Spread Plate Method Research Articles

Bacteriological and physiochemical evaluation of locally produced tiger nut drink (kunu aya) sold in Owerri metropolis Imo state Nigeria

The public heath implication of consumption of contaminated and unregulated hawked drinks has received great attention in recent times. This work investigated succession parameters of micro-organisms and physiochemical characteristics of tiger nut (Cyperus esculentus) drink hawked in Owerri metropolis over a 72 hour period. Samples of freshly produced drinks were purchased from seven major locations (Ekeonuwa, Control, Ikenegbu, Futo campus, Ihiagwa, Obinze and Naze junction) in Owerri metropolis. Microbial evaluation of the drink samples was carried out using spread plate method. Physiochemical analysis of tiger nut drinks were carried out using standard A.O.A.C methods. The results indicated the presence of Staphyloccus sp, Microccus sp, Enterobacter sp, Bacillus subtilis, Bacillus cereus, Salmonella sp, Escherichia coli and Corynebacterium sp. These contrasted from organisms isolated from laboratory prepared control. The mean total bacterial counts indicated tiger nut drink sold around Naze recorded the highest total heterotrophic bacterial count (109 CFU/ml), tiger nut drinks sold at FUTO campus, Obinze and Ihiagwa recorded the highest total fecal coliform count (104 CFU/ml) and total Salmonella-Shigella count (104 CFU/ml), while the laboratory prepared recorded the least heterotrophic bacterial count (103 CFU/ml), zero total fecal coliform count and total Salmonella-Shigella count recorded. The pH and titratable acidity over a 72 hour period indicated that all the drinks were acidic, where the street sold drinks recorded the highest acidity compared to the laboratory prepared drink. Very low microbial count observed for the laboratory prepared tiger nut drinks underscores the importance of Good Manufacturing Practices (GMP). It is recommended that regulatory government agencies for food and drink products should step up campaign for adoption/implementation of GMP by local vendors/producers.

Electrospun Polyvinylpyrrolidone-Based Dressings Containing GO/ZnO Nanocomposites: A Novel Frontier in Antibacterial Wound Care.

In recent years, the rapid emergence of antibiotic-resistant bacteria has become a significant concern in the healthcare field, and although bactericidal dressings loaded with various classes of antibiotics have been used in clinics, in addition to other anti-infective strategies, this alarming issue necessitates the development of innovative strategies to combat bacterial infections and promote wound healing. Electrospinning technology has gained significant attention as a versatile method for fabricating advanced wound dressings with enhanced functionalities. This work is based on the generation of polyvinylpyrrolidone (PVP)-based dressings through electrospinning, using a DomoBIO4A bioprinter, and incorporating graphene oxide (GO)/zinc oxide (ZnO) nanocomposites as a potent antibacterial agent. GO and ZnO nanoparticles offer unique properties, including broad-spectrum antibacterial activity for improved wound healing capabilities. The synthesis process was performed in an inexpensive one-pot reaction, and the nanocomposites were thoroughly characterized using XRD, TEM, EDX, SEM, EDS, and TGA. The antibacterial activity of the dispersions was demonstrated against E. coli and B. subtilis, Gram-negative and Gram-positive bacteria, respectively, using the well diffusion method and the spread plate method. Bactericidal mats were synthesized in a rapid and cost-effective manner, and the fiber-based structure of the electrospun dressings was studied by SEM. Evaluations of their antibacterial efficacy against E. coli and B. subtilis were explored by the disk-diffusion method, revealing an outstanding antibacterial capacity, especially against the Gram-positive strain. Overall, the findings of this research contribute to the development of next-generation wound dressings that effectively combat bacterial infections and pave the way for advanced therapeutic interventions in the field of wound care.

Bioactivity of chitosan nanoparticles synthesized by a novel route towards Escherichia coli and Bacillus subtilis

Chitosan nanoparticles (CSNP) with an average size of 25.67 nm were synthesized via a novel wet chemical route and characterized using scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. Comparative antibacterial assays of Chitosan NP suspensions prepared in water (at neutral pH) and in dilute acetic acid and chitosan gel prepared in dilute acetic acid/hydrochloric acid (all at concentrations up to 1%) were performed against Escherichia coli ( E. coli) and Bacillus subtilis ( B. subtilis) bacteria using spread plate method. A parallel viability test was conducted to confirm the presence of surviving cells in the bulk test volume. Minimum bactericidal concentration (MBC) of chitosan gel was 0.5 mg/ml for the bulk chitosan dissolved in 0.05% v/v aq. acetic acid and 0.1 mg/ml for chitosan dissolved in 0.01% v/v aqueous (aq.) hydrochloric acid. In comparison, Chitosan NP were found to be growth promoter at neutral pH and exhibited cell protective efficacy in presence of aq. acetic acid. The biocidal activity of chitosan gel in acidic media was higher when prepared in strong inorganic acid, that is, aq. HCl in comparison with the gel prepared in a relatively weak organic acid that is, aq. CH3COOH at the same concentration. Antibacterial action also showed pH dependence with higher activity at lower pH. However, respective aq. acids also gave comparable bactericidal action; indicating that chitosan may not have any inherent antibacterial property and basically it acts as a growth promoter.

The Effect of Rhizospheric Fungal Species of Tomato (Solanum lycopersicum L.), on In-Vitro Growth of the Tomato Early Blight Pathogen

Tomato (Solanum lycopersicum L.) is a globally consumed crop, susceptible to various biotic and abiotic stresses. Among biotic stresses, plant diseases, particularly Early Blight caused by Alternaria spp. leads to heavy losses in the field and post-harvest stages. The conventional use of synthetic agrochemicals to control these diseases poses environmental risks, underscoring the need for sustainable alternatives. Hence, this study aimed to assess the potential of rhizospheric fungi in controlling Alternaria spp. Infected tomato leaves, stem parts, and fruits were collected from commercial cultivations in four agro-ecological regions in Sri Lanka (two from mid-country in the wet zone, and two from the intermediate zone). All samples were washed using running tap water, and 1cm2 sections, including the edges of lesions, were cut, surface sterilized, placed on potato dextrose agar (PDA), and incubated at room temperature until colonies appeared. Fungal colonies that emerged from lesions, were sub-cultured onto fresh PDA, and pure cultures were obtained using the hyphal-tip purification method. Alternaria isolates were identified morphologically and confirmed the pathogenicity by Koch’s postulate method. Species-level identification involved genomic DNA extraction and sequencing ITS regions. The soil dilution plate technique was used to isolate rhizospheric fungi from two agroecological regions. Composite samples (10 g) of soil attached to the roots of 5 uprooted healthy plants, from each field were used. Soil suspensions were inoculated onto PDA using the spread-plate method and incubated at room temperature for 5 days. A total of 27 isolates were obtained and morphologically identified up to the genus level after preparing pure cultures. The dual-culture plate technique was used to assay percentage inhibition, and according to Tukey’s pairwise comparison, Trichoderma sp. and Purpureocillium lilacinum exhibited significantly high (p≤0.05) inhibition of Alternaria colony growth, (68.25±1.18% and 64.35±3.31% respectively). Microscopic observations of inhibition zones between Alternaria and antagonistic rhizospheric fungal colonies, including Trichoderma sp. and P. lilacinum showed the presence of mycoparasitism-related structures, such as haustoria, coils, loops, and knobs. Qualitative assay for fungal chitinase enzyme production demonstrated high activities in P. lilacinum, Trichoderma sp. and Acremonium sp., whereas in a quantitative assay for fungal glucanase enzyme, testing the ability of fungal culture filtrates to release 1µg of glucose per minute, from Beta-glucanase solution, P. lilacinum, Trichoderma sp., and Paecilomyces sp. showed significantly high (p≤0.05) activities (105.14± 3.62, 100.11±4.09 and 99.83 ±6.44 μg glucose/L respectively). In conclusion, this study highlights the potential of rhizospheric fungi like P. lilacinum and Trichoderma sp. as effective biocontrol agents against Alternaria spp. and these findings lay the groundwork for future applications in enhancing tomato crop resilience against pathogens and promoting sustainable and eco-friendly agricultural practices.
 
 Keywords: Rhizospheric fungi, Mycoparasitism, ITS region, Chitinase enzyme, Glucanase enzyme

Biosynthesis of Silver Nanoparticles From Marine Actinobacterium Micromonospora sp. and Their Bioactive Potential.

Background The biosynthesis of nanoparticles represents a rapid, environmentally friendly, cost-effective, and straightforward technology. This approach allows for the production of nanoparticles with a wide range of chemical compositions, sizes, shapes, high uniformity, and scalability. One of the principal advantages of biogenic nanoparticles is their water solubility and compatibility with biological systems. Biologically synthesized nanoparticles have demonstrated superior efficiency compared to conventionally synthesized particles. Among biosynthesis, microbial-mediated biosynthesis is a promising one that has a selectively reducing ability on specific metal ions through electron transfer. Objectives Evaluation of antimicrobial and antioxidant activity of silver nanoparticle synthesized by actinobacteria Micromonospora sp. which is isolated from marine environment. Materials and methods In this study, actinobacteria were isolated from the marine sediment using the spread plate method. The isolates were identified based on morphological observation, cell wall amino acids, sugar analysis, and micromorphological analysis. The silver nanoparticle synthesis from microbes and their inhibition against clinical pathogens have been evaluated by the disc diffusion method.Antioxidant efficiency was evaluated in termsof total antioxidant activity through ammonium molybdenum assay. Results A total of five isolates were isolated from the sediment sample. The cell-free extract of MBIT-MSA4 can synthesize silver nanoparticles that have potential antimicrobial activity against the clinical pathogens Streptococcus mutans at a zone of inhibition 6 mm, 10 mm inhibition zone of Klebsiella pneumonia,and 8 mm zone of inhibition of Staphylococcus aureus. Also, it has significant antioxidant activity up to 73% of free radical inhibition. Conclusion Marine microbial-mediated biosynthesized silver nanoparticles have potential antimicrobial activity against S.mutansand methicillin-resistantStaphylococcus aureus(MRSA) and inhibit the oxidation process through antioxidant activity. Thisenhanced efficient biosynthesisednanoparticle has significantly reduced the concentration of free radicals caused by pathogens.

Simulated drug disposition in critically ill patients to evaluate effective PK/PD targets for combating Pseudomonas aeruginosa resistance to meropenem.

Bacterial infections, including those caused by Pseudomonas aeruginosa, often lead to sepsis, necessitating effective antibiotic treatment like carbapenems. The key pharmacokinetic/pharmacodynamic (PK/PD) index correlated to carbapenem efficacy is the fraction time of unbound plasma concentration above the minimum inhibitory concentration (MIC) of the pathogen (%fT > MIC). While multiple targets exist, determining the most effective one for critically ill patients remains a matter of debate. This study evaluated meropenem's bactericidal potency and its ability to combat drug resistance in Pseudomonas aeruginosa under three representative PK/PD targets: 40% fT > MIC, 100% fT > MIC, and 100% fT > 4× MIC. The hollow fiber infection model (HFIM) was constructed, validated, and subsequently inoculated with a substantial Pseudomonas aeruginosa load (1 × 108 CFU/mL). Different meropenem regimens were administered to achieve the specified PK/PD targets. At specified intervals, samples were collected from the HFIM system and subjected to centrifugation. The resulting supernatant was utilized to determine drug concentrations, while the precipitates were used to track changes in both total and drug-resistant bacterial populations over time by the spread plate method. The HFIM accurately reproduced meropenem's pharmacokinetics in critically ill patients. All three PK/PD target groups exhibited a rapid bactericidal response within 6 h of the initial treatment. However, the 40% fT > MIC and 100% fT > MIC groups subsequently showed bacterial resurgence and resistance, whereas the 100% fT > 4× MIC group displayed sustained bactericidal activity with no evidence of drug resistance. The HFIM system revealed that maintaining 100% fT > 4× MIC offers a desirable microbiological response for critically ill patients, demonstrating strong bactericidal capacity and effective prevention of drug resistance.

In Vitro and In Silico Analysis of the Antifungal Activity of Beta-cyclodextrin and Cinnamon Oil Inclusion Complexes against Fusarium oxysporum f. sp. cubense-TR4

The Panama Disease-TR4 is a fatal disease of banana plants caused by the fungus Fusarium oxysporum f. sp. cubense-TR4 (Foc-TR4). Cinnamon oil has known antifungal properties, while beta-cyclodextrin has been proven to protect essential oils from degradation through encapsulation. The study aims to determine the effectiveness of cinnamon oil, and its beta-cyclodextrin complex against Foc-TR4, through their characterization, in vitro antifungal screening, and in silico docking analysis. The characterization of the complex yielded an encapsulation efficiency of 61.18%, an average particle size of 98.27 μm, while its calorimetry confirmed complex formation. Cinnamon oil, beta-cyclodextrin, and the complex were tested against Foc-TR4 through spread plate method against Ivagard (dialkyl dimethyl ammonium chloride) in triplicate. Among the treatments, the complex exhibited significantly weaker antifungal activity, with only cinnamon oil comparable with Ivagard at α = 0.05. Cinnamaldehyde displayed favorable binding energies of -6.1 and -4.9 kcal/mol against the Foc-TR4 proteins, and -3.7 kcal/mol against beta-cyclodextrin. This implies that cinnamaldehyde can spontaneously form stable complexes with the Foc-TR4 proteins or beta-cyclodextrin. These findings suggest the potential of cinnamaldehyde, in cinnamon oil and even in the inclusion complexes, as a viable antifungal treatment against Foc-TR4.

Isolation of a potential rock phosphate solubilizing Aspergillus sp. towards development of biofertilizer

Phosphorus (P) is one of the key bio elements that limits agricultural production. Although Sri Lanka is endowed with rock phosphate deposits, practical means to utilize this resource are limited. Phosphate-solubilizing fungi (PSF) play an important role in enhancing the bioavailability of soil phosphorus to plants. In this study, fungi from eight soil samples in Kaikawala, Sri Lanka, were isolated using spread plate method and analyzed for its phosphate solubility capacity. One fungal isolate with significant halo zones on Pikovskaya’s agar (PVK) plates, containing 0.5% tricalcium phosphate, was identified. After purification, the isolate was transferred to three media setups: PVK with 0.5% apatite (ERP) as the phosphate source, PVK with 0.5% tricalcium phosphate as a positive control, and PVK without P source as a negative control and analyzed for the solubilization index (SI). DNA sequences of the internal transcribed spacers with 5.8S region of ribosomal DNA (ITS1-5.8S-ITS2) was analyzed with universal primer pair ITS4-F/ITS5-R to determine the identity of the species. Fungal isolate PSF01 showed a phosphate solubilizing activity on both PVK media, ERP (1.07 ± 0.03 cm SI) and tricalcium phosphate (1.07 ± 0.01 cm), with similar effects (p > 0.05). However, the SI in the negative control was 1.00 ± 0.00 cm without halo zone (p < 0.05). The fungal strain is fast growing with initially white but quickly changing to black colonies after producing conidial spores on potato dextrose agar with distinctive conidial heads and pale-yellow lower surface characterized as Aspergillus. Sanger DNA sequencing identified the fungus as Aspergillus niger (99%) and a phylogenetic tree was constructed using MEGA11 software using reference data obtained from NCBI GenBank data to identify the isolated PSF01. Consequently, our preliminary studies demonstrate the importance of examining more soil samples to identify PSF for sustainable agricultural applications in the future.

Screening and characterization of endophytic bacteria isolated from celery with the potential to promote plant growth

Abstract. Nuriyanah, Widowati T, Masnang A, Nurjanah L, Novilasari D, Lekatompessy SJR, Simarmata R. 2023. Screening and characterization of endophytic bacteria isolated from celery with the potential to promote plant growth. Biodiversitas 24: 6897-6904. Endophytic bacteria that inhabit plant tissue contribute to the enhancement of host plant growth by producing secondary metabolites. They can improve plant growth by phytohormone modulation and nutrient uptake. In addition, endophytic bacteria can increase plant health and tolerance by antibiotic and hydrolytic enzyme production. This study aims to screen and characterize endophytic bacteria of celery as plant growth promoter agents. Thirty endophytic bacteria were isolated successfully from part of the celery plant using the spread plate and plant piece method which grew at Nutrient Agar media. Based on the characterization assay, 30 isolates may produce IAA with various concentrations of 0.21-7.18 mg/L. In addition, 3 of 30 isolates had phosphate solubilizing activity, in different indexes ranging from 0.71-1.43. Twenty-one isolates were able to grow in an N-free medium, indicating the capability of isolates to fix nitrogen qualitatively. The hydrolytic enzymes assay resulted in 14 amylolytic, 17 proteolytic, and 12 cellulolytic activities. In addition, nine of 30 isolates show multi-activity in amylase, protease, and cellulase enzyme production. Based on the screening results, there are four potential isolates (SLBg 1.2; SLBg 1.5; SLAg 1.1, and SLAg 1.5) as promising plant-growth promoters and were molecularly identified as members of the Bacillus genus. These isolates can be developed as biofertilizers for supporting the cultivation of celery plants.

Synthesis and Antibacterial Activity of Grafted Poly(Vinyl Chloride) Polymer against Gram-Positive and Gram-Negative Bacteria

Infectious diseases caused by microorganisms have gained worldwide attention in recent years. According to data compiled by the World Health Organization, the number of deaths resulting from infectious diseases is on the rise. In light of these dangers, the study of antibacterial materials has become increasingly vital. In this research, an antibacterial polymer was developed using poly(vinyl chloride) (PVC) and 4,4-diamminodiphenylmethane (DDM). The produced polymer’s chemical structure and thermal properties were investigated using Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and thermo-gravimetric analysis. The antibacterial activity of the resulting PVC-g-DDM polymer was effective in killing both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The antimicrobial efficacy was tested using a spread plate method, demonstrating its potential utility in a variety of applications like biomedical, coatings, water purification systems, and others. Antimicrobial resistance is increasing, especially among bacteria that have acquired resistance to multiple therapeutics. To fully optimize and explore the polymer’s potential and its usage, more research is needed.

Potential of antagonistic activity from associated bacteria from healthy and bleaching acropora corals of Blitar Waters, East Java, Indonesia

Global warming leads to high coral bleaching phenomena in marine ecosystems. The bleach condition of corals can cause a disease that is enhanced by opportunistic or pathogenic microorganisms. This research aimed to investigate the antagonistic interactions between healthy and bleached coral-associated bacteria. Isolation of coral-associated bacteria was carried out using the spread plate method in half-strength Zobell 2216E medium. Antagonistic activity was observed using a double-layer method, with each layer of medium inoculated with healthy or bleached coral-associated bacteria. The interactions between healthy and bleached coral isolates were analyzed using correspondence analysis. Twenty healthy and 11 bleached coral isolates were found in the six coral samples. Approximately 14 bacterial isolates from healthy corals demonstrated antagonistic (inhibitory) activity against 11 bacterial isolates from bleaching coral samples, with isolate AcD.14 from bleached coral, inhibited by 57.14% of the active isolates from healthy corals. Correspondence analysis resulted in isolate AcD.16, a bleaching coral bacterium with sufficient pathogenicity to elicit an antagonistic response from healthy coral bacteria. This study showed that there is high potential for finding bio-control agents for coral diseases using their natural microbiomes from healthy corals.

Isolation and Characterization Morphology of Nitrogen-Fixing Bacteria from Mangrove Sediment

Mangrove ecosystems are coastal areas with ecological and socioeconomic functions. This area has a function as a nutrient provider for aquatic biota. The mangrove ecosystem also plays important role in nutrient cycling in coastal areas. The process of converting nitrogen into nutrients needed by plants (ammonium (NH4+) or nitrate (NO3+)) is called nitrogen fixation. The agents that play a role in this process are bacteria. The mangrove sediment has an abundant microbial communities which responsibility to this process. Sampling was carried out in the mangrove area of Tirang Beach, Semarang using the purposive sampling method. There are three sampling points, such as in the estuary, residential areas, and ponds. The isolation process was carried out using the spread plate method on solid NFB (Nitrogen Free Bromthymol) selective media, and purification using the quadrant streak plate method on the media. There are the most bacterial colonies in mangrove locations next to settlements, range from 2-11 bacterial colonies, and the least in mangrove locations near water bodies is 6 bacterial colonies. After purification, eight pure bacterial colonies have been identified and have similar morphological characteristics, white color, circular shape, entire margin, and flat elevation.

CELLULASE ACTIVITY OF RIZOSFER BACTERIA OF KETAPANG PLANTS (Terminalia catappa L.)

Cellulose, a natural biopolymer produced by plants, undergoes enzymatic breakdown by cellulase, cleaving the monomer's β-1,4 glycosidic bonds. Cellulolytic bacteria, producers of cellulase, can be isolated from plant rhizospheres, like Ketapang (Terminalia catappa L.), abundantly found in the Biology Department at Universitas Sriwijaya, South Sumatra, Indonesia. This study aimed to isolate cellulolytic bacteria from Ketapang rhizosphere soil and assess their cellulase activity. The spread plate method on selective Carboxy Methyl Cellulose Agar (CMC agar) was used for bacterial isolation, and cellulase activity was measured using the DNS method (3.5-dinitro salicylic acid). Results yielded 22 isolates of cellulolytic bacteria capable of growth on selective CMC agar. Thirteen isolates, identified through a screening test, produced cellulase, forming clear zones post-addition of 0.1% Congo Red and 1 M NaCl. Cellulolytic activity indices for these isolates ranged from 0.02 to 2.6 mm. The highest indices (IAS) and cellulase activities were observed in BS10, BS4, and BS22, with IAS values of 2.64, 2.12, and 1.71, and cellulase activities of 354.99 U/ml, 9.42 U/ml, and 9 U/ml, respectively. Identification results suggested that isolate BS10 bore similarities to the genus Zoogloea, while isolates BS4 and BS22 showed similarities to the genus Bacillus.

Groundwater Purification Using Bio-Sand Filter Modified with Iron Oxide-Coated Sand and Activated Carbon

Groundwater is the most used natural resource which serves different diverse purposes and alternative water supply for households mainly in rural areas. However, it is polluted by contaminants such as heavy metals, total hardness and coliform bacteria. One such promising water treatment technology for households in rural areas is the Bio-sand Filter (BSF) because of its low cost and efficient removal of contaminants. Although BSF is efficient to remove contaminants, there is still needed to improve the flow rate and the performance of removing pollutants from groundwater. Therefore, this study focused on the development of a laboratory-scale Modified Bio-sand Filter (MBSF) that was combined with Iron-oxide Coated Sand (IOCS) with Granular Activated Carbon (GAC) made from coconut shells to purify groundwater. Laboratory scale of filters were performed in this study. Groundwater was collected from household’s well water in Kean Svay district, Kandal province. The filters were operated by filling the water source with 10 liters per day and operated for 20 days. Heavy metals were measured by Atomic Absorption Spectroscopy (AAS) and Arsenator. Total hardness was analyzed by Ion Chromatography (IC). Target coliform bacteria such as E. coli and total coliform were cultured by spread plate method. Flow rate was observed by setting time for 1 minute and measure the volume of effluent water. By the results, all physicochemical parameters of MBSF were within the standard limit of drinking water. MBSF showed significantly better removal efficiency for arsenic with 100% than BSF while MBSF can also remove Mn and Fe which was under the drinking water standard of WHO and MIME. Total hardness was achieved more than 80% in MBSF, while BSF with only 40%. MBSF also performed slightly better than BSF as well in removing both E. coli and total coliform with more than 90% reduction. The flow rate of the MBSF was faster about two and a half times than the flow rate of BSF with the average of 0.15 L/min for MBFS and 0.06 L/min for BSF. The results from this study contributed to the improvement of household water treatment method for purifying groundwater efficiently in rural areas.

Benzalkonium Chloride and Benzethonium Chloride Effectively Reduce Spore Germination of Ginger Soft Rot Pathogens: Fusarium solani and Fusarium oxysporum.

Ginger soft rot is a serious soil-borne disease caused by Fusarium solani and Fusarium oxysporum, resulting in reduced crop yields. The application of common chemical fungicides is considered to be an effective method of sterilization, and therefore, they pose a serious threat to the environment and human health due to their high toxicity. Benzalkonium chloride (BAC) and benzethonium chloride (BEC) are two popular quaternary ammonium salts with a wide range of fungicidal effects. In this study, we investigated the fungicidal effects of BAC and BEC on soft rot disease of ginger as alternatives to common chemical fungicides. Two soft rot pathogens of ginger were successfully isolated from diseased ginger by using the spread plate method and sequenced as F. solani and F. oxysporum using the high-throughput fungal sequencing method. We investigated the fungicidal effects of BAC and BEC on F. solani and F. oxysporum, and we explored the antifungal mechanisms. Almost complete inactivation of spores of F. solani and F. oxysporum was observed at 100 mg/L fungicide concentration. Only a small amount of spore regrowth was observed after the inactivation treatment of spores of F. solani and F. oxysporum in soil, which proved that BAC and BEC have the potential to be used as an alternative to common chemical fungicides for soil disinfection of diseased ginger.

Isolation and identification of bacteria from engine oil polluted soil from mechanic workshops in Okija Town

This research was conducted for the isolation and identification of bacteria from engine oil polluted soil from mechanic workshops in Okija village. Two different mechanic workshops within Okija were selected and soil samples were collected differently based on top soil and depth soil from each site. Then 0.1ml was taken from the dilution 10-3 to 10-6 diluents and inoculated into a Bushnell Haas agar (containing: (g/l) 0.2, MgSO4; 0.02, CaCl2; 1.0, KH2PO4; 1.0, K2HPO4; 1.0, NH4NO3; 0.05, FeCl3; 20.0, Agar and 5ml of fresh engine oil as sole carbon source) and was incubated at 370C for 5 -7 days. Spread plate method involving the use of serial dilutions was employed for the isolation of bacteria. The culture was examined for bacterial growth and subsequently cultured the specific bacteria growth on nutrient agar plates with 2ml of fresh engine oil for 24 hours to determine engine oil degradability by the isolated bacteria. The numbers of viable bacterial count were 20, 26, 18 and 28 as expressed in colony forming units per milliliter (cfu/ml). The bacterial count was furthermore expressed in percentage per the dilution factor 10-3; S1Top, S1Depth, S2Top and S2Depth were 21.74%, 28.26%, 19.56% and 30.44% respectively. The bacteria species isolated were Serratia, Micrococcus, Pseudomonas and Bacillus species. Bacillus was the most dominant. There was presence and activity of bacteria species in the depth soil than top soil. The bacterial species isolated were able to degrade fresh engine oil especially Bacillus species. From, the data obtained it was found that depth soil has more presence and activity of bacterial species because there is less disturbance in the depth soil than top soil. Bacillus species and Pseudomonas species are most adapted to conditions present in soils polluted with used engine oil and hence could be exploited in bioremediation activities in cases of accidental oil spillage.

The Bacteriological Quality of Raw Cow Milk Obtained from Herdsmen in Okigwe, Imo State, Nigeria

This study was conducted to examine the bacteriological quality of cow’s raw milk obtained from herdsmen at Okigwe, Imo States. Standard microbiological procedure was used to carry out this research. A total number of five samples were gotten from different streets in Okigwe metropolis in sterile specimen containers. Ten fold serial dilution was carried with each of the sample with the 4th tube to 6th tube being inoculated on Nutrient agar, Salmonella- Shigella agar and MacConkey agar by spread plate method and incubated at 37oC for 24 hours. The microorganisms were identified were Staphylococcus aureus, Corynebacterium sp, Salmonella typhi, Streptococcus sp, E. coli, Lactobacillus sp and Salmonella typhi. The total viable count for raw cow milk ranged from 6.6 x105CFU/ml to 1.01 x 106CFU/ml while total coliform count was from 6.2x106 CFU/ml to 1.7x106 CFU/ml. They frequency of occurrence of Lactobacillus sp was highest with a value 85 and the lowest frequency was Salmonella typhi II with a value of 2. The raw cow milk was observed to harbor some pathogenic microorganisms which can cause infections when ingested.

In vitro susceptibility test of Cladosporium cladosporioides isolates from Argentinian tomato crops against commercial fungicides

Tomato cultivation is an important agricultural activity in northeast of Argentina, and disease control is indispensable for its production. The purpose of the current study was to identify two fungi isolated from tomato plants cultivated in greenhouses, with symptoms of leaf mould disease and a strain of the genus Cladosporium from a culture collection, and evaluate their in vitro susceptibility to four commercial fungicides. Macro and microscopic examination, molecular characterisation and sequence analysis were applied for identification. Broth dilution and spread plate methods were used to determine the minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC). The active ingredients of the products were azoxystrobin+difenoconazole, trifloxystrobin+tebuconazole, chlorothalonil and metalaxyl-M+mancozeb. The results were processed using the Kruskal-Wallis method. The isolates were identified as Cladosporium cladosporioidesa-c; consequently, lesions found on tomato plants did not corrspond to Cladosporium fulvum. There was a significant statistical difference between the obtained values. Qualitatively, the three strains had a similar behaviour for chlorothalonil (MIC values: 0.25 - 0.5 μg/ml, MFC values: 4 μg/ml). In all cases, tests with metalaxyl-M+mancozeb yielded higher values than those achieved for chlorothalonil (MIC values: 8 μg/ml, MFC values: 8- 32 μg/ml). trobilurin-formulated fungicides were less effective against C. cladosporioidesa-b (MIC values: 16-256 μg/ml, MFC values: >64 μg/ml). C. cladosporioidesc was the most sensitive isolate. The information about the presence of a non-frequent fungus and its fungicide susceptibility, would be useful for establishing control strategies and enhance production.

Isolation and Screening Microorganisms from Black Soldier Fly Larvae (Hermetia illucens) that Producing Amylase, Protease and Cellulase

The Black Soldier Fly (BSF) has gained significant attention for its ability to decompose various types of organic waste and produce valuable enzymes related to its digestive system. These enzymes hold immense potential for applications across diverse industrial sectors, with promise in the livestock industry. This study aimed to extract and assess amylase, protease, and cellulase enzymes from microorganisms present in BSF larvae fermentation. The isolation technique employed was the spread plate method, followed by rigorous screening for the presence of these enzymes. The results showed 46 isolates of 26 bacterial isolates and 20 yeast isolates. In the amylase enzyme screening, a substantial 25 bacterial and 19 yeast isolates exhibited positive amylase activity. For cellulase, 20 bacterial and 14 yeast isolates displayed positive results. In the case of protease, 16 bacterial and 12 yeast isolates demonstrated protease enzyme activity. Notably, nine isolates exhibited the remarkable capability to produce multiple enzymes, including eight bacterial and one yeast isolate. These results showcase the rich enzymatic potential of BSF-associated microorganisms, offering exciting prospects for their application in various industrial sectors, especially in enhancing the efficiency and sustainability of livestock production

Study on the factors of hydrogen sulfide production from lignite bacterial sulfate reduction based on response surface method

Bacterial sulfate reduction (BSR) is one of the key factors leading to the anomalous accumulation of hydrogen sulphide in coal mines. Environmental factors such as temperature and pH play a crucial role in the metabolism and degradation of coal by sulfate-reducing bacteria (SRB). In this study, coal samples were selected from Shengli Coal Mine, and SRB strains were isolated and purified from mine water using a dilution spread-plate anaerobic cultivation method. Based on single-factor experiments and response surface methodology (RSM), the impact of temperature, pH, oxidation–reduction potential (ORP), chemical oxygen demand to sulfate ratio (COD/SO42−) on the generation of hydrogen sulphide during brown coal BSR was analyzed. The results showed that the anaerobic degradation of coal by SRB was inhibited by either too high or too low a temperature to produce hydrogen sulfide, and the greatest production of hydrogen sulfide occurred at a temperature of about 30 °C; The greatest production of hydrogen sulfide occurred at an initial ambient pH of 7.5; COD/SO42− ratio of around 2.0 is most conducive to hydrogen sulphide generation; the lower ORP value is more favorable for hydrogen sulfide generation. The optimal conditions obtained by RSM were: temperature of 30.37 °C, pH of 7.64 and COD/SO42− of 1.96. Under these conditions, the hydrogen sulfide concentration was 56.79 mg/L, the pH value was 8.40, the ORP value was −274 mV, and the SO42− utilization rate was 58.04%. The RSM results showed that temperature, ambient pH and COD/SO42− had a significant effect on hydrogen sulfide production, and the degree of effect was: ambient pH > temperature > COD/SO42−.