Vegetable oil Spills are becoming frequent and are potentially more challenging than petroleum hydrocarbon spills. Microbial lipases occupy a place of prominence among biocatalysts are often used for remediation of vegetable oil-polluted sites. This work was carried out to isolate microorganisms from oil-polluted sites and screen them for their lipolytic activity. Microorganisms were isolated from eight experimental soil samples contaminated with different types of vegetable oil, soil from an oil mill in Ibadan, and normal uncontaminated soil as a control. The isolates were characterized, identified and those common to at least one of the experimental sites and oil mill sites were screened for their lipolytic activity. Data obtained were analysed using Duncan Multiple Range Test. Seventy three microorganisms were isolated from the polluted soil and identified as species of Bacillus (16), Pseudomonas (12), Flavobacterium (6), Alcaligenes (2), Proteus (3), Micrococcus (1), Aspergillus (9), Penicillium (6), Saccharomyces (4), Geotrichum (1), Kluveromyces (1). Bacillus subtilis, Bacillus licheniformic, Pseudomonas cepacia, Pseudomonas fluorescens, Flavobacterium sp., Alcaligenes sp. and Candida parapsilosis which were common to at least one of the experimental site and oil mill site were preliminarily screened for lipolytic activity and all nine confirmed by presence of halos around the colonies. These screened organisms have potential for the degradation of fatty waste. They could therefore be employed in environmental clean-up of vegetable oil spill site.
Vegetable oils are oil extractions from plants and fruits such as palm nut, sunflower, soybean, coconut, rapeseed, canola, olive, castor and corn [
Like mineral oils, vegetable oils can vary significantly and when released to the marine environment for instance will behave differently according to their individual characteristics. These characteristics depend on factors at the time of cultivation of the feed stock for example, climate; degree of processing; type and specific nature of the oil, sea state and weather conditions at the time of the spill. In many cases, the influence of vegetable oil characteristics on its behaviour in the environment is not well-studied or understood. Consequently, the behavior and fate of specific vegetable oils is somewhat more difficult to predict than that of mineral oils.
Vegetable oils comprised primarily of triacylglycerols or fatty acids, which, in their fresh state, may be broken down by bacteria, fungi and yeast. This breakdown is as a result of lipases, a class of hydrolases primarily responsible for hydrolysis of acylglycerides. Many microorganisms such as bacteria, yeasts, molds and a few protozoa are known to secrete lipases for the digestion of lipid materials [
These organisms produce veritable development sources of lipases with different enzymological properties and specificities but molds are known to be more potent lipase producer [
Lipase producers have been isolated mainly from soil or spoiled food material that contains vegetable oil. Lipase production from a variety of bacteria, fungi and actinomycetes has been reported in several works [
Microorganisms are therefore useful hi protecting the environment. It is significant to isolate microbes of high potential for the biodegradation of vegetable oil. Therefore, this study was undertaken to isolate, identify, characterize and screen microorganisms from vegetable oil contaminated soil samples.
Soil samples were collected from a vegetable oil factory: oil mills, challenge area of 7˚21"N 3˚53"E Ibadan during the raining season; and soil from the nursery section of department of Botany university of Ibadan thoroughly mixed with eight different types of edible oil purchased at different retail markets in Ibadan metropolis.
Hundred milliliters and 200 mls of each of the oil samples were mixed thoroughly with 2 kg of the soil sample collected from the plant nursery of the Department of Botany. Two kilograms of the uncontaminated soil was collected also at the same site. These various samples were placed in bowls for 2 months.
Isolation of all the isolates was carried out using the method of [
Bacterial isolates were identified using morphological procedures and biochemical tests, with reference to Bergey’s Manual of Systematic Bacteriology [
The isolated fungi were identified according to their micro-morphology, as well as colour and morphology of sporulating structures, reference was made to compendium of soil fungi. The Yeasts were identified and confirmed using API 20 Aux kit.
Lipolytic activities of the test isolates were initially screened for on olive oil agar medium. The screening was carried out by a modified method of [
In another instance, the isolates were further confirmed for lipolytic activity using a medium containing, 15 g peptone, 0.l g Cacl2, 5 g NaCl all weighed and dissolved in sequence by addition of 800 ml of water and the pH adjusted to 7.5 with NaOH. Thereafter, 15 g of agar was added. The volume was made up to 1 liter. The mixture was sterilized by autoclaving for 15 minutes at 121˚C. One percent (V/V) high refined olive oil purified by membrane filtration and 5 g Aniline blue dissolved in distilled water and filter-sterilized.
Thirty-four bacterial isolates were obtained from the soil sample in the bowls contaminated with different types of edible oil. Thirty fungal isolates and yeasts inclusive were also obtained, as shown on
Isolate Code/Source of Isolation | Organisms Isolated |
---|---|
GN1 (Groundnut oil) | Bacillus licheniformis |
GN2 | Bacillus subtilis |
GN3 | Bacillus pumilus |
GN4 | Flavobacterium sp. |
GN5 | Micrococcus luteus |
OO1 (Olive oil) | Bacillus licheniformis |
OO2 | Flavobacterium sp. |
OO3 | Alcaligenes sp. |
OO4 | Pseudomonas putida |
OO5 | Bacillus alvei |
OO6 | Pseudomonas fluorescens |
OO7 | Bacillus megaterium |
OO8 | Pseudomonas cepacia |
CS1 (Cottonseed oil) | Bacillus alvei |
CS2 | Bacillus licheniformis |
CS3 | Pseudomonas putida |
CS4 | Flavobacterium sp. |
SF1 (Sunflower oil) | Flavobacterium sp. |
SF2 | Pseudomonas putida |
SF3 | Pseudomonas cepacia |
SF4 | Bacillus alvei |
PO1 (Palm oil) | Bacillus alvei |
PO2 | Proteus mirabilis |
PO3 | Bacillus subtilis |
SO1 (Soya oil) | Pseudomonas putida |
SO2 | Pseudomonas flurescense |
SO3 | Pseudomonas fluorescens |
PK1 (Palmkernel oil) | Flavobacterium sp. |
PK2 | Proteus vulgaris |
PK3 | Pseudomonas putida |
PK4 | Bacillus licheniformis |
VO1 Vegetable oil (king) | Bacillus licheniformis |
VO2 | Bacillus subtilis |
VO3 | Pseudomonas putida |
Isolate Code/Source of Isolation | Organisms Isolated |
---|---|
GN1 (Groundnut oil) | Saccharomyces cerevisae |
GN2 | Mucor meihei |
GN3 | Aspergillus niger |
OO1 (Olive oil) | Penicillium expansum |
OO2 | Aspergillus niger |
OO3 | Rhizopus oryzae |
CS1 (Cottonseed oil) | Mucor racemosus |
CS2 | Saccharomyces rouxii |
CS3 | Aspergillus terreus |
CS4 | Penicillium funiculosum |
SF1 (Sunflower oil) | Saccharomyces rouxii |
SF2 | Aspergillus niger |
SF3 | Penicillium herquei |
PO1 (Palm oil) | Candida valida |
PO2 | Mucor racemosus |
PO3 | Candida parapsilosis |
SO1 (Soya oil) | Aspergillus niger |
SO2 | Penicillium herquei |
SO3 | Rhizopus oryzae |
SO4 | Saccharomyces rouxii |
SO5 | Streptomyces nitrosporeus |
PK1 (Palmkernel oil) | Penicillium expansum |
PK2 | Aspergillus terreus |
PK3 | Kluyveromyces africanus |
PK4 | Aspergillus niger |
PK5 | Streptomyces nitrosporeus |
VO1 (Vegetable oil) | Aspergillus niger |
VO2 | Mucor meihei |
VO3 | Aspergillus terreus |
VO4 | Aspergillus niger |
Type of soil/Frequency of occurrence | ||
---|---|---|
Isolate Name | Artificially contaminated soil | Contaminated soil from oil mill site |
Bacillus licheniformis | 5 (14.7) | 1 (11.1) |
Bacillus subtilis | 3 (8.8) | 2 (22.2) |
Bacillus pumilus | 1 (2.9) | - - |
Flavobacterium sp. | 5 (14.7) | 1 (11.1) |
Micrococcus luteus | 1 (2.9) | - - |
Alcaligenssp | 2 (5.9) | 1 (11.1) |
Pseudomonas putida | 5 (14.7) | - - |
Bacillus alvei | 4 (11.8) | - - |
Pseudomonas fluorescens | 2 (5.9) | 2 (22.2) |
Bacillus megaterium | 1 (2.9) | - - |
Pseudomonas cepacia | 2 (5.9) | 2 (22.2) |
Proteus mirabilis | 1 (2.9) | - - |
Proteus vulgaris | 2 (5.9) | - - |
Total | 34 (100) | 9 (100) |
Values in parentheses represent percentage of occurrence.
Type of soil/Frequency of occurrence | ||
---|---|---|
Isolate | Artificially-contaminated | Contaminated soil from oil mill site |
Saccharomyces cerevisae | 1 (3.3) | - |
Aspergillus niger | 7 (23.3) | - |
Penicillium expansum | 2 (6.7) | - |
Rhizopus oryzae | 2 (6.7) | - |
Aspergillus terreus | 3 (6.7) | - |
Penicillium funiculosum | 1 (3.3) | - |
Penicillium herquei | 2 (6.7) | - |
Penicillium atrovenetum | - | 1 (33.3) |
Mucor meihei | 2 (6.7) | - |
Mucor racemosus | 2 (6.7) | - |
Saccharomyces rouxii | 3 (10) | - |
Candida valida | 1 (3.3) | - |
Candida parapsilosis | 1 (3.3) | 1 (33.3) |
Streptomyces nitrosporeus | 2 (6.7) | - |
Kluyveromyces africanus | 1 (3.3) | - |
Geotrichum candidum | - | 1 (33.3) |
Total | 30 (100) | 3 (100) |
Values in parentheses represent percentage of occurrence.
Several researchers have isolated various microbial species from oil contaminated soils [
Different screening strategies have been proposed for the determination of lipase activity, assays using agar plates are highly recommended, because it is an easier method with lower cost [
For this study only isolates common to both purposely contaminated soil and oil mill soil were screened for lipase production.
Sample | Microbial group/volume of edible oil/count (log10cfu/ml) | |||
---|---|---|---|---|
Heterotrophic bacterial count | Heterotrophic fungal count | |||
Description | 100 ml | 200 ml | 100 ml | 200 ml |
Groundnut oil | 4.491g | 3.8451f | 4.6812c | 3.0000g |
Olive oil | 5.2021d | 5.0792b | 3.7782e | 3.3010e |
Soya oil | 5.1761e | 0.0000h | 4.6021d | 3.2788e |
Palm kernel oil | 6.3222a | 6.1461a | 4.2788e | 4.1461b |
Palm oil | 4.5798f | 4.2305e | 4.7324b | 4.2305a |
Cottonseed oil | 5.9638b | 4.8692c | 4.8633a | 3.6021d |
Vegetable oil | 5.3424c | 4.3802d | 4.8865a | 4.0000c |
Sunflower oil | 5.3424c | 2.0000g | 3.7782e | 3.3010e |
Values with different superscripts are significantly different (P ≤ 0.05) with respect to Duncan’s Multiple Range Test.
Microbial group | Total count (log10cfu/ml) |
---|---|
Bacteria | 5.44 |
Fungi | 5.74 |
Isolates | Lipolytic activity |
---|---|
Bacillus subtilis | + |
Pseudomonas cepacia | + |
Bacillus licheniformis | + |
Pseudomonas fluorescens | + |
Alcaligenes sp. | + |
Flavobacterium sp. | + |
Candida parapsilosis | + |
Key: + Positive Presence of halos around the colonies.
The microbial isolates in this study proved capable of lipase production. Indeed, lipolyticactivity has been observed for pure cultures of all the microbial genera isolated in this study [
The formation of opaque zones around the colonies of the selected isolates is an indication of lipase production by the organisms [
This study revealed that 73 microorganisms in all belonging to different genera were isolated from different sources and all proven to be capable of lipase production. Hence this gives opportunity to explore or investigate these microbial lipases which are an important group of biotechnological valuable enzymes and could be better explored for fatty waste degradation.
Popoola, B.M. and Onilude, A.A. (2017) Microorganisms As- sociated with Vegetable Oil Polluted Soil. Advances in Microbiology, 7, 377-386. https://doi.org/10.4236/aim.2017.75031