Plate Assay Method Research Articles

Characterization of raw starch hydrolysing and detergent-compatible amylase from newly isolated Bacillus subtilis from bakery chimney

Amylases are highly needful product in food, beverage, and detergent industry. With the objective of isolating α-amylases producing strains, samples were collected from local bakery chimneys and kitchens, Chennai, Tamil Nadu, India. The potential α-amylase producers were isolated and screened by Starch-Iodine plate assay method. The optimized parameters such as 1.5% of starch concentration in the media with 7.2-pH at 35 °C for 36 h enhanced the production of α-amylase. The strain showing high potential, identified by 16s rRNA sequencing was found to be Bacillus subtilis. The Bacillus subtilis strain produced extracellular α-amylase with activity of 1330.14 U/mL. Further partial purification by ammonium sulphate precipitation followed by dialysis and column chromatography enhanced the specific activity to 1695.306 U/mg. The molecular weight of enzymes was found to be around 36 kDa. The enzyme stability and activity characterization revealed that the enzyme was thermotolerant which was able to retain more than 70% activity at 70 °C, 80 °C and 90 °C. The purified enzyme was also found to be Ca2+ independent. The hydrolysis of raw starch from rice, wheat and corn flour using the purified α-amylase from Bacillus subtilis showed significant activity of 72.8%, 76.1% and 65.2% with 4 h incubation respectively. The potentiality of the enzyme as an additive with commercial detergent was also determined to be effective. This study favourably attracts the attention towards industrial applications.

Hypocholesterolemic potential of Bacillus amyloliquefaciens KAVK1 modulates lipid accumulation on 3T3-L1 adipose cells and high fat diet-induced obese rat model.

The significance of microorganisms occurring in foods is predominantly targeted due to their application for identifying a novel range of the bacterial spectrum. Diverse microbial species are capable of exhibiting potential pharmacological activities like antimicrobial and anticancer. Microbial strains capable of reducing obesity-related syndromes have also been reported. In the present study, the hypocholesterolemic efficacy of Bacillus amyloliquefaciens isolated from dairy products was scrutinised by in vitro (3T3-L1 adipose cells) and in vivo (high-fat diet-induced obese Wistar albino rats) methods. Potential cholesterol-lowering isolates were screened using a plate assay method and optimised by physical parameters. Molecular identification of the topmost five cholesterol-lowering isolates was acquired by amplification of the 16S rRNA gene region. Bacillus amyloliquefaciens strain KAVK1, followed by strains KAVK2, KAVK3, KAVK4, and KAVK5 were molecularly determined. Further, cholesterol-lowering strains degraded the spectral patterns determined by the side chain of a cholesterol molecule. The anti-lipase activity was demonstrated using the porcine pancreatic lipase inhibitory method and compared with the reference compound Atorvastatin. Lyophilised strain KAVK1 revealed maximum pancreatic lipase inhibition. Strain KAVK1 attenuated lipid accumulation in 3T3-L1 adipose cell line predicted by Oil Red O staining method. Significant reduction of body weight and change in lipid profile was recognised after the supplement of KAVK1 to obese rats. Histopathological changes in organs were predominantly marked. The result of this study implies that the cholesterol-lowering B. amyloliquefaciens KAVK1 strain was used to treat hypercholesterolemia.

Pseudomonas aeruginosa from a Tertiary Teaching Hospital: A Study of AmpC and Biofilm-producing Clinical Isolates

Abstract Introduction: Opportunistic Pseudomonas aeruginosa is one of the most prevalent bacteria with a broad spectrum of human-associated infections. The World Health Organization (WHO) claims that Pseudomonas aeruginosa infection is a typical healthcare-associated disease of critical or high concern. It is well known for being inherently resistant to many antibiotics and has the ability to produce biofilm. In the present study, we aimed to investigate P. aeruginosa with reference to biofilm and AmpC β-lactamases producing isolates in a tertiary care hospital. Materials and Methods: An observational cross-sectional study was performed at the Department of Microbiology, KIMS, KVV, Karad, and Krishna Hospital & Medical Research Center, Karad. (KH & MRC). A total of 180 isolates were subjected to this test to find out which produced the AmpC β -lactamase enzyme by the cefoxitin-cloxacillin double-disc synergy test method and biofilm production by tissue culture plate assay method. Results: Our study found a total of 97 isolates (53.89%) of AmpC production and biofilm production accounted for 151 (83.89%). Conclusion: AmpC- beta-lactamases are responsible for increasing the resistance of P. aeruginosa to several beta-lactam antibiotics and this study also showed that the clinical isolates of P. aeruginosa had a higher propensity to form biofilm and that there was a direct association between biofilm formation and antibiotic resistance.

Biochemical characterization of YoAlp®: a sheep-fermented milk obtained with autochthonous starter cultures

Fermented milks are a source of bioactive peptides with different potential benefits on human health and may be considered as functional foods. Sheep milk and fermented milk have been collected and a biochemical characterization, by a proteomic approach, GC/MS and microtiter plate assay methods, have been conducted to evaluate their peptide, fatty acid and aromatic profile, and to assess potential health promoting effects. Furthermore, a comparison between sheep-fermented milk (SFM) made with commercial starter cultures and YoAlp®, a sheep-fermented milk obtained using local strains of lactic acid bacteria, has been performed. Peptide’s profile comparison shows a higher number of amino acidic frequencies using autochthonous starter cultures than commercial ones. Among these peptides, 20.78% and 29.87%, respectively, are supposed to be potentially bioactive. Furthermore, in both products, the fatty acid profile was similar to that of origin sheep milk, and concerning aromatic profile, YoAlp® shows yogurt typical aromatic assets. Considering bioactivity, ACE inhibitor activity is high for both samples. Similar values, as expected by peptide profile analysis, have been obtained. Even in the case of antioxidant capacity, peptide profile bioactivity prediction has been confirmed by the assay showing a DPPH inhibition higher for SFM than for YoAlp®, but this difference is not statistically significant. Local strains of lactic acid bacteria seem to work as well as the commercial, preserving biodiversity and typicality. However, further analyses are needed to understand microbial proteolytic activities and to investigate gastric digestion resistance of bioactive peptides.

Production of Industrially Important Cellulases and Pectinases using Lignocellulosic Biomass

Cellulases and pectinases are the two main industrially important enzymes that find significant applications in food, feed, pharmaceutical, textile and environmental sectors. In the present study, Trichoderma asperellum S14 was used for enzymes production in liquid state fermentation and for enzymatic hydrolysis of lignocellulosic biomass. Prior to liquid state fermentation, Trichoderma asperellum S14 was qualitatively screened for the pectinolytic and cellulytic activity by using plate assay method. Alkaline pretreatment (1% NaOH) was used for the pretreatment of lignocellulosic biomasses including; wheat bran, wheat straw, rice straw, rice husk and sugarcane bagasse. Maximum cellulase activity of 4.3 U/mL was observed at pH 4.8 and 30ºC after 72 hours of incubation from Trichoderma asperellum S14 using alkali pretreated sugarcane bagasse as a substrate. While, maximum pectinase activity 9.2 U/mL obtained at pH 4.8 and 30ºC after 72 hours of incubation from Trichoderma asperellum S14 using alkali pretreated wheat bran. Enzymatic hydrolysis was also performed using crude enzymes extract at 50ºC for 72 hours. Maximum hydrolysis (70%) was observed in case of pretreated sugarcane bagasse using Trichoderma asperellum S14 as fungal source for enzyme production. Lignocellulosic biomasses are considered as the best feedstock used in second generation biofuel production because of their renewable resources as well as inexpensive and environment friendly nature. Present study shows the potential of Trichoderma asperellum S14 to degrade variety of lignocellulosic biomass efficiently and could be used to hydrolyze complex lignocellulosic materials towards the production of second generation biofuel.

Effect of Acetic Acid on Clinical Isolated Pseudomonas aeruginosa Biofilm in Chronic Suppurative Otitis Media: In vitro Study

Background: Forming biofilms on bacteria can inhibit the penetration of antimicrobial agents and avoid the immune defence system. It becomes one of the factors causing therapy failure and chronicity of infection. Pseudomonas aeruginosa is the most common bacteria found in Chronic Suppurative Otitis Media (CSOM), which has the virulence ability to form biofilm structures. Some studies have reported that acetic acid can inhibit and eradicate biofilm complexes and is thought to be an alternative to additional therapy against bacterial infections that form biofilms. Objective: to explain the effect of acetic acid inhibiting and eradicating Pseudomonas aeruginosa biofilm in CSOM. Methods: This study used an experimental in vitro laboratory with a post-test-only control group method. Samples were taken from the secretions of the mastoid cavity of CSOM patients. The inhibitory effect of acetic acid was observed by administering acetic acid to Pseudomonas aeruginosa. In contrast, the effect of eradicating biofilm was observed by administering acetic acid to Pseudomonas aeruginosa which had already formed a biofilm. The observations in this study were using the microtiter plate assay method and were measured with an ELISA reader. Data analysis used the One-Way Anova test and multiple comparisons (Tukey HSD Test). Result: The inhibitory effect of acetic acid on the growth of Pseudomonas aeruginosa biofilm was obtained (p=0.000) with significant results (p <0.05) between the positive control group and the concentration group of 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, and 5%. The Minimum Biofilm Inhibitory Concentration (MIBC) value of acetic acid in forming Pseudomonas aeruginosa biofilms was 0.16%. The effect of acetic acid eradication on Pseudomonas aeruginosa biofilms (p=0.000) with significant results (p<0.05) between the positive control group and the concentration group of 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, and 5%. While the minimum value of acetic acid Biofilm Eradication Concentration (MEBC) for Pseudomonas aeruginosa biofilm eradication was 0.08%. Conclusion: Acetic acid inhibits the formation and eradication of Pseudomonas aeruginosa biofilms in CSOM.

Evaluation of Disinfectant Efficacy against Biofilm-Residing Wild-Type Salmonella from the Porcine Industry.

Salmonella enterica is a causative pathogen of Salmonellosis, a zoonosis causing global disease and financial losses every year. Pigs may be carriers of Salmonella and contribute to the spread to humans and food products. Salmonella may persist as biofilms. Biofilms are bacterial aggregates embedded in a self-produced matrix and are known to withstand disinfectants. We studied the effect of glutaraldehyde and peracetic acid, two active substances frequently used in disinfectant formulations in the pig industry, on representative biofilm-residing wild-type Salmonella collected from pig housings in the United Kingdom (UK). We screened biofilm production of strains using the microtiter plate (MTP) assay and Congo Red Coomassie Blue (CRCB) agar method. Previously published stainless-steel coupon (SSCA), polyvinylchloride coupon (PCA), and glass bead (GBA) assays were used for disinfection studies. The mean reduction in the tested wild-type strains met the criterion of ≥4 log10 CFU at a disinfectant concentration of 0.05% with SSCA and GBA, and 0.005% with PCA for peracetic acid, along with 0.5% for glutaraldehyde with all three assays on the mean. At these concentrations, both tested disinfectants are suitable for disinfection of pig housings against Salmonella. When evaluating the efficacy of disinfectants, biofilms should be included, as higher disinfectant concentrations are necessary compared to planktonic bacteria.

Isolation and identification of multi-drug resistance Acinetobacter baumannii isolated from clinical samples at Baghdad, Iraq

An opportunistic bacterium called Acinetobacter baumannii has significantly increased the frequency of infections in recent years. With only a limited number of “traditional” virulence factors, It is infections have spread rapidly through hospitals across the globe. The present study aimed to work out the relationship between the multi-drug resistance (MDR) of A. Baumannii and biofilm formation. A total of 150 samples were collected from various clinical sources from different age groups and gender patients in Ghazi AL Hariri Hospital and Baghdad Teaching laboratories in Medical City in Baghdad, Iraq from December 2021 to March 2022. Microscopical inspection and cultural features on various culture media, including culturing on selective medium CHROMagar, were used to identify bacterial isolates. The characteristics of the isolates were then established by some biochemical tests. Identification was confirmed using the Vitek-2 system with an accuracy of 99%, which revealed that only (50) isolates were given identical morphological characteristics and biochemical tests belonging to Acinetobacter baumannii isolates. 28 (56%) isolates were collected from burns. 10 isolates (20%) and 9 isolates (18% were collected from wound and sputum cultures of A. baumannii , respectively, while only 3 isolates (6%) were from urine culture. The susceptibility test for all the fifty clinical isolates of A. baumannii was performed against 10 different antibiotics. The results showed that A. baumannii isolates were Multidrug-resistant (MDR) (98%), while the other (2%) of the isolates were extensively drug-resistance (XDR) to themajority of antibiotics tested. All 50 isolates in the present study were subjected to the micro-titer plate (MTP) assay method (96 walls). The results indicated that strong biofilm was detected in 40 (80.0%) of the tested isolates. Thirty bacterial isolates were were found to be MDR and had strong biofilm production.

Detection of biofilm formation in multi-drug resistant Staphylococcus aureus among clinical isolates in Zaria metropolis, Kaduna, Nigeria

Antimicrobial resistance are associated with several virulence factors such as biofilm. The biofilm formation ability of S. aureus complicates effective therapeutic management of infections. In this research, we intend to observe biofilm formation in S. aureus using two in vitro phenotypic methods: tissue culture plate and Congo red agar as well as biofilm associated genes Ica A B & D and 16SrRNA. A total of 150 presumptive coagulase positive staphylococcal isolates from all specimens (blood, urine, high vaginal swab, wound swab, ear swab, endocervical swab) submitted to the microbiology laboratory of the selected hospitals were collected. Seventy-three (73) or 48.7% of the isolates were identified as coagulase positive Staphylococcus using microbiology standard methods (tube coagulase test, growth on mannitol salt agar & Dnase agar). Using microgen staph identification kit, the isolates were further characterized into various Staph spp. Disc agar diffusion method was used for eleven (11) antibiotics susceptibility test while vancomycin screening agar and BMD (Broth Micro dilution) MIC test was used for vancomycin susceptibility evaluation. Indirect observation of biofilm was performed using congo red agar (CRA) and Microtitre plate assay method. PCR and sequencing were conducted on the isolates to molecularly detect the presence of 16SrRNA, IcaA, IcaB, IcaD genes. Susceptibility of S aureus (21%) isolates tested against 12 different categories of antibiotics shows 4(27%) not multidrug resistant (MDR), 7(47%) were MDR and 4 (27%) were extensively drug resistant (XDR). High percentage (74%) of Multiple antibiotic resistant index at ≥0.3 suggested that the isolates originated from an environment where antibiotics are often used. Biofilm assessment of MDR S aureus isolates revealed that 9(82%) and 11 (100%) of the isolates were biofilm formers using Congo Red Agar method and Microtiter plate assay respectively. All isolates amplified with 16SrRNA primer signifying all are S. aureus, 100% of the isolates amplified with IcaA, 90% isolates amplified with IcaD, and 50% isolates amplified with IcaB. We can conclude that there is high prevalence of Biofilm forming S. aureus strains in the hospitals studied. This result should be born in mind by clinicians while prescribing medication for S. aureus positive conditions. Hospital managements should be proactive in preventing the preservation of the biofilm formers in their hospital environment through adequate sanitary and disinfection protocols. It is also necessary for clinician to note that nitrofurantoin, linezolid and vancomycin were most effective against the biofilm producing S. aureus and microtitre plate assay method was observed to be most reliable assay in the study of biofilm.

Screening for the Production of MetNase and Its Optimum Conditions from Streptomycete Isolate

MetNase might be a valuable source for different medical applications. MetNase has the potential to be used against a wide range of tumour cell types as an anticancer agent. The purpose of this research is to screen and develop optimal conditions of streptomycetes isolates for MetNase production. For this study, 34 randomly selected streptomyces isolates from soil and marine water were used. The isolates that can produce MetNase were qualitatively selected using a quick plate assay method. When the pH was set to 7.0, the incubation temperature was set to 30 °C, and the carbon and nitrogen sources were glucose and peptone, respectively, the highest levels of MetNase synthesis were attained.

Exploring the Identity and Properties of Two Bacilli Strains and their Potential to Alleviate Drought and Heavy Metal Stress

Naturally available plant growth-promoting rhizobacteria (PGPR) have 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzymes, and are capable of processing the plant-borne ACC by converting it into α-ketobutyrate and ammonia. Thus, the PGPRs help in the depletion of ethylene levels, and enhance abiotic stress tolerance in plants. In the present study, two rhizobacterial strains, i.e., Bacillus cereus and B. haynesii, isolated from Vigna mungo and Phaseolus vulgaris, were used. These strains were taxonomically identified by 16S rDNA sequencing as B. cereus and B. haynesii, with NCBI accession numbers LC514122 and LC 514123, respectively. The phylogeny of these strains has also been worked out based on homology, with data available on NCBI GenBank. The strains were screened for their plant growth-promoting traits, and quantified in the same way. The enzymatic activity and molecular weight of the ACC deaminase obtained from both bacterial strains have also been determined. An in vitro drought tolerance study was done by using PEG 6000. These bacterial strains exhibited higher ACC deaminase activity (~5 to 6 µmol/mL), exopolysaccharide yield (15 to 18 mg/10 mL protein), and indole acetic acid (27–32 µg/mL). These characteristics indicate that the bacterial strains under present study may be helpful in enhancing the drought tolerance of the crops with enhanced yield. Bacillus cereus has been found to be a tolerant strain to As, Ba, and Ni, based on the plate assay method, and so it has the potential to be used as biofertilizer in fields affected by these metals.

Biodecolourization of Textile Effluents using Ligninolytic Enzymes Produced from Selected Bacterial and Fungal Isolated From Soil Contaminated Textile Waste

ABSTRACT Textile effluent contains enormous chemicals with detrimental environmental effects on both fauna and flora due to its chemical compositions. In this study, the effect of lignolytic enzymes produced by microorganisms for the treatment of textile effluent was examined using standard microbiological techniques. The potential of the isolates to produce laccase (L), lignin peroxidases (LiP) and manganase peroxidase (MnP) was investigated using streak plate method and assay methods. Finally, the L, LiP and MnP enzymes produced with the optimal processing parameter were used to decolorize textile effluent singly and in combination for ten (10) days. Fourteen (14) microbial isolates which include eight (8) bacterial and four (4) fungi were isolated from soil contaminated with textile effluents. Aspergillus terreus and Aspergillus niger showed higher production of laccase with 8.0 mm diameter zone of inhibition. Bacillus licheniformis and Bacillus subtilis had the widest zone of inhibition (12.0 and 8.0 mm) respectively. Only Aspergillus flavus however had the potential to produce lignin peroxidase (with 10 mm zones of clearance) of all the fungi isolated in this study. Laccase caused the highest decolourization of 72.5% comparable to 71.1% observed for the three enzyme combination while LiP has 57.0%. This finding established the potential use of bacterial ligninocellulolytic enzymes for the decolourization of textile effluent.

Isolation and Characterization of Polyhydroxybutyrate (PHB) Producing Microorganisms from Karad Region, Maharashtra

World population is increasing day by day and to satisfy their various needs, plastic material become part and parcel of life. Millions of tons of these non degradable plastics accumulate in the environment per year and cause hazardous effects on flora and fauna. Hence there was ever pending search for bioproduct which can substitute this nonbiodegradable plastic. Polyhydroxybutyrates (PHB) are drawing much attention as they are biodegradable, environmentally friendly, and also biocompatible bioplastics. In the present study an attempt was made to isolate and characterize the PHB producing microorganisms from Karad region of Maharashtra. Total 75 isolates obtained from soil, spent wash, pond water, dairy effluent, cow dung, soil and organic waste samples were enriched using mineral salt medium and then screened primarily for PHB producers by Sudan Black B plate staining method followed by secondary screening using Nile blue plate assay method. As a result of screening, 11 PHB producing potential isolates were obtained which were then studied for morphological, biochemical and enzymatic characters. By referring Bergeys Manual of Determinative Bacteriology and 16 S r RNA technique these 11 PHB producing isolates were identified and found from genera Bacillus, Candida, Citrobacter and Klebsiella, Staphylococcus and Rhodococcus.

Analysis of Staphylococcus epidermidis biofilm bormation in several types of intravenous fluids based on time

An intravenous catheter is a medical device used to inject intravenous fluid into the body. This procedure can lead coagulase-negative staphylococci (CoNS) bacteria such as Staphylococcus epidermidis penetrates the skin and forms biofilm on the catheter. Biofilms bring serious problem such as antibiotic resistance, the long-term effects increase the length of staying in the hospital, cost, morbidity, and mortality. This research aimed to analyze the biofilm formation of S. epidermidis in several types of intravenous fluids based on time. This was a laboratory experimental research using microtiter plate assay method and crystal violet coloring. Three 96-well microplates were inoculated with S. epidermidis in ringer lactate, 10% dextrose, 5% dextrose, normal saline, and gelafusal, each plate was incubated at three different times of 24, 48, and 72 h respectively. The results show that the optical density (OD) values of all intravenous fluids with bacteria within 24 and 48 h of incubation time did not show significant differences compared to negative controls, while the 72 h treatments of 10% dextrose, 5% dextrose, and normal saline showed significant differences. This indicates that biofilms of S. epidermidis were not formed in intravenous fluids within 24 and 48 h of incubation time, however this bacterium started forming biofilm in 10% dextrose, 5% dextrose, and normal saline within 72 h of incubation time. In conclusion, the length of incubation time may influence biofilm formation.

Production of Bioplastic by Local Strains of Bacillus subtilis using Watermelon Peels as Substrate

The amount of environmental contamination brought on by the careless disposal of plastic garbage has increased to 400 million tons every year on a global scale. These synthetically generated traditional polymers are not easily biodegradable. This work was therefore undertaken to isolate Bacillus subtilis from soil with potential to produce bioplastic: Poly-ß-hydroxybutyrate (PHB). Samples of soil were collected from various locations (BG = Botanical Garden, FARD = Fine Art Refuse Dumpsite, SHRD = Suleiman Hall Refuse Dumpsite, FVM = Faculty of Veterinary Medicine animal paddock) within Ahmadu Bello University, Zaria, Nigeria. Spread plate technique was used to isolate B. subtilis on nutrient agar, and the isolates' cultural, morphological, and biochemical properties were identified. The isolates of B. subtilis were screened for PHB production using two different methods: the plate assay method and slide technique using Sudan black B dye. The PHB was then produced using submerged fermentation with watermelon peel as sole source of carbon in the production medium. The PHB was extracted using Sodium-hypochlorite method and the quality of the PHB was determined using FT-IR analysis. Four isolates of Bacillus subtilis were obtained from the soil samples (50 %), one each out of the two samples (50 %) per unit location (BG2, FARD3, SHRD2, and FVM4). The screening revealed that all the isolates were PHB producers. The B. subtilis isolate SHRD2 from the students’ dormitory was found to produce the highest PHB yield of 0.98 g/L from the watermelon substrate, whereas isolate from the animal paddock (FVM4) yielded the lowest quantity of the PHB (0.12 g /L). The biopolymer's (bioplastic) identity was confirmed to be PHB based on the peaks in the FT-IR spectra, which displayed wave numbers for a variety of functional groups, including -O-H, C-H, C-O, and C=O. It was concluded that the local isolates of B. subtilis have potentials for PHB production using watermelon peels as source of carbon and energy.

Interactions among Relevant Non-Saccharomyces, Saccharomyces, and Lactic Acid Bacteria Species of the Wine Microbial Consortium: Towards Advances in Antagonistic Phenomena and Biocontrol Potential

The topic of microbial interactions is of notable relevance in oenology, being connected with their impact on microbial biodiversity and wine quality. The interactions among different couples of microorganisms, in particular yeasts and lactic acid bacteria representative of the must/wine microbial consortium, have been tested in this study. This interaction’s screening has been implemented by means of plate assays, using culture medium, grape juice, and wine agar as substrates. Different antagonistic phenomena have been detected, belonging to the following interaction categories: yeast-yeast, yeast-bacteria, bacteria-yeast, and bacteria-bacteria. In general, the inhibitory activity has been observed in all three media agar used as substrates, resulting in more frequent on culture medium, followed by grape juice and, finally, wine. Specifically, the work is one of the first reports demonstrating the reciprocal interactions between non-Saccharomyces yeasts (NSY) and malolactic bacteria. The findings shed new light on the co-inoculation of the yeast starter culture with malolactic bacteria, as well as the biocontrol potential of Lactic Acid Bacteria (LAB) strains. Highlighted microbial interactions are relevant for the management of alcoholic fermentation, malolactic fermentation, and the development of distinctive aroma profiles, control of spoilage yeasts, and the selection of tailored mixed starter cultures. In addition, the plate assay method could be a fast, cheap, and suitable method to exclude negative interactions among Saccharomyces spp., NSY, and malolactic bacteria during trials from regional spontaneous fermentations with the aim to select tailored mixed starter cultures.

English

This study was aimed at evaluating the effects of aqueous extracts of Lentinula edodes (SHI), Pleurotus pulmonarius (PUL), and Pleurotus sajor-caju (PSC) on the growth of eight yeast species and the exo-enzyme production by Candida species after exposure to the extracts, which were prepared with the cold extraction methodology and tested against yeasts by the broth microdilution method. Proteinase and phospholipase were produced in vitro with the plate assay method using bovine serum albumin and egg yolk, respectively. The antifungal activity of the filtered mushroom extracts at different dilutions was confirmed against non-albicans Candida and Rhodotorula species. Exposing Candida isolates to extracts significantly reduced phospholipase production (p < 0.001). The antifungal activity against yeasts varied among different mushroom extracts. Moreover, SHI and PUL extracts may modulate the activity of phospholipase produced by Candida albicans. However, further studies are required to evaluate the inhibitory effect and its mechanism, as well as complementary studies with a higher number of samples tested. Key words: Mushrooms extracts, yeasts, Candida species, antifungal activity, proteinases, phospholipases.

Hydroxamate type of Siderophore production by rhizobacterial strains isolated from root nodules of Vigna trilobata

21 rhizobacterial strains were isolated from root nodules of Vigna trilobata (L-Verdc) plants raised in soils collected from geographically different areas in Andhra Pradesh, India. The identification of rhizobacteria was done by biochemical and by 16 S rDNA sequencing analysis along with accession numbers (Rhizobium sp. MRR 106, KC428655 and Rhizobium sp. MRR 123, KC503884). Detection of siderophore production by microorganisms in solid medium is the universal Chrome Azurol’ S (CAS). Agar plate assay method was used for this study. Out of 21 strains, only 7 showed the siderophore production. Of the 4 siderophore positive, two strains Rhizobium sp. MRR 106 and Rhizobium sp. MRR 123 are hydroxamate type and rest of them are catechol type. Siderophore production was observed at 24 h of incubation and it reached maximum at 144 h of incubation. Qualitative and quantitative estimation of hydroxamate type of siderophore production shows much difference along with iron concentrations up to 20 to 80 μM in Rhizobium sp. MRR 106 and Rhizobium sp. MRR 123. Various carbon and nitrogen sources greatly influenced the siderophore production. Among them, glucose (1%) and glycine (0.1%) were found to be the best for siderophore production.

Standardization of used engine oil concentration for analysis of biodegradation potential of Pleurotus sp. MP 5 MCC 1815 - A Scanning Electron Microscope based analysis

To broaden the understanding of mycoremediation mechanisms of white rot fungus Pleurotus Sp. MP 5MCC 1815, an attempt was made in the present work for standardization of used engine oil (UEO) foranalysis of its biodegradation potential. Standardization of UEO concentration proceeded via radial plateassay method which revealed that MP 5 displayed highest average colony growth rate (cm/day) thanPleurotus ostreatus MTCC 1804 (Positive test control) recorded as 5.9±0.1 cm/day (P0.05 under t-test) at 2% (v/v) UEO on 7th day) as compared to that of 0.5, 1, 1.5 and 2.5% UEO (P0.05 under one-way anova),therefore 2% UEO was standardized for further studies. SEM based analysis was later performed in orderto study the surface characterization of the degraded oil sample which indicated the surface changes occurredduring degradation in fungal treated oil sample and were compared to that of control.

Bioremediation Potential of Selected Rhizosphere Fungi of Tridax Procumbens Linn. and Chromolaena odorata (L.) R.M. King & H. Rob

Co-metabolism between plants and rhizosphere microbes is the mainstay of rhizoremediation of contaminated soils. The aim of the current study was to isolate and screen selected rhizosphere micro-fungi of two Asteraceae (Tridax procumbens and Chromolaena odorata) collected from the wild in University of Lagos, Akoka, Lagos State for bioremediation potential. Rhizosphere fungi were isolated, identified and evaluated for crude oil myco-remediation. The inhibitory effect of different concentrations of crude oil on mycelial growth of the most abundant fungi was determined with poisoned plate assay method. The most abundant and recurring fungi around C. odorata and T. procumbens were determined using the serial dilution and plating methods. Analysis of the rhizosphere soil of T. procumbens and C. odorata showed they were sandy loam type. C. odorata soil had higher moisture, organic carbon, and acid content than T. procumbens. Aspergillus flavus and Trichoderma harzianum were the most abundant and recurring fungi in C. odorata and T. procumbens, respectively. Thirty-nine micro-fungi belonging to twenty genera were isolated from the test plants’ rhizosphere. A. flavus and T. harzianum tolerated 2.50 to 10.00% crude oil contamination assessed with their mycelial growth inhibition reducing with time. A. flavus and T. harzianum caused a 68.45% and 86.71% reduction of crude oil contamination respectively, in a time dependent manner. The filamentous fungi ~A. flavus and T. harzianum can potentially be used to simultaneously ameliorate crude oil contaminated soils in conjunction with C. odorata and T. procumbens, respectively in the innovative technology termed rhizoremediation.