Oil Agar Research Articles

MOLECULAR CHARACTERIZATION AND LIPOLYTIC ACTIVITY OF YARROWIA LIPOLYTICA ISOLATED FROM YOGHURT CREAM

Objective: Yarrowia lipolytica is an aerobic dimorphic yeast that produces various metabolites such as enzymes, organic acids, proteins, surfactants and has industrial potential in some biotransformation and bioremediation reactions. To be a preliminary study to industrial productions, in present study it was aimed to isolate Y. lipolytica strain from yoghurt cream, perform its molecular characterization and investigate the lipolytic activity. Material and Method: A total of 10 samples were taken from the homemade yoghurt cream obtained by fermented milk from local dairy producers in Ankara. Yeast-Peptone-Glycerol (YPG) broth and Yarrowia lipolytica Distinctive (YLD) agar media were used for isolation of the species. Species-level identification was carried out by ITS-Polymerized chain reaction analysis. Lipolytic activity was determined with Rhodamine-B/Olive Oil Agar nutrient medium procedure. Result and Discussion: Yeast colonies were isolated from homemade yoghurt creams by growing in YPG broth. Yeasts were stained with Gram staining method and Gram-positive stained ones were inoculated on YLD agar. At the end of the incubation period, the brown pigmented colony growing on YLD agar was selected as Yarrowia species. According to the molecular characterization results the brown pigmented colony was identified as Yarrowia lipolytica with a total of 358 bases, 100% sequence matching ratio and 100% similarity ratio and the strain was determined as lipase positive. The development of the Y. lipolytica strain isolated in our study as a producer culture that can be used in industrial production should be supported by further studies to benefit from its various properties.

Bio-remedial Effect of Specific Micro-organisms (Bacteria) on Used Engine Oil: A Case Study of Some Mechanic Workshops in Maiduguri Metropolitan Council (MMC)

The potential of three micro-organisms (Pseudomonas, Streptococcus and Bacillus sp) were isolated from hydrocarbon contaminated soil and were evaluated for their biodegradation ability. The rate of biodegradation of the engine oil in the soil samples were exposed to used engine oil with different exposure rates of 5,10,15 and 20 years were studied for a period of three (3) weeks under greenhouse experiment. The soil samples were obtained from four different mechanic workshops in M.M.C and they were plated on nutrients agar and oil agar medium to isolate the bacterial species from the spilled soil samples. All the micro-organisms used in this study showed their abilities to remediate soil exposed to used engine oil and the remediated soil samples were able to support the growth of Maize ( Zea mays) after 10 years effective growth

Characterization of moulds associated with spoilage of bread soldin Ilaro, Yewa-South, Nigeria

Bread is an important staple food; however, it is highly susceptible to microbial spoilage due to postproduction contamination and improper storage. This study aimed at determining moulds associated with bread spoilage and their hydrolytic activities. Fifteen (15) brands of bread were sampled and moulds that are responsible for their spoilage were isolated using spread plate technique. The isolates were identified on the basis of colonial and microscopic characteristics. Characterization of the moulds was on the basis of their hydrolytic activities. The mould counts of the bread ranged from 1.0 x 104 cfu/g to 7.7 x 106 cfu/g. The highest count was recorded in sample JB and the least count was in sample PB. The moulds associated with spoilage of the bread samples were; Aspergillus niger, Aspergillus flavus, Mucor sp., Rhizopus sp., and Penicillium sp. The predominant organism was Aspergillus niger. The moulds showed varying degrees of hydrolytic activities on milk, starch and olive oil agar media. On the basis of their hydrolytic activities, all the moulds played vital roles in the spoilage of bread. Bread, a ready-to-eat food could be a source of food-related illnesses due to moulds associated with its spoilage. Keywords: Bread, spoilage, hydrolytic activities illnesses.

ISOLATION AND CHARACTERIZATION OF OIL UTILIZING BACTERIA ISOLATED FROM DRILLING OILY BASE MUD WASTE IN THE EGYPTIAN WESTERN DESERT

The main aim of this study is to isolate, screen and identify the most potent oil degrading bacteria (ODB) isolated from oil base mud (OBM) waste resulted from group of selected oil and gas drilling and exploration wells. Another aim is to determine the metabolic fingerprint of the most potent ODB. To achieve these aims, eleven samples from the waste of oily base mud were collected from different drilling sites at western desert. Total bacterial counts at 22 oC and 30 oC were determined for giving a full picture about the presence of natural bacterial flora in the collected samples. Moreover, ODB were determined on oil agar media containing 0.1% oil (V/V) using spread plate method. The results showed that, the average of total bacterial counts at 22 oC and 30 oC were 1.4x104 and 1.6x104 CFU/g, respectively. Moreover, the oil degrading bacteria (ODB) counts were fluctuated between 1.0×102 and 6.1x103 CFU/g in all collected samples. After six screening steps, the most potent oil degrading bacteria were identified using BIOLOG as follows Enterobacter hormaechei, Enterobacter cloacae and Bacillus subtilis. The only difference in the metabolic fingerprints view between two bacterial strains that, Enterobacter hormaechei (H17) are able to utilize D-serine while and Enterobacter cloacae (H32) not. It can be concluded that, metabolic fingerprints of the most potent oil degrading bacteria provide full information about the microbial metabolic activities that may be helpful in determination of favorable conditions which aid in biodegradation experiments.

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF LIPASE FROM BACILLUS SP. FROM KITCHEN GREASE

Objective: The objective of this work was to isolation, purification and characterization of solvent tolerant lipase from Bacillus sp. The objective of this work was to isolation, purification and characterization of solvent tolerant lipase from Bacillus sp. from kitchen grease for a variety of applications including organic synthetic reactions and preparation of enantiomerically pure pharmaceuticals.Methods: Lipase producing isolates were screened from kitchen grease on a selective medium rhodamine B olive oil agar, and tributyrin agar was used to screen the lipase and esterase producing an organism, respectively. The isolate identified using 16S rDNA sequencing method and enzyme activity was quantitatively assayed. Lipase production was characterized in different conditions.Results: The isolate showed highest lipase activity was which later was identified as Bacillus sp. using 16S rDNA sequencing method. The lipase was purified using ammonium sulfate precipitation. The isolate showed excellent tolerance to methanol, ethanol, acetonitrile, and moderate tolerance to butanol. The increased biomass concentration, maximum production, and activity were achieved at 37°C in 24 h incubation, then gradual reduction in production was observed. The maximum activity of lipase enzyme was obtained at pH between 6 and 9.Conclusion: The isolate produce solvent tolerance lipase enzyme and it can be a promising candidate of solvent tolerance lipase enzyme for variety of industrial applications.

Hydrocarbonoclastic potentials of Enterobacteriaceae isolated from the crude oil polluted Iko river estuary and freshwater ecosystem of the Niger Delta Region of Nigeria

Hydrocarbonoclastic potentials of Enterobacteriaceae isolated from the crude oil polluted Iko river estuary and freshwater ecosystem of the Niger Delta was investigated. The isolation of crude oil utilizing bacterial isolates in water sample was carried out by surface spreading technique using diluents prepared with quarter strength Ringers solution and cultured on oil agar medium (OAM). Of the 32 potential hydrocarbon utilizing bacteria from crude oil impacted aquatic ecosystem only eight isolates identified as; Citrobacter amalonaticus–Y1 (FSW); Proteus mirabilis strain–I(FSW), Pseudomonas fluorescens–N (FSE); Citrobacter farmeri–Y12 (FSE), Citrobacter amalonaticus strain–Y2 (ESWS1), Enterobacter sp.–Y8 (ESWS3), Proteus mirabilis strain–K (ESWS1), Proteus penneri strain–O(ESES3) were found to demonstrate strong hydrocarbonoclastic potentials with variable levels of low pH and increase optical density and free carbon iv oxide production. The study revealed that Citrobacter amalonaticus strain–Y2 (ESWS1) of the family Enterobacteriaceae is a good candidate for bioaugmentation technique of bioremediation.

The Abilities of Four Species of Nigerian Aquatic Phycomycetes to Utilize Petroleum and Petroleum Products as Sole Carbon Sources

Studies were carried out on the abilities of four aquatic phycomycetes species isolated from crude oil polluted marine , brackish and fresh water environments in Nigeria to utilize refined Petroleum and Petroleum products as sole sources of carbon. The said species of fungi included Brevilegnia indica, Protoachyla paradoxa, Saprolegnia bhargavi and Thraustotheca clavata. They were grown on fungal culture media which contained mineral salts solution, refined petroleum, kerosene and diesel as sole sources of carbon and agar as a solidifying agent. Two concentrations of each of the resulting oil agar media were used vis 1% and 2%. The resultant medium was then used to culture each of the test fungi in triplicates. The resultant culture plates were then incubated at 25 OC and left for daily observation. The test fungi that grew on each medium were observed for their abilities to emulsify the refined petroleum or its products (diesel and kerosene). Control experiments were also set up using Malt Extract Agar medium. Brevilegnia indica grew on 1% petroleum and diesel growth media but did not grow on kerosene medium. Protoachyla paradoxa grew minimally on diesel medium at 1% and 2% compositions. Saprolegnia bhargavi did not grow on the petroleum agar medium or petroleum products media. Thraustotheca clavata grew minimally on the kerosene medium at both 1% and 2% compositions. The four test fungi emulsified the diesel agar medium at both 1% and 2% compositions. Brevilegnia indica emulsified only petrol at 1% while Non of the isolates emulsified the kerosene medium at both 1% and 2% compositions. Oil globules were also observed in the vegetative hyphae of the test fungi which grew on the experimental culture media. This means that such fungi either degraded or accumulated the petrol or petroleum products in their systems.

Potential in heavy oil biodegradation via enrichment of spore forming bacterial consortia

It is quite challenging to recover heavy crude oil due to its high viscosity and long chain hydrocarbon contents. In this study the isolation of thermophilic bacterial consortia from crude oil contaminated soil samples and their ability to degrade heavy crude oil were investigated. Thermophilic spore forming bacteria were inoculated with heavy oil in minimal salt media. Maximum growth was observed on 21st day of incubation at 40 °C. Samples were analyzed for biosurfactant production, where a substantial decrease in surface tension and interfacial tension was not observed. A well-assay was used to check the potential of bacterial consortia for oil-clearing zones on oil agar plate with bacterial cultures and their cell-free filtrates. 16S rRNA sequencing of 27 isolates from oil contaminated soil showed that isolates belonged to three classes: Alpha-proteobacteria, gamma-proteobacteria and Bacilli. Scanning electron microscopy showed that the oil utilizing bacteria were rod-shaped with rough surface and fimbria. Gas chromatography analysis of treated oil after 21 days showed that bacterial consortia efficiently degraded heavy crude oil to light hydrocarbons ranging from C11–C27, from initial C37+ carbon chain of untreated controls. These bacterial consortia showed potential in heavy crude oil biodegradation by breaking it down to smaller hydrocarbon composition.

English

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Oil spilled soil samples collected from Dharmapuri and Salem districts were screened for a novel lipase producing bacteria by screening in Tributyrin agar plate and Rhodamine olive oil agar and lipase screening liquid medium. Among the two hundred isolates thirty two isolates exhibited high lipolytic activity on TBA plate assay and twenty isolate exhibited high lipolytic activity on ROA plate assay The isolate BLP 141 produces lipase more than 12 U/ml in the further lipase screening liquid medium was selected as a novel lipase producing bacteria and identified by morphological and biochemical characteristics as Pseudomonas. The 16S rRNA gene amplified by bacterial universal primer 27F and 1492R yielded 1478 bp was analysed by BLAST confirmed it as Pseudomonas gessardii and was submitted in GenBank with accession number KJ547711.

Isolation and Characterization of Petroleum Hydrocarbon Degrading Bacteria from the Bohai Sea, China

Fourteen petroleum hydrocarbon degrading bacteria strains were isolated from oil-contaminated site. Isolated strains were able use diesel oil as sole carbon and energy source. Bacterial strain HD1 was selected due to the luxuriant growth on oil agar. The oil degradation rate of strain HD1 was analyzed using UV-spectrometry-based methods. The result showed that the rate of diesel oil degradation of 75% was observed after 14days of cultivation.

Biodegradation of spent engine oil by bacteria isolated from the rhizosphere of legumes grown in contaminated soil

Biodegradation of spent engine oil (SEO) by bacteria isolated from the rhizosphere of Cajan cajan and Lablab purpureus was investigated. It was with a view to determining most efficient bacterial species that could degrade SEO in phytoremediation studies. Hydrocarbon degrading bacteria were isolated and identified by enrichment culture technique using oil agar supplemented with 0.1% v/v SEO. Total heterotrophic and oil utilizing bacterial count showed the occurrence of large number of bacteria predominantly in the rhizosphere soil, ranging between 54×108 - 144×108 CFU/g and 4×108- 96×108 CFU/g respectively. Percentage of oil utilizing bacteria ranged between 0% (uncontaminated non rhizosphere soil) to 76% (contaminated rhizosphere). Turbidimetrically, five bacterial species namely Pseudomonas putrefacience CR33, Klebsiella pneumonia CR23, Pseudomonas alcaligenes LR14, Klebsiella aerogenes CR21, and Bacillus coagulans CR31 were shown to grow maximally and degraded the oil at the rate of 68%, 62%, 59%, 58%and 45% respectively. Chromatographic analysis using GC-MS showed the presence of lower molecular weight hydrocarbons in the residual oil (indicating degradation) after 21 days, whereas the undegraded oil (control) had higher molecular weight hydrocarbons after the same period. The species isolated were shown to have high ability of SEO biodegradation and therefore could be important tools in ameliorating SEO contaminated soil. DOI: http://dx.doi.org/10.3126/ije.v3i2.10515 International Journal of the Environment Vol.3(2) 2014: 63-75

Occurrence of hydrocarbon degrading bacteria in soil in Kukawa, Borno State

Soil samples were collected from five sites covering petroleum exploration station in Kukawa, Kukawa Local Government Area of Borno State, Nigeria between October, 2012 and February, 2013 at two different depths (0-10cm and 10-20cm) to enumerate and identify hydrocarbon degrading bacteria in the soil. Total aerobic heterotrophic bacteria (TAHB) were enumerated on Nutrient agar (NA), and Hydrocarbon utilizing bacteria (HUB) enumerated on Oil agar (OA). The bacterial isolates were identified using morphological and biochemical tests. It was observed that the microorganisms (TAHB, and HUB) were more densely populated at 10cm depth. (TAHB: 5.3×108 - 11.4×108cfu/g, and HUB: 2.4×105 - 5.3×105 cfu/g, than at 20 cm depth (TAHB: 3.0×108 - 5.7×108 cfu/g, and HUB: 2.1×105 - 4.8×105 cfu/g). The HUB was identified as species of Bacillus, Pseudomonas, Klebsiella, Lactobacillus, Micrococcus, Corynebacterium, and Actinomyces. Bacillus, and Pseudomonas species were more constantly isolated than other isolates and they constitute 100% of total bacterial isolates. The potential of hydrocarbon utilizing bacteria isolated to degrade hydrocarbon was studied. Nineteen (19) bacterial species was screened, Bacillus subtilis, Pseudomonas aeruginosa, Bacillus cereus, Klebsiella pneumoniae, Micrococcus leteus,and Lactobacillus casei, utilized and degrade crude oil at considerably high rates after 21 days of incubation. The degradation efficiency was confirmed by GC-MS analysis, which indicated that the bacterial isolates utilized most of the crude oil components particularly straight chain alkanes and cycloalkanes DOI: http://dx.doi.org/10.3126/ije.v3i2.10503 International Journal of the Environment Vol.3(2) 2014: 36-47

Psychrotrophic Lipase Producers from Arctic Soil and Sediment Samples

Culturable microorganisms were successfully isolated from soil and sediment samples collected in 2011 on the northern coast of Hornsund, West Spitsbergen. A total of 63 single colony isolates from three sampling sites obtained were subjected to temperature dependence study to assess whether they are obligate psychrophilic or psychrotrophic strains. From initial temperature screening, only 53 psychrotrophic isolates were selected that are capable of growing between 4-28 degrees C. The rest that were capable of tolerating higher temperatures up to 37 degrees C were not included in this study. These isolates were chosen for lipase enzyme screening confirmation with the standard plate assay of olive oil and fluorescent dye Rhodamine B. Six lipase positive isolates were also subjected for subsequent lipase enzyme plate screening on tributyrin, triolein, olive oil and palm oil agar. Lipase production by these six isolates was further assayed by using colorimetric method with palm oil and olive oil as the substrate. These isolates with promising lipase activity ranging from 20 U/ml up to 160 U/ml on palm oil and olive oil substrate were successfully identified. Molecular identification by using 16S rRNA revealed that five out of six isolates were Gram-negative Proteobacteria and the other one was a Gram-positive Actinobacteria.

Screening of microbes for lipases specific for saturated medium and long-chain fatty acids of milk fat

Over 250 microbial strains were screened for lipase activity specific towards saturated medium and long-chain fatty acids. Strains showing trimyristin (C14:0) hydrolysis on agar medium were further streaked on tricaprylin (C8:0) and on olive oil (mostly C18:1) agar to identify strains hydrolysing C12–C15 fatty acids with modest or no activity towards short-chain and unsaturated fatty acids. The twenty strains showing the desired specificity were grown as liquid cultures and analysed for p-nitrophenyl (p-NP) myristate hydrolysis in the presence of Triton X-100. Cells from the cultures showing the highest activity towards p-NP myristate were analysed for specificity towards p-NP esters of different fatty acid chain lengths. On the basis of the p-NP ester profiling, four lipase samples were selected for hydrolysis experiments on butter oil. Of these, the sample obtained from the cultivation of Didpodascus capitatus met the screening criteria: C12:0–C16:0 specificity and no preference towards C18:0 and C18:1 fatty acids.

Production and partial characterization of biosurfactant produced by crude oil degrading bacteria

Production of biosurfactant by crude oil degrading bacteria for use in microbial enhanced oil recovery was investigated. Crude oil utilizing bacteria were isolated from soil by enrichment method on oil agar at 30 °C for 5 days. The isolates were identified and screened for biosurfactant production using blood haemolysis and emulsification tests. IR and GC–MS analyses were carried out to detect the type of biosurfactant. The biosurfactant was purified and its stability at various pH, temperature and salinity levels was studied. The organisms were identified as: Achromobacter xylosoxidans subspecies xylosoxidans, Bacillus licheniformis, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Sphingomonas paucimobilis and Micrococcus kristinae. Emulsification test (E24) revealed that Serratia marcescens had the highest emulsification index of 87%. GC–MS indicated the biosurfactants as lipopeptides. The biosurfactant can be used in EOR under various environmental conditions.

Identification of lipase producing thermophilic bacteria from Malaysian hot springs

Lipase enzyme is an important group of biocatalyst for biotechnological application. One of the major industrial microorganism is the Bacillus species which are in abundance in the hot springs. The aim of this research was to isolate and characterize lipase producing bacteria from five hot springs in Malaysia. Eight bacterial strains from five hot springs were characterized by both biochemical and molecular techniques. Four strains were lipase producing thermophilic bacteria (coded as A1, A3, A4 and A14). The presence of lipase enzyme was confirmed by an orange fluorescence halos around the bacteria in rhodamine B olive oil agar plate. Based on molecular analysis, A1, A3 and A4 were assigned to the Bacillus and Geobacillus sp., and isolate A14 is found to be a nucleoside permease gene from Anoxybacillus flavithermus WK1.

Identification Of Lipase – Producing Psychrophilic Yeast, Leucosporidium Sp.

Cold-active enzymes have recently received great attention due to their potential applications in a broad range of industrial, agricultural and medical processes. One of the enzymes is lipase (triacyglycerol acylhydrolases E.C 3.1.1.3) which is unique in catalyzing the hydrolysis of triacylglycerols into free fatty acids and glycerol. In this particular research, an obligate psychrophilic microorganism was isolated from Casey Station, Antarctica. The growth of this microorganism has been tested at different temperatures, 4C, 27C and 37C. At 4C, the microorganism was able to grow whereas at 27C and 37C, there was no growth at all. The presence of lipase enzyme in this microorganism was detected by halo zone on palm oil (substrate) agar plates. Identification of this microorganism was done based on its morphological, biochemical and molecular characteristics. For the morphology analysis, two types of microscopy observation were carried out: phase contrast microscopy and Scanning Electron Microscopy (SEM). Both observations showed budding structures. This suggested that this particular microorganism is psychrophilic yeast. Biochemical tests were done based on its capability to ferment and assimilate sugar. In addition, assimilation of nitrate was also tested. In molecular approach, the genomic DNA (gDNA) of this microorganism was successfully extracted and the extracted gDNA was used for amplification via polymerase chain reaction (PCR) technique using Internal Transcribed Spacer (ITS) primers. The PCR product obtained was sequenced and submitted for Basic Local Alignment Search Tool (BLAST) at National Center for Biotechnological Information (NCBI). The analysis showed that this microorganism contained ITS sequences (highest identity with Leocosporidium sp.).

A rapid and efficient method for directed screening of lipase-producing Burkholderia cepacia complex strains with organic solvent tolerance from rhizosphere

Lipase from Burkholderia cepacia strain is one of the most versatile biocatalysts and is used widely in many biotechnological application fields including detergent additives, the resolution of racemic compounds, etc. Based on the known whole genomic information of B. cepacia strain, both ampicillin and kanamycin were added to the TB-T medium to screen B. cepacia complex stains from rhizosphere soil samples. The selected colonies from the modified TB-T medium were then qualitatively determined the ability to produce extracellular lipase on the rhodamine B-olive oil agar plates. A total of 35 lipolytic pseudo-B. cepacia complex strains were isolated and the positive rate of lipolytic bacteria was 65%. Among them, 15 pseudo-B. cepacia complex strains showed tolerance to benzene, n-hexane and n-heptane at concentration of 10% (V/V) and were identified by the recA gene sequence. All of the 14 lipolytic bacteria were identified as B. cepacia complex strains except that the recA gene sequence of one lipolytic bacterium, strain ZMB009, was not obtained.

Instability of petroleum oil degradation by induction of mutation in 45 kb and 60 kb plasmids

A strain of Pseudomonas sp. was isolated from oil‐contaminated soil on petroleum oil agar. This isolate used different alkenes and had two plasmids on gel agarose. These plasmids are probably involved in oil degradation. The mutant strain, which were obtained by sub‐culturing in nutrient broth (oil−), lost one plasmid (60 Kb) and had deletion in another plasmid (45 Kb). This result suggests that the ability to degrade petroleum oil must depend on these plasmids. The wild isolate has maximum OD of 2.5 for growth on petroleum oil, however the mutant did not have any significant growth on petroleum. GC Profiles of petroleum oil after degradation by wild and mutant were investigated.

Oil as a substrate for Mortierella species

Sunflower oil poured onto compost in November induced massive growth of Mortierella species. Mortierella hyalina was isolated into culture at 4 or 18 °C on weak media but not at 23–25 °C or on 2% malt agar. It grew well on olive oil agar, with its hyphae aggregating inside oil drops. The nature of nutrients utilized in normal soils and composts by Mortierella is not known but lipid levels there would appear to be low. Many Mortierella species are able to convert diverse carbon sources into lipids and are thus known as oleaginous fungi. Their biotechnological applications are briefly discussed.