Serial Dilution Plate Research Articles

In-vitro and in-silico studies based discovery of 2-aryl-N-(4-morpholinophenyl)thiazol-4-amines as promising DNA gyrase inhibitors

Background: DNA gyrase is an important enzyme for the survival of bacteria. Many DNA gyrase inhibitors are in clinical practice. However, these inhibitors also encompass certain toxic and drug/food interactions. This warrants the development of a new template as DNA gyrase inhibitors. Therefore, this study aimed to deliver morpholine-based thiazoles (5a-5l) as safer DNA gyrase inhibitors.Methods: The 5a-5l were prepared by reacting compound 3 with various aryl thioamides. The structures of 5a-5l were ascertained by their spectral records. The 5a-5l were subjected to their antibacterial activity potential (serial plate dilution method), DNA gyrase inhibiting activity, and toxicity analysis (MTT assay) against HepG2 & Vero cell lines. The in-silico studies (pharmacokinetic parameters and molecular docking) of 5a-5l were likewise performed.Results: It was surprisingly observed that the MIC values of 5a-5l were equal to the MIC values of ciprofloxacin (12.5 µg/ml) against the tested bacteria, whereas the DNA gyrase inhibitory activity (IC50 in µg/ml) of 5h (3.52), 5g (3.76), 5f (3.88), 5e (4.08), 5l (4.11), 5b (4.28), 5k (4.28), 5i (4.30), and 5d (4.32) was equal/better than ciprofloxacin (4.32). The MTT assay also implied the non-cytotoxic nature of 5a-5l against HepG2 & Vero cell lines up to 200 µg/ml concentration. The docking outcomes indicated a similar binding pattern of 5a-5l and ciprofloxacin at the active site of DNA gyrase, wherein 5a-5l displayed a better binding affinity for the active site. The in-silico toxicity data employing the ProTox-II web server indicated no hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, or cytotoxic effect of 5a-5l. Also, the SwissADME software supported the drug-likeliness properties and high gastrointestinal absorption of 5a-5l.Conclusion: Compounds 5h, 5g, 5f, 5e, 5l, 5b, 5k, 5i, and 5d are potent DNA gyrase inhibitors with promising safety profiles.Keywords: Discovery; Morpholine; Thiazole; DNA gyrase; MTT assay; Docking

Prevalence of aflatoxigenic fungi and contamination in soils and maize grains from aflatoxin-hot spot areas in Tanzania

Maize is a major food security crop contributing over 50 % of total food requirement in Tanzania. However, number of factors limit its production and quality. Aflatoxins contamination mostly produced by two Aspergillus species, A. flavus and A. parasticus compromise food safety, public health and economic concern globally. Despite its harmful effects little is documented on the aflatoxin- producing fungi on prevalence’s and contamination levels in soils and maize. The present study investigated Aflatoxigenic occurrence and aflatoxins contamination extents in seven districts. A total of 126 soil and 126 maize samples were collected and analyzed for presence, dominance, and levels of aflatoxin contaminations. Serial dilution and direct plating techniques were used for fungal isolation from soil and maize respectively and macro morphology characterization were used for identification. Aflatoxin levels were detected by High-Performance Liquid Chromatography. A. flavus (38.1 %), A. parasiticus (22.2 %) and A. niger (16.7 %) were found to be the most dominant linked to aflatoxins B1 contamination. Total Aflatoxins were detected in Bahi (72.2 %, with mean of 87.9 μg/kg), Chemba (61.2 %, 74.2 μg/kg), and Babati (56.3 %, 65.8 μg/kg) indicating these were more prone to aflatoxins and higher aflatoxigenic prevalence. Aflatoxin B1 was detected most in Bahi (22.4 %, 402.4 μg/kg), Chemba (13.4 %, 241 μg/kg), and Babati (11.1 %, 199.2 μg/kg). Fungal counts ranged 3.4 ×105 to 6.9 ×107 CFU/g for contaminated samples. Aflatoxigenic species varied significantly (P<0.001) between cropping systems and the locations. Aflatoxigenic occurrence and levels were shown to be influenced by the cropping systems, as evidenced by the soils exhibiting a higher frequency than maize grains. Compared to mixed cropping, the mono-cropping system exhibited the greatest levels of aflatoxigenic population and aflatoxin contamination, indicating that farming practices affect contaminations and the need for improved cropping system practices.

Isolation and Identification of the Ammonium and Nitrite Oxidizing Bacterial Strains for Nitrogen Treatment in Shrimp Pond in Quang Ninh Province, Vietnam

The objective of this study was to isolate and identify the nitrifying bacteria from wastewater in the aquaculture system at Quang Ninh province and initially evaluate their ability to treat nitrogen in water and determine the strain of bacteria and identify the strain of bacteria. Nitrifying bacteria were isolated by serial dilution and agar plates and then purified by agar slant until axenic colonies were obtained. The study has successfully isolated 3 bacterial strains capable of ammonium oxidation (AOB-1, AOB-2, AOB-3) and 3 bacterial strains with nitrite oxidation ability (NOB-1, NOB-2, NOB-3) from shrimp farming wastewater in Quang Ninh province, Vietnam. These strains demonstrate potential for ammonium and nitrite oxidation, making them applicable for nitrogen treatment in sewage. Strains with nitrite oxidation ability showed a potential high capability for nitrogen removal from water. The decoding of the 16S rRNA gene sequences, compared using Blastn (NCBI) indicated similarities with Bacillus subtilis (AOB-1), Pantoea agglomerans strain JCM1236 16S RNA (AOB-2), Enterobacter hormaechei strain ES1 16S RNA (AOB-3), Arthrobacter nicotianae (NOB-1), Acinetobacter lactucae strain JVAP01 16S RNA (NOB-2) and Enterobacter asburiae (NOB-3). Therefore, the bacteria that contained 6 strains can be considered to apply for wastewater treatment in shrimp farming.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

The roles of extrinsic neurons in modulating neuroimmune crosstalk during acute enteric infection with Citrobacter rodentium

Background: The intestinal tract is one of the most densely innervated organs in our body, containing extrinsic sensory and sympathetic neurons. With their cell bodies located outside of the gut, these neurons directly detect enteric pathogens and release immunomodulatory neurotransmitters or neuropeptides to communicate with immune cells in the gut. Specifically, sympathetic neurons secrete neurotransmitter norepinephrine, which activate β2 adrenergic receptors (β2ΑR) on immune cells to regulate immune functions. Recent studies from our lab, as well as others, have begun to highlight the neural regulation of immune responses during enteric infections. However, the precise pathways responsible for orchestrating these effects remain elusive. Hypothesis: We sought to determine whether extrinsic sensory and sympathetic neurons are implicated in host protective responses against enteric pathogen Citrobacter rodentium (C. rodentium). Methods: Mice were inoculated by oral gavage with C. rodentium (108 colony-forming units) or sterile luria broth. Bacterial colonization was quantified by homogenization of distal colon tissues and plating serial dilutions onto MacConkey agar. Relevant tissues were collected for qPCR, flow cytometry or histology. Genetically engineered Arc Targeted Recombination in Active Population (TRAP) mice were used to identify neurons that were activated during infection. These ArcTRAP mice received single intraperitoneal (i.p.) injection of 4-hydroxytamoxifen post-infection to “trap” active neurons, marking them tdTomato+. 6-hydroxydopamine (6OHDA) was used to chemically ablate sympathetic innervations to the colon via 3 consecutive daily i.p. injections. Results: At day 4 post-infection, there were significantly more active tdTomato+ neurons in the rostral ventrolateral medulla (RVLM) of the infected mice compared to the non-infected controls. This increase was more pronounced as the infection peaked at day 10, indicating that more neurons were being activated in the RVLM in response to C. rodentium in the gut. 6OHDA treatment significantly increased bacterial burden in distal colon at day 10 post-infection. This was associated with decreased tyrosine hydroxylase expression compared to the vehicle controls. Subsequently, qPCR revealed that there was increased pro-inflammatory cytokines expression in the colon of sympathectomized infected mice compared to the infected controls. Similarly, β2AR knockout significantly increased bacterial burden in distal colon at day 3 and 10 post-infection. Increased pro-inflammatory cytokines expression suggests an immunomodulatory role for β2AR during C. rodentium infection. Conclusions: These studies provided novel insights into the neuroimmune crosstalk that is crucial for acute enteric infections with C. rodentium. Specifically, these experiments may reveal how extrinsic neurons shape local immune responses in the gut. NIH NIAID R01AI150647; Graduate Student Support Program (UC Davis: School of Veterinary Medicine). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluating the efficiency of indigenous soil bacterial strains for heavy metal removal

Significant quantities of toxic heavy metals are regularly deposited into soil environments every day as a result of industrial and human activities, as well as natural emissions. Today, biological methods for reducing heavy metal pollution are becoming increasingly common as an innovative approach to combating heavy metal pollution. The purpose of this study was to identify soil bacteria capable of removing heavy metals and to explore the potential of native bacteria in the bioremediation process. During this experiment, various soil samples were collected from several sites (river bank Rvs1, bus station Bt2, agricultural site Ag3, landfill Gr4 and construction site Cn5). Five bacterial strains were isolated from soil samples using a conventional serial plate dilution method. Morphologically distinct colonies were selected and purified during incubation. Pure cultures of bacterial colonies were established and maintained as slant cultures. Depending on preliminary physical, chemical and morphological characteristics, isolates RvsS1, Bt2, Ag3, Gr4 and Cn5 were compared with the bacterial species: “Proteus”, “Staphylococcus”, “Pseudomonas”, “Bacilli” and “Klebsiella” respectively. Bacterial isolates were examined for their uptake of Cu, Cd and Pb in flask bioreactors. Strains RV1, Bt2, Ag3 and Gr4 removed 48.7%, 25.6%, 59.8% and 23.5% of lead, respectively. The removal efficiency of isolate Cn5 was 35.68% for lead and 48.53% for copper. Thus, the data demonstrate that the use of indigenous bacteria is potentially effective in the bioremediation of heavy metals. Keywords: BIOREMEDIATION, MICROORGANISMS, HEAVY METALS, LOCAL BACTERIA, ISOLATES, STRAINS, ABSORPTION

GC-MS analysis of hospital waste soil and isolation of antimicrobial agent producing bacteria

The current study, deals with the GC-Mass analysis of soil and antibiosis activity of soil bacteria, isolated from district hospital at Kabir Chaura, Varanasi, India. Standard serial dilution plate procedures were used to isolate the bacteria. Gram staining was used to identify the isolates morphologically. Strain KC14 was Gram-negative and all other isolates were Gram-positive. Isolated bacteria were tested against six human pathogens viz., Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Shigella flexneri and Bacillus subtilis. Primary and secondary screening were done by perpendicular streaking and seed overlay techniques. Based on the results most potential isolates were selected for tertiary screening. The GC-MS of soil samples revealed some major medical pollutants such as para-hydroxy acetanilide, 2-methyl-5-nitro-imidazole and 2-acetoxybenzoic acid. The KC14 isolate was chosen for further studies after demonstrating positive results against S. aureus with a maximum antagonistic activity of 17.3 ± 0.5 zone of inhibition. The molecular identification of KC14 isolate was determined by using 16S rRNA sequencing. The obtained nucleotide sequences were subjected to BLASTN in NCBI, and found similar to the available sequence of Acinetobacter junii. The nucleotide sequences have been deposited in GenBank with accession no. OP265396. The active compound was partially purified by ethyl acetate and tested its activity against S. aureus. This study defines the composition of hospital waste soil and identified a Gram-negative bacterium producing antibacterial activity against important human pathogens.

Effects of rainfall intensity and slope gradient on soil sealing and crusting, erosion, and phosphorus solubilizing bacteria

Soil erosion significantly affects various soil properties and processes, yet its impacts on soil microbial populations remains unclear. Understanding the effects of soil erosion on soil microbial properties enables the formulation of recommendations to protect soils and reduce their vulnerability. This study aimed to determine the effects of rainfall intensity and slope on soil crusting, erosion and bacterial communities in soils with various texture and mineralogy. Three kaolinitic soils with low to medium clay content (K1, K2, and K3) and three smectitic soils with high clay content (S1, S2, and S3) were subjected to simulated rainfall at three intensities (45, 70 and 100 mm/h) and two slopes (5° and 8°). Infiltration rate was measured using a graduated cylinder in 2-minute rotations of the simulator. Runoff and sediment yield were collected in a beaker. Crust strength was measured using a cone penetrometer, and its thickness was determined with a vernier caliper. Bacterial isolates were obtained by plating serial dilutions in nutrient agar and incubating them at 25°C for 24–48 h48 hous. The phosphate solubilising bacteria were then identified. The smectitic soils formed the strongest crusts with S2 exhibiting significantly (p < 0.05) higher crust strength (18.54 Kpa) at an intensity of 45 mm/h and slope of 8° On the other hand, soil K3 (kaolinitic) had the lowest crust strength (5.4 Kpa) at an intensity of 100 mm/h and slope of 8° Soil loss increased with higher rainfall intensity and steeper slopes for the kaolinitic soils with K1 being most erodible (468.2 kg/ha) at an intensity of 100 mm/h and slope of 8° The number of phosphate solubilising bacteria decreased with increasing rainfall intensity. Furthermore, this study revealed that soil properties played a more significant role in shaping bacterial composition and phosphate solubilisation than rainfall intensity alone. These findings highlight the complex interactions between soil erosion, bacterial communities, and soil properties, which are crucial for devising effective soil conservation strategies.

Simulated microgravity facilitates stomatal ingression by Salmonella in lettuce and suppresses a biocontrol agent

As human spaceflight increases in duration, cultivation of crops in spaceflight is crucial to protecting human health under microgravity and elevated oxidative stress. Foodborne pathogens (e.g., Salmonella enterica) carried by leafy green vegetables are a significant cause of human disease. Our previous work showed that Salmonella enterica serovar Typhimurium suppresses defensive closure of foliar stomata in lettuce (Lactuca sativa L.) to ingress interior tissues of leaves. While there are no reported occurrences of foodborne disease in spaceflight to date, known foodborne pathogens persist aboard the International Space Station and space-grown lettuce has been colonized by a diverse microbiome including bacterial genera known to contain human pathogens. Interactions between leafy green vegetables and human bacterial pathogens under microgravity conditions present in spaceflight are unknown. Additionally, stomatal dynamics under microgravity conditions need further elucidation. Here, we employ a slow-rotating 2-D clinostat to simulate microgravity upon in-vitro lettuce plants following a foliar inoculation with S. enterica Typhimurium and use confocal microscopy to measure stomatal width in fixed leaf tissue. Our results reveal significant differences in average stomatal aperture width between an unrotated vertical control, plants rotated at 2 revolutions per minute (2 RPM), and 4 RPM, with and without the presence of S. typhimurium. Interestingly, we found stomatal aperture width in the presence of S. typhimurium to be increased under rotation as compared to unrotated inoculated plants. Using confocal Z-stacking, we observed greater average depth of stomatal ingression by S. typhimurium in lettuce under rotation at 4 RPM compared to unrotated and inoculated plants, along with greater in planta populations of S. typhimurium in lettuce rotated at 4 RPM using serial dilution plating of homogenized surface sterilized leaves. Given these findings, we tested the ability of the plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis strain UD1022 to transiently restrict stomatal apertures of lettuce both alone and co-inoculated with S. typhimurium under rotated and unrotated conditions as a means of potentially reducing stomatal ingression by S. typhimurium under simulated microgravity. Surprisingly, rotation at 4 RPM strongly inhibited the ability of UD1022 alone to restrict stomatal apertures and attenuated its efficacy as a biocontrol following co-inoculation with S. typhimurium. Our results highlight potential spaceflight food safety issues unique to production of crops in microgravity conditions and suggest microgravity may dramatically reduce the ability of PGPRs to restrict stomatal apertures.

Isolation, Identification and Screening of Humic Acid Producing Fungi from Soil Environment of Oil palm (Elaeisguineensis) Associated with Empty Fruit Bunches

The demand for sustainable and sustainable biotechnological processes has developed as a result of a growing interest in converting agricultural waste into valuable products. In this study, fungal strainswere isolated from soil environment ofoil palm empty fruit bunchesandscreened for humic acid production. In Osun state, Nigeria, soil sample was taken from a local palm oil mill. Different colonies of fungi were obtained for further research using the serial dilution plating procedure. Plate screening was used to evaluate the fungal biomass as well as colony diameter of the fungal isolates, and a submerged fermentation test was used to measure the amount of humic acid concentration the isolates produced. Fungal isolates were identified by using molecular methods and morphological analysis. The fungi species Aspergillusniger, Rhizopusstolonifer, and Penicilliumchrysogenum were isolated from the soil environment. When compared to Rhizopusstolonifer, Aspergillusniger grew more superiorly in terms of zone diameter (26.5 1.50 mm) and biomass (2.23 0.16 g), while Penicilliumchrysogenum showed no growth. Throughout the fermentation process, Aspergillusnigeralso produced a greater quantity of humic acid, with concentrations varying from 0.70 mg/l to 2.20 mg/l. Aspergillusniger strain HR18's existence was confirmed by molecular analysis. This experiment proved that Aspergillusniger can efficiently produce humic acid from OPEFB, providing a useful method for waste utilization and sustainable practices.

Effects of Novel Dental Composites on Streptococcus mutans Biofilms.

With the phase-out of amalgam and the increase in minimally invasive dentistry, there is a growing need for high-strength composite materials that can kill residual bacteria and promote tooth remineralization. This study quantifies how antibacterial polylysine (PLS) and re-mineralizing monocalcium phosphate monohydrate (MCPM) affect Streptococcus mutans biofilms and the strength of dental composites. For antibacterial studies, the MCPM-PLS filler percentages were 0-0, 8-4, 12-6, and 16-8 wt% of the composite filler phase. Composite discs were immersed in 0.1% sucrose-supplemented broth containing Streptococcus mutans (UA159) and incubated in an anaerobic chamber for 48 h. Surface biomass was determined by crystal violet (CV) staining. Growth medium pH was measured at 24 and 48 h. Biofilm bacterial viability (CFU), exo-polysaccharide (water-soluble glucan (WSG) and water-insoluble glucan (WIG)), and extracellular DNA (eDNA) were quantified. This was by serial dilution plate counting, phenol-sulfuric acid microassay, and fluorometry, respectively. The biaxial flexural strengths were determined after water immersion for 1 week, 1 month, and 1 year. The MCPM-PLS wt% were 8-4, 8-8, 16-4 and 16-8. The normalized biomass, WSG, and WIG showed a linear decline of 66%, 64%, and 55%, respectively, as the PLS level increased up to 8%. The surrounding media pH (4.6) was all similar. A decrease in bacterial numbers with the 12-6 formula and a significant reduction with 16-8 compared to the 0-0 formulation was observed. The eDNA concentrations in biofilms formed on 12-6 and 16-8 formulations were significantly less than the 0-0 control and 8-4 formulations. Doubling MCPM and PLS caused a 14 and 19% reduction in strength in 1 week, respectively. Average results were lower at 1 month and 1 year but affected less upon doubling MCPM and PLS levels. Moreover, a 4% PLS may help to reduce total biomass and glucan levels in biofilms on the above composites. Higher levels are required to reduce eDNA and provide bactericidal action, but these can decrease early strength.

Isolation of Rhizospheric Bacteria from Soil and Their Antifungal Activity Against Helminthosporium oryzae the Causal Agent of Brown Spot of Rice

The brown spot of rice caused by Helminthosporium oryzae is a serious disease that affects the yield of rice crops. Brown spots of rice have been reported in almost all rice-growing countries. Therefore, the present study was to explore the management of brown spots of rice. through an antagonistic activity using rhizospheric bacteria. The method employed for soil sample collection was persuaded for bacterial isolation through the serial dilution plate technique. For the results of morphological characteristics of bacterial strain from soils of rice crop collected from Hyderabad. The results of the present study show that many colonies made by bacteria and which were consisted of different sizes, shapes, and colors. Whereas the results of the 15 isolates strains of rhizobacteria, only 06 bacterial strains IAP-1, IAP-2, IAP-04, IAP-09, IAP-12, and IAP-14 were found more effective in antagonizing H.oryzae, under in-vitro conditions. Antagonistic activity of rhizobacteria against H. organize showed significant inhibitory effects. The rhizobacteria showed the primordial result against against H. oryzae and will be helpful as a formulation not only for commercial purposes but also for beneficial biological control against the Brown spot of rice

Development of state of art test setup to measure thermal conductivity of soils infused with treated and un‐treated natural microorganisms

AbstractSoil is a natural heterogeneous material and from its formation process, heat transfer capacity (thermal conductivity) of soil is playing vital role in many engineering applications. The aim of this research study is to enhance thermal conductivity using natural and treated bacteria from soil samples procured from four different regions namely Dholera, Navsari, Sabarmati River, and Madhavpur of Gujarat, India. To fulfill this objective, thermal conductivity test set‐up was indigenously developed based on guarded hot plate method and is calibrated up to 150°C temperature. The colonies of microorganisms available in soil were studied by serial dilution and spread plate technique. Further, influence of microorganisms on soil structure aggregation pre and post treatment was analyzed by microscope. Four types of microorganisms were introduced in the soil and the thermal conductivity of sample is measured for curing period of 7, 14, 21, and 28 days. The results indicate increment in k value for thermal conductivity in clayey soil up to 26.67% and decrement in sandy soil is up to 45.45%, while most optimum results were observed for 7 days curing period. Present research demonstrates use of microbes available in various soils to enhance heat transfer mechanism through proper identification and treatment.

Soil Microbial Biomass, Microbial Population and Diversity in Maize-Banana Based Agroforestry System in Kisii County, Kenya

Soil microbes are involved in many important ecosystem processes including nutrient acquisition, biogeochemical cycling and soil aggregation. Soil microbial diversity affects the soil belowground dynamics and fate of carbon and nutrients. Soil microbes are important for agricultural and plant production systems, hence understanding the effects of agroforestry systems on the soil microbes, is necessary in order to improve on soil health and fertility. The objective of the study was to determine the soil microbial biomass, microbial populations and microbial diversity in maize-banana based agroforestry system. The study was conducted at Kenya Agricultural and Livestock Research Organization farm in Kisii County. The experiment was laid out in a randomized complete block design with maize and banana intercropped with agroforestry trees. The treatments were; Maize, banana (MMBB), Maize-banana, Calliandra (MBCC), Maize (MM), banana (BB), Maize-banana, Leucaena (MBLL), Maize-banana, Sesbania (MBSS) and Maize, fertilizer (MMF). Soil samples were collected from the agroforestry fields using a soil auger. Soil microbial biomass was measured using the chloroform fumigation extraction. Fungi and bacteria were enumerated by serial dilution plate method.Shannon diversity index (H’) and Simpson diversity index (1 - D) were used for the calculation of species diversity. SAS (version 9) statistical software was used for analysis. The treatments with agroforestry tree species had significantly higher soil microbial biomass (MBSS-86.33, MBCC-52.66 and MBLL- 47.0MgC/Kg) populations of bacteria (MBSS-197, MBCC-128.0 and MBLL-111.25x108cfu g-1soil) and fungi (MBSS-50.83, MBCC-29.167 and MBLL-14.0x105cfu g-1 soil) and diversity of bacteria (MBSS- (H' =1.61, D = 1), MBCC- (H' =1.04, D = 0.83), MBLL (H' = 0.52, D = 0.5) and fungi MBSS (H' =1.39, D = 1) MBCC (H' =1.04, D= 0.83), MBLL (H' =1.56, D = 0.93). MBSS increased microbial biomass, microbial populations and microbial diversity significantly an indication of improved soil health and hence recommended for adoption by farmers.

Characterization of Antibiotic-producing Actinomycetes Isolated from River Tana and Lake Elementaita in Kenya

The rise of antibiotic-resistant bacteria has become a global health concern, necessitating the search for novel sources of antibiotics. Actinomycetes, a group of microorganisms, have been known for their ability to produce bioactive compounds with antimicrobial properties. This study aimed to isolate, identify, and characterize antibiotic-producing Actinomycetes from River Tana and Lake Elementaita. Samples were collected from the study sites, and Actinomycetes were isolated using serial dilution and spread plate techniques. The isolates were characterized based on their morphological and biochemical properties. Furthermore, their antibacterial activity against Escherichia coli, Staphylococcus aureus, and Salmonella typhi was evaluated using the agar well diffusion method. The zones of inhibition were measured (mm), and analysis was done to compare the activity of the isolates using Kruskal Wallis test and medians compared using Wilcoxon with Bonferroni correction at alpha = 0.05 in SAS version 9.4. Analysis of DNA sequences was done using the BLAST program and a phylogenetic tree was constructed using MEGA X version 11. Biochemical tests revealed positive results for catalase, indole, oxidase, and citrate utilization, while coagulase and methyl red tests were negative. In terms of antibacterial activity, 54.5% of the isolates showed activity against E. coli, 45.5% against S. aureus, and 45.5% against S. typhi. Isolate LEL2201 had significant (p &lt; 0.05) higher zone of inhibition against S. aureus (inhibition zone of 25.0mm), while isolate RT2201 exhibited the highest activity against E. coli and S. typhi (inhibition zone of 8.5 mm and 8.6 mm, respectively). Molecular characterization through 16S rRNA gene sequencing identified the isolates as belonging to the Actinobacterium order. Phylogenetic analysis revealed their similarity to known Actinomycetes species including Actinomycetales bacterium, Streptomyces intermedius and Streptomyces flavomacrosporus from various countries. The findings of this study demonstrate the presence of antibiotic-producing Actinomycetes in River Tana and Lake Elementaita. Thus, further investigations are warranted to identify and characterize the specific antibacterial compounds produced by these isolates.

Isolation Of Bacterial Strains And Production of Enzymes For Biodegradation of Biomedical Cotton Waste

In recent times, biodegradation of used hospital cotton by using the bacterial strain is a promising and interesting field attracting worldwide researchers. It was observed that biodegradation of biomedical waste like cotton etc., is becoming a huge problem for both developed as well as developing countries. To overcome this problem, bacterial species were isolated using serial dilution and agar plating methods from different soil sources. Pure colony or single colony was isolated by the streak plate method. The bacterial species were identified with the help of various physical characteristics, staining, and biochemical activities. Strain improvement was done by UV and EtBr to know the best culture for amylase production. Purification of the crude amylase was done by dialysis, and its activity was calculated by DNS assay. Protein concentration was measured by Lowery’s method. The purified enzyme was characterized for the effect of temperature, pH, activator, and inhibitors. The bacterial colonies were isolated and named as IS2015 01 to IS2015 16, the total enzyme activity of the purified enzyme was calculated to be 0.96 mg/ml, and the amount of protein in the purified sample was calculated to be 1.47 mg/ml. Amylase purified here was found stable in a pH range of 7 - 9 and temperature range of 28°C - 37°C. The activity was found to be enhanced under the influence of cations such as Ca2+, Mg2+ and retarded under the influence of anions such as EDTA and SDS. The isolated bacterial strain and enzyme were found effective in degrading the biomedical cotton waste. In conclusion, the isolated bacterial strain and enzyme may be effectively used to degrade the biomedical cotton.

Bacteriological analysis of hospital sewage of district Hospital Ratnagiri

Hospital sewage is a waste drained out from the various clinical facilities of the hospital. The hospital sewage contains pathogens which pose severe public health threat. Most hospitals lack sewage treatment facilities and discharge the sewage untreated directly in to the public sewer. The study was aimed to analyze the hospital sewage of District hospital Ratnagiri for Coliform count by MPN and serial plate dilution method, and identification of the Bacterial isolates by conventional method from the sewage. Physico-chemical parameters like Temperature, pH, TDS, COD, BOD5, samples were determined which affect the bacterial flora of the sewage. Presumptive and Confirmatory MPN test and Serial plate dilution was performed. Coliform were isolated and identified using Gram stain, Colony characteristics and Biochemical characteristics. Temperature was 28(+1.22)°c, pH 6.52(+0.10), TDS 251(+1.41) mg/l, COD 96.9(+0.82) mg/l and BOD 69.5 (+0.58) mg/l. TDS was higher, COD and BOD values were less as compare to the permissible limits. Presumptive MPN was &gt;1.8 x 103, Confirmatory MPN was 3.3x101 per 100 ml and coliform count by serial dilution was 6x106 CFU/ml on Blood agar and 2x106 CFU/ml on MacConkey’s agar which indicated higher counts of coliform in the sewage. Common bacteria like Escherichia coli, Pseudomonas aeruginosa, Klebsiella peumonae, Chromobacter violaceum and Bacillus subtilis were isolated. The study reveals presence of significant coliform load in the hospital sewage which poses risk of infection to the community.

Isolation and characterization of potential multiple extracellular enzyme-producing bacteria from waste dumping area in Addis Ababa

Extremozymes are innovative and robust biocatalysts produced by various microorganisms from harsh environments. As thermophilic organisms can only develop in a few places, studying them in geothermal environments has provided new insights into the origins and evolution of early life and access to significant bio-resources with potential biotechnology applications. The work aimed to isolate and identify likely multiple extracellular enzyme-producing thermophilic bacteria from an Addis Ababa landfill (Qoshe). The streaking approach was used to purify 102 isolates acquired by serial dilution and spread plate method. The isolates were morphologically and biochemically characterized. Thirty-five cellulases, 22 amylase, 17 protease, and nine lipase-producing bacteria were identified using primary screening methods. Further secondary screening using Strain safety evaluation; two bacterial strains (TQ11 and TQ46) were identified. Based on morphological and biochemical tests, they were found to be gram-positive and rod-shaped. Furthermore, molecular identification and phylogenic analysis of selected promising isolates confirmed the identity of the isolates, Paenibacillus dendritiformis (TQ11) and Anoxybacillus flavithermus (TQ46). The results indicated that, multiple extracellular enzyme-producing thermophilic bacteria isolated from a waste dumping area in Addis Ababa offer useful features for environmental sustainability in a wide range of industrial applications due to their biodegradability and specialized stability under extreme conditions, increased raw material utilization, and decreased waste.

Formulation of Effective Microorganism [EM] to Analyse its Impact on Municipal Wastewater Management

Discharging wastewater from households, industries, and other sources into the environment without treatment hurts the ecosystem as well as people’s quality of life by causing negative impacts on all living species, including health concerns to humans such as diarrhoea which kills 3,50,000 Indian children annually and only 33% of India’s urban wastewater is treated. The majority of energy is dedicated solely to the aeration process, which regulates the degradation of organic matter (sludge) found in wastewater, which is one of the most critical issues in wastewater treatment. As a result, the goal of this research is to develop long-term strategies for degrading organic materials using Effective Microorganisms Technology (EM). EM, also known as Effective Microorganism, is a carefully selected and proportioned liquid culture of fermenting microorganisms. In this study, three microorganisms were isolated from various sources, including soil, yogurt, and milk by serial dilution and spread plate technique, each isolate was made into a pure culture after incubation. They were identified by the VITEK method as Pseudomonas aeruginosa, Lactobacillus sporogenes, and Candida albicans. The microbial consortium was then formed by incubating all three organisms in a nutrient broth that had been modified. Each microorganism culture and formulated microbial culture were added to sewage samples taken in Erlenmeyer flasks. All samples were tested for physico-chemical parameters such as pH, BOD, COD, TDS, TSS, SO4 ²– and Cl- after a three-day incubation period at room temperature. The standard deviation method was used for statistical analysis and Microsoft Power BI was used to visualize the results. Lactobacillus sporogenes was the only culture that showed a significant reduction in all parameters except one when compared to other cultures that showed a reduction in most parameters except a few. As a result, Lactobacillus proved to be effective in wastewater treatment when compared with other cultures. Further research is required to determine its true potential.

INDOLE ACETIC ACID AND EXOPOLYSACCHARIDE PRODUCTION OF NITROGEN FIXING BACTERIA ISOLATED FROM NUTMEG TREE

The constraint of plant cultivation in the tropics is low nitrogen (N) content in soil. The application of nitrogen fixing bacteria (NFB) is not only fulfill the N demand but also provide the phytohormones Indole acetic acid (IAA) and exopolysaccharides (EPS). The objective of the experiment was to was to isolate the NFB from the rhizosphere of nutmeg tree and characterized their IAA and EPS production in a liquid culture. The NFB was isolated by serial dilution plate method on Tryptic Soy Agar. In order to test the ability of various NFB isolates to produce IAA and EPS, all isolates were grown in Tryptic Soy Broth for 72 hours at room temperature. The results showed that at the end of experiment the media acidity was shift from neutral to alkaline. The population of five isolates of NFB was approximately 8-9 log10 (of CFU/mL). The IAA production in liquid culture of all isolates was similar but the EPS production was varied and depend on the isolates, The highest EPS content was in PL1 culture and the lowest was in the culture of PL5. This experiment verified that the NFB isolated from nutmeg rhizosphere enable to produce IAA and EPS.