The overwhelming demand of oil and fats to meet the ever increasing needs for biofuel, cosmetics production, and other industrial purposes has enhanced a number of innovations in this industry. One such innovation is the use of microorganisms as alternative sources of oil and fats. Organic solid waste that is causing a big challenge of disposal worldwide is biodegradable and can be utilized as substrate for alternative oil production. The study evaluated the potential of isolated yeast-like colonies to grow and accumulate oil by using organic solid waste as substrate. Of the 25 yeast-like colonies isolated from the soil samples collected from three different suburbs in Kampala district, Uganda, 20 were screened positive for accumulation of lipid but only 2 were oleaginous. The NHC isolate with the best oil accumulation potential of 48.8% was used in the central composite design (CCD) experiments. The CCD experimental results revealed a maximum oil yield of 61.5% from 1.25 g/L cell biomass at 10 g/L of solid waste and temperature of 25°C. The study revealed that organic solid waste could be used as a substrate for microbial oil production.
Vegetable oils are the most traded, with palm oil being the most preferred due to its wide application in food, cosmetics, and biofuel industries. As the high demand of vegetable oil is being met, on the other hand, there is depletion of natural resources, a rise in poor cultivation practices, and eventually altered global climatic conditions that pose a threat to food security [
In Kampala, the capital city of Uganda, approximately 28,000 tons of solid waste are collected monthly, of which about 90% are organic [
Seven soil samples were collected from wastes dumping sites in Kyambogo University, Banda, and Kireka, suburbs within Kampala District, Uganda. The samples were obtained approximately 2–8 cm below the soil surface and were stored in sterile transparent polythene bags at room temperature prior to transfer to the laboratory.
The yeast cells were isolated on potato dextrose agar (PDA) (Conda, Madrid, Spain) plates which contained dextrose 20.0 g/L, infusion from potato 200.0 g/L, bacteriological agar 15.0 g/L, and 50.0 mg chloramphenicol supplement. The supplement was initially dissolved in 3.0 mL of absolute ethanol prior to being added to the medium base. The soil samples (5.0 g) were suspended in 9.0 mL of sterilized deionized water. The suspensions were thoroughly mixed for one minute at 2600 rpm using a vortex mixer (Stuart, Florida, USA) and subsequently followed by a tenfold serial dilution using sterile-buffered peptone water broth (Conda, Madrid, Spain). From each sample suspension, a dilution from 10−4 to 10−7 was chosen and an aliquot of 0.3 mL from each was spread onto PDA. The plates were incubated (ESCO Isothermal Incubator, Singapore) at 30°C for a period of 3 days. Yeast-like colonies were isolated and subsequently cross-streaked onto fresh PDA plates.
A loop full of each isolate suspension was inoculated into 100 mL nitrogen limiting medium contained in 250 Erlenmeyer flask. The medium contained the following in g/L: glucose 70, (NH4)2SO4 0.1, KH2PO4 0.4, MgSO4·7H2O 1.5, ZnSO4 0.0043, CaCl2 0.003, MnCl2 0.0012, CuSO4 0.0005, and yeast extract 0.795. The samples were incubated in an incubator shaker (Excella E25, New Brunswick Scientific, USA) at 150 rpm and 30°C for 4 days. The samples were then analyzed for their cell dry weight and percentage lipid content as described by Leasing [
The oil was structurally characterized by a Fourier transform infrared spectrophotometer (FTIR) (Perkin Elmer, Massachusetts, USA) as described by Wacoo et al. [
The solid waste for the production of microbial oil was collected without sorting waste disposal sites of a market, a residential place, and a restaurant. The waste was sorted and the organic solid waste was sun-dried and subsequently ground into a powder prior to being used in the production experiment. The powder waste was used as a substitute of glucose in the nitrogen-limiting medium mentioned in Section
The sorted waste was first hydrolyzed using concentrated sulphuric acid followed by a method described by Saeman et al. [
Central composite design was used to efficiently identify the optimum values of the temperature and solid waste which could lead to high oil accumulation. A two-factor five-level central composite design was therefore used to study the effect of solid waste and temperature on the oil yield of the selected yeast. Thirteen experiments were sufficiently used to estimate the second-order regression coefficients for the two variables as shown in (
Levels of independent variables.
Independent variables | Lower level | Upper level |
---|---|---|
Solid waste (g) | 5 | 10 |
Temperature (°C) | 25 | 35 |
In the current study, 25 oleaginous microorganisms with yeast-like colonies were isolated on PDA containing chloramphenicol antibiotics (Table
Isolation and screening for lipid-producing yeasts.
Sampling place | Isolate | Cell dry weight (g/L) | Lipid accumulation (%) |
---|---|---|---|
Kyambogo University | GC2A | 2.94 | 5.8 |
GC2B | 7.0 | 3.3 | |
GC2C | 2.23 | 3.6 | |
GC2D | 7.03 | 8.1 | |
GC2E | 2.57 | 2.7 | |
GC2F | 2.66 | 0.4 | |
GC2G | 3.21 | 3.4 | |
GC2H | 1.64 | 7.3 | |
GC2J | 2.24 | 8.0 | |
GC2K | 3.58 | 22.6 | |
GC2M | 2.18 | 11.5 | |
GC2N | 9.49 | 0.4 | |
GC1A | 4.85 | 0.0 | |
NHA | 5.80 | 0.0 | |
NHB | 3.57 | 0.0 | |
NHC | 1.27 | 48.8 | |
NHD | 5.11 | 6.3 | |
NHE | 5.40 | 11.1 | |
NHF | 6.04 | 4.8 | |
NHG | 2.98 | 7.0 | |
NHH | 6.69 | 0.0 | |
NHJ | 1.57 | 0.0 | |
NHK | 4.77 | 11.9 | |
| |||
Banda | BCCA | 7.21 | 5.5 |
BCCB | 2.22 | 16.2 | |
| |||
Kireka | KA | 2.41 | 11.6 |
All the results are means ± standard deviation of triplicate analysis.
All the isolated yeast-like colonies were screened for accumulation of lipid as described in Section
The microbial oil from NHC isolate was characterized as described in Section
FTIR spectra of (A) sunflower oil, (B) refine coconut oil, (C) crude palm oil, and (D) oil extracted from yeast (current study).
The microbial oil was quite similar to coconut and sunflower oil at position 1655 cm−1 and this peak was attributed to
Glucose has been reported as the best substrate for microbial oil production, since it can be assimilated by most oleaginous microorganisms to produce oil [
Characterization of solid waste collected for microbial oil production.
The corresponding results and the predicted values from the CCD are shown in Table
Central composite design and the corresponding experimental results and predicted values.
Runs | Factors | Microbial oil yield | ||
---|---|---|---|---|
Solid waste [ | Temperature [ | Actual (%) | Predicted (%) | |
1 | 7.5 | 30 | 21.15 | 21.29 |
2 | 7.5 | 22.9289 | 53.2 | 54.38 |
3 | 7.5 | 30 | 21.4 | 21.29 |
4 | 5 | 25 | 61.5 | 62.79 |
5 | 7.5 | 30 | 20.8 | 21.29 |
6 | 5 | 35 | 28.3 | 29.75 |
7 | 3.96447 | 30 | 50.7 | 48.43 |
8 | 7.5 | 30 | 21.11 | 21.29 |
9 | 10 | 35 | 28 | 26.24 |
10 | 7.5 | 30 | 22.05 | 21.29 |
11 | 7.5 | 37.0711 | 24.6 | 25.32 |
12 | 10 | 25 | 35.9 | 34.3 |
13 | 11.0355 | 30 | 23.3 | 25.57 |
As shown in Table
Equation (
The correlation obtained from the regression analysis (Table
ANOVA statistical results for the response surface quadratic model.
Source | Sum of squares | df | Mean square | | | |
---|---|---|---|---|---|---|
Model | 2396.26 | 5 | 479.25 | 152.12 | <0.0001 | Significant |
| 831.23 | 1 | 831.23 | 263.84 | <0.0001 | |
| 522.44 | 1 | 522.44 | 165.83 | <0.0001 | |
| 160.02 | 1 | 160.02 | 50.79 | 0.0002 | |
| 554.22 | 1 | 554.22 | 175.91 | <0.0001 | |
| 442.52 | 1 | 442.52 | 140.46 | <0.0001 | |
Residual | 22.05 | 7 | 3.15 | |||
Lack of fit | 21.14 | 3 | 7.05 | 30.96 | 0.0032 | Significant |
Pure error | 0.91 | 4 | 0.23 | |||
Cor. total | 2418.32 | 2 |
The predictability plot as a function of the amount of solid waste and temperature is shown in Figure
The graph of the predicted response developed as a function of solid waste concentration and temperature.
This study has not only demonstrated that oleaginous microorganism can be programmed for maximum cell biomass generation and oil production but also revealed the ability of the oil-producing microorganism to turn nuisance organic solid waste into valuable oil. Utilization of solid waste as a substrate for production of oil by oleaginous microorganism is a novel process that establishes the economic value of wastes besides proving a solution to the waste disposal challenge.
The authors declare that there are no conflicts of interest.
Fortunate Laker, Arnold Agaba, and Andrew Akatukunda contributed equally to this work and are joint first authors.
The authors would like to acknowledge Uganda Industrial Research Institute (UIRI) for support in carrying out this research and would also like to thank Okoth Thomas of Chemistry Department (UIRI) for his contribution towards sample analysis.