 80.8%) were recorded from strains Ab9-ES and Ab33-ES, respectively. Bioemulsifier yields of 4.52 g/L and 4.31 g/L were obtained from strains Ab9-ES (XB9) and Ab33-ES (YB33), respectively. Fourier-transform infrared spectroscopic analysis revealed the glycoprotein nature of the bioemulsifiers. The bioemulsifiers formed stable emulsions only in the presence of edible oils. Maximum emulsifying activities of 79.6% (XB9) and 67.9% (YB33) were recorded in the presence of sunflower oil. The bioemulsifiers were found to be stable at a broad range of temperature (4-121 °C), moderate pH (5.0-10.0) and salinity (1-6%). In addition, bioemulsifier XB9 showed maximum emulsifying activities (77.3%, 74.5%, and 74.9%) at optimum temperature (50 °C), pH (7.0), and NaCl concentration (3%), respectively. On the contrary, YB33 demonstrated highest activities (73.6%, 72%, and 61.2%) at optimum conditions of 70 °C, pH 7.0, and NaCl concentration of 5%, respectively. Findings from this study suggest the potential biotechnological applications of the bioemulsifiers, especially in the remediation of oil-polluted sites.

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater.

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77% ± 1.53% and 75.99% ± 3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2 M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60 °C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5-9.5) and temperature (55-65 °C) when incubated for 3 h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4 °C and 25 °C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.

Microbial degradation of 2,4-dichlorophenoxyacetic acid: Insight into the enzymes and catabolic genes involved, their regulation and biotechnological implications.

A considerable progress has been made to understand the mechanisms of biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D biodegradation pathway has been elucidated in many microorganisms including Cupriavidus necator JMP134 (previously known as Wautersia eutropha, Ralstonia eutropha and Alcaligenes eutrophus) and Pseudomonas strains. It generally involves the side chain removal of 2,4-D by α-ketoglutarate-dependent 2,4-D dioxygenase (tfdA) to form 2,4-dichlorophenol (2,4-DCP); hydroxylation of 2,4-DCP by 2,4-DCP hydroxylase (tfdB) to form dichlorocatechol; ortho or meta cleavage of dichlorocatechol by chlorocatechol 1,2-dioxygenase (tfdC) to form 2,4-dichloro-cis,cis-muconate; conversion of 2,4-dichloro-cis,cis-muconate to 2-chlorodienelactone by chloromuconate cycloisomerase (tfdD); conversion of 2-chlorodienelactone to 2-chloromaleylacetate by chlorodienelactone hydrolase (tfdE) and, finally, conversion of 2-chloromaleylacetate to 3-oxoadepate via maleylacetate by chloromaleylacetate reductase and maleylacetate reductase (tfdF), respectively, which is funnelled to the tricarboxylic acid cycle. The latest review on microbial breakdown of 2,4-D, other halogenated aromatic pesticides, and related compounds was compiled by Haggblom, however, a considerable progress has been made in this area of research since then. Thus, this review focuses on the recent advancement on 2,4-D biodegradation, the enzymes, and genes involved and their biotechlogical implications.

Kinetics of heavy metal inhibition of 1,2-dichloroethane biodegradation in co-contaminated water.

Sites co-contaminated with heavy metals and 1,2-DCA may pose a greater challenge for bioremediation, as the heavy metals could inhibit the activities of microbes involved in biodegradation. Therefore, this study was undertaken to quantitatively assess the effects of heavy metals (arsenic, cadmium, mercury, and lead) on 1,2-DCA biodegradation in co-contaminated water. The minimum inhibitory concentrations (MICs) and concentrations of the heavy metals that caused half-life doubling (HLDs) of 1,2-DCA as well as the degradation rate coefficient (k(1)) and half-life (t(½)) of 1,2-DCA were measured and used to predict the toxicity of the heavy metals in the water microcosms. An increase in heavy metal concentration resulted in a progressive increase in the t(½) and relative t(½) and a decrease in k(1). The MICs and HLDs of the heavy metals were found to vary, depending on the heavy metals type. In addition, the presence of heavy metals was shown to inhibit 1,2-DCA biodegradation in a dose-dependent manner, with the following order of decreasing inhibitory effect: Hg(2+) > As(3+) > Cd(2+) > Pb(2+). Findings from this study have significant implications for the development of bioremediation strategies for effective degradation of 1,2-DCA and other related compounds in wastewater co-contaminated with heavy metals.

Quantitative assessment of the toxic effects of heavy metals on 1,2-dichloroethane biodegradation in co-contaminated soil under aerobic condition.

1,2-Dichloroethane (1,2-DCA) is one of the most hazardous pollutant of soil and groundwater, and is produced in excess of 5.44×109 kg annually. Owing to their toxicity, persistence and potential for bioaccumulation, there is a growing interest in technologies for their removal. Heavy metals are known to be toxic to soil microorganisms at high concentrations and can hinder the biodegradation of organic contaminants. In this study, the inhibitory effect of heavy metals, namely; arsenic, cadmium, mercury and lead, on the aerobic biodegradation of 1,2-DCA by autochthonous microorganisms was evaluated in soil microcosm setting. The presence of heavy metals was observed to have a negative impact on the biodegradation of 1,2-DCA in both soil samples tested, with the toxic effect being more pronounced in loam soil, than in clay soil. Generally, 75 ppm As³âº, 840 ppm Hg²âº, and 420 ppm Pb²âº resulted in 34.24%, 40.64%, and 45.94% increase in the half live (t½) of 1,2-DCA, respectively, in loam soil, while concentrations above 127.5 ppm Cd²âº, 840 ppm Hg²âº and 420 ppm of Pb²âº and less than 75 ppm As³âº was required to cause a >10% increase in the t½ of 1,2-DCA in clay soil. A dose-dependent relationship between degradation rate constant (k1) of 1,2-DCA and metal ion concentrations was observed for all the heavy metals tested, except for Hg²âº. This study demonstrated that different heavy metals have different impacts on the degree of 1,2-DCA degradation. Results also suggest that the degree of inhibition is metal specific and is also dependent on several factors including; soil type, pH, moisture content and available nutrients.