Identification of locally isolated entomopathogenic Fusarium species from the soil of Changa Manga Forest, Pakistan and evaluation of their larvicidal efficacy against Aedes aegypti

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases. Current reliance of dengue vector control is mostly on chemical insecticides. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control through chemical insecticides. These chemical insecticides also have negative environmental impacts on animals, plants and human health. Myco-biocontrol agents are naturally occurring organisms and are found to be less damaging to the environment as compared to chemical insecticides. In the present study, entomopathogenic potential of local strains of fungi isolated from soil was assessed for the control of dengue vector. Local fungal isolates presents better alternative to introducing a foreign biocontrol strain, as they may be better adapted to environmental conditions of the area to survive and may have more entomopathogenic efficacy against target organism. Larvicidal efficacy of Fusarium equiseti and Fusarium proliferatum was evaluated against Aedes aegypti. Local strains of F. equiseti (MK371718) and F. proliferatum (MK371715) were isolated from the soil of Changa Manga Forest, Pakistan by using insect bait method. Larvicidal activity of two Fusarium spp. was tested against forth instar larvae of A. aegypti in the laboratory, using concentrations 105, 106, 107 and 108 conidia /ml. LC50 values for F. equiseti after 24h, 48h, 72h and 96h of exposure were recorded as 3.8x 108, 2.9x107, 2.0x107, and 7.1x106 conidia /ml respectively while LC50 values for F. proliferatum were recorded as 1.21x108, 9.6x107, 4.2x107, 2.6x107 conidia /ml respectively after 24h, 48h, 72h and 96h of exposure. The results indicate that among two fungal strains F. equiseti was found to be more effective in terms of its larvicidal activity than F. proliferatum against larvae of A. aegypti.

Sustainable Production of Bioethanol by Zymomonas mobilis and Saccharomyces cerevisiae using Rice Husk and Groundnut Shell as Substrates

Background of Study: Plant waste such as rice husk and groundnut shell are generated in large amounts, these waste presents a tremendous pollution to the environment. Worldwide, these wastes are often simply dumped into landfills and oceans or used as animal feeds. The recovery of food processing wastes as renewable energy sources represents a sustainable option for the substitution of fossil energy in order to minimize environmental damages and to meet energy demands of the growing population. Aim: To produce bioethanol from rice husk and groundnut shell using local strains of Zymomonas mobilis and Saccharomyces cerevisiae. Place and Duration of Study: Conducted at the Microbiology Laboratory of Abubakar Tafawa Balewa University Bauchi, Bauchi state, Nigeria, between April to June, 2021. Methods: Groundnut shell and Rice husk were collected from local milling center. The wastes were powdered, sieved and used as carbon source. Proximate composition of the subsrate was done and the total carbohydrate was determined by difference. The sum of the percentage moisture, ash, crude lipid, crude protein and crude fibre was subtracted from 100. Zymomonas mobilis and Saccharomyces cerevisiae were isolated from rotten sweet oranges and locally fermented beverage (‘kunun-zaki’) respectively by growing them on Malt Yeast Peptone Glucose Agar (MYPGA) after which they were further screened for their ability to tolerate ethanol and they serve as organisms for fermentation. The enzyme α- amylase was used for hydrolysis. The fermented substrates were distilled at 78oC and the distillate was collected as bioethanol in a conical flask. UV-VIS spectrophotometer was used to determine the absorbance of each concentration (0, 0.2, 0.4, 0.6 and 0.8cm3) of reducing sugar content of the hydrolysates and the bioethanol produced by developing a standard curve at a wavelength of 491nm and 588nm respectively. The concentration of reducing sugar and bioethanol was determined using a reference line from the Standard curve. Results: Proximate analysis done shows that rice husk have 70.09% carbohydrates while groundnut shell has 65.09% carbohydrates. Groundnut shell yielded the highest reducing sugar of 5.096%. Rice husk yielded the lowest quantity of reducing sugar with a total yield of 2.962%. Maximum concentration of bioethanol of 0.971% was produced from the combination of Saccharomyces cerevisiae and Zymomonas mobilis from groundnut shell. The lowest concentration of 0.121% of bioethanol was produced when Saccharomyces cerevisiae was used on rice husk hydrolysates. The synergistic relationship of Saccharomyces cerevisiae and Zymomonas mobilis yielded the maximum bioethanol when compared with the yield obtained when the organisms were used singly. Zymomonas mobilis produced highest bioethanol content when the organisms are used single. Conclusion: This study demonstrates the potentiality of local strains of Saccharomyces cerevisiae and Zymomonas mobilis isolated from rotten sweet orange and locally fermented beverage (‘kunun-zaki’) to produce bioethanol by fermenting the rice husk and groundnut shell hydrolysates.

Extreme Clusters of Grains in Random Microstructures of Polycrystals

It was experimentally observed that in polycrystalline materials under low macro loading of the specimen the first sites of failure initiation take place in the specific clusters of few grains. In some grains of these extreme clusters, the local (meso-) strains and stresses are high enough to cause first damages or plastic slips. In the stochastic microstructure of polycrystals, the formation of an extreme cluster is random and rare. Nevertheless, they govern the failure process initiation and can severely affect the reliability of polycrystalline machine parts. It is time and resource consuming to search and investigate extreme clusters on the real specimens of polycrystalline materials experimentally. A theoretical tool is desirable. Here we present the powerful computational method to look for extreme clusters, to investigate their possible patterns, and to evaluate the absolute maximums of local strains/stresses that can be achieved in these clusters. The experimentally observed clusters consist of few (3-4) preferably oriented neighboring grains or even of one big supergrain. The strain and stress bursts arise due to an interaction of the grains. One can expect that in bigger clusters, larger local bursts of fields can be generated. We found the typical forms of the extreme clusters (small and big) in four different polycrystals with grains of a weak and strong anisotropy for the case of uniaxial tension. In all regarded cases, the extreme clusters have the forms of the symmetrical patterns. In big clusters of highly anisotropic grains, the maximum of mesostrain exceeds the macrostrain by several times. In clusters of weakly anisotropic grains, the local strain concentration is rather moderate (tens of percents).

Low cycle fatigue behavior of circumferentially notched specimens made of modified 9Cr–1Mo steel at elevated temperature

During service, notched designed components such as steam generators in the nuclear power plant usually experience fatigue damage at elevated temperatures, due to the repeated cyclic loadings during start-up and shut-down operations. Under such extreme conditions, the durability of these components is highly-affected. Besides, to assess the fatigue life of these components, a reliable determination of the local stress-strain at the notch-tips is needed. In this work, the maximum strains of circumferentially notched cylindrical specimens were calculated using the most commonly known analytical methods, namely Neuber's rule, modified Neuber's rule, Glinka's rule, and linear rule, with notch root radius of 1.25, 2.5, and 5 mm, made of modified 9Cr–1Mo steel at 550 °C, and subjected to nominal stress amplitudes of ±124.95, ±149.95, and ±174.95 MPa. The calculated local strains were compared to those obtained from Finite Element Analysis (FEA). It was found that all the analytical approximations provided unreliable local strains at the notch-tips, resulting in an overestimation or underestimation of the fatigue life. Therefore, a mathematical model that predicts the fatigue lives for 9Cr–1Mo steel at elevated temperature was proposed in terms of the applied stress amplitude and the fatigue stress concentration factor. The calculated fatigue lifetimes using the proposed model are found to be in good agreement with those obtained experimentally from the literature with relative errors, when the applied stress amplitude is ±149.95 MPa, are of 1.97%,–8.67%, and 13.54%, for notch root radii of 1.25, 2.5, and 5 mm, respectively.

Studies of some genes of Bacillus subtilis MN99 local strains against plant diseases

The objective of this study was the isolation and characterization of Bacillus subtilis local strains from the soil in Mongolia. These local strains of B. subtilis are showed to have high antagonistic activities against some plant pathogenic fungi and bacteria. Six strains of B. subtilis were isolated and characterized morphologically, physiologically and biochemically according the Bergey’s Manual of Systematic Bacteriology. In order to identify species of the isolated strains, we amplified and sequenced 16S rRNA gene, essential funtinal genes bmyB, spoVG and srfAA, which are related to antagonistic activity of these strains. The sequences were aligned using CLASTALW multiple sequence alignment tool. Phylogenetic tree was drawn according to Maximum likelihood”method and “Tamura-Nei” model using “MEGA-X version 10.2.6 program. Among all isolates of B. subtilis MN99 and 7/24 strains had higher antagonist activity against plant diseases. According to partial sequence of srfAA (620bp) gene of MN99, the local strain belongs to B. subtilis and partial sequence of bmyB (370bp), spoVG (22bp) gene of MN7/24 strain showed that the it belongs to B. atrophaeus species. All local strains of B. subtilis had bacillomycin synthesis gene, and B. subtilis MN99 strain had only surfactine synthesis gene, while did not have spore formation and hemolysis gene SpoVG.

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

Molecular Cloning and Expression of Haloacid Dehalogenase Gene from a Local Pseudomonas aeruginosa ITB1 Strain and Tertiary Structure Prediction of the Produced Enzyme

Organohalogens are widely utilized as pesticides, herbicides, solvents, and for many other industrial purposes. However, the use of these compounds caused some negative impacts to the environment due to their toxicity and persistency. In the light of this, some microbes have been identified and employed to perform dehalogenation, converting halogenated organic compounds to non-toxic materials. In this research, we successfully cloned and sequenced the haloacid dehalogenase gene from a local Pseudomonas aeruginosa ITB1 strain, which is involved in the degradation of monochloroacetate. First, the haloacid dehalogenase gene was amplified by PCR using a pair of primers designed from the same gene sequences of other P. aeruginosa strains available in the GenBank. The cloned gene in pGEM-T in E. coli TOP10 was sequenced, analyzed, and then sub-cloned into pET-30a(+) for expression in E. coli BL21 (DE3). To facilitate direct sub-cloning, restriction sequences of EcoRI (G/AATTC) and HindIII (A/AGCTT) were added to the forward and reversed primers, respectively. The expressed protein in E. coli BL21 (DE3) appeared as a 26-kDa protein in SDS-PAGE analysis, which is in good agreement with the size predicted by ExPASy Protparam. We obtained that the best expression in LB liquid medium was achieved with 0.01 mM IPTG induction at 30°C incubation for 3 hours. We also found that the enzyme is more concentrated in the pellet cells as inclusion bodies. Furthermore, the in-silico analysis revealed that this enzyme consists of 233 amino acid residues. This enzyme’s predicted tertiary structure shows six β-sheets flanked by α-helixes and thus belongs to Group II haloacid dehalogenase. Based on the structural prediction, amino acid residues of Asp7, Ser121, and Asn122 are present in the active site and might play essential roles in catalysis. The presented study laid the foundation for recombinant haloacid dehalogenase production from P. aeruginosa local strains. It provided an insight into the utilization of recombinant local strains to remediate environmental problems caused by organohalogens.