Depending on their fatty-acid profile, microbial oils have many industrial applications such as manufacturing biodiesel, nutraceuticals, essential oils and soaps. Our study aimed to extract microbial oil from Aspergillus niger and to study the biochemical characteristics of the extracted oil. Another approach of this study was to find a sustainable way of growing A. niger by using agro waste as a nutrient source; for this purpose, a growth medium was prepared from waste onion peels. Our findings indicate that A. niger oil could contain high amounts of unsaturated fatty acids as well as can be used as an antimicrobial agent against Staphylococcus aureus. Moreover, the growth medium prepared was suitable to culture A. niger. Using onion peels will not only help reduce the upstream-processing cost of microbial oil production but will also contribute to adequate management of the agro waste, consequently, reducing its negative impact on the environment.
Research Article
Extraction of microbial oil from Aspergillus niger and preparation of a sustainable growth medium for the same
https://doi.org/10.21203/rs.3.rs-2884031/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this latest preprint version
microbial oil
Aspergillus niger
onion peel
sustainable
culture medium
oleaginous
Various products, ranging from cooking oil and nutraceuticals to biodiesel and cosmetics, require plant-based oils in their production or processing. Using plant-based resources for oil extraction competes with food production and may eventually lead to the depletion of these resources. To overcome this issue, we need to find sustainable alternatives to these feedstocks. One of the ways is to extract lipids from oleaginous microorganisms—a wide range of bacteria, yeasts and fungi that can accumulate more than 20% oil in their dry cell weight—and the oil extracted from them is called microbial oil or single-cell oil (Ratledge 2013; Wynn and Ratledge 2005). Of all the oleaginous microbes, filamentous fungi are found to be more beneficial concerning the kind of microbial oil they accumulate which include palmitic acid, linoleic acid, linolenic acid and stearic acid with ~ 50% corresponding to unsaturated fatty acids.(Patel et al., 2020; Zhu and Bonito 2020). Aspergillus niger is one of the filamentous oleaginous fungi and several studies on the fungus cultured on different growth media showed significant accumulation of single-cell oil (Alrubayae and Kadhim 2020; Andre et al., 2010). Different types of oleaginous microbes can be isolated from different sources such as soil, seawater and animal feed (Li et al., 2011). But these places are also a habitat for various classes of other organisms as well and therefore, it becomes a strenuous task to isolate oleaginous microbes from non-oleaginous ones. As A. niger is pathogenic to certain plants and the most common hosts include onion bulbs, peanuts, Indian gooseberry, and coconuts, (Schuster et al., 2002) the fungus can be easily isolated from these infected plant or fruit sources. For these reasons, in our study, we used A. niger as the source of microbial oil.
In eukaryotic oleaginous microbes, accumulation of lipids in storage form is triggered under nutrient-limiting conditions, especially nitrogen source, and in presence of excess carbohydrate source. Under such conditions, citric acid accumulation takes place which is eventually converted to acetyl Co-A. The starting point of this effect is the cleavage of adenosine monophosphate to inosine monophosphate (due to lack of nitrogen). The unavailability of adenosine monophosphate inactivates the adenosine monophosphate-dependent isocitrate dehydrogenase. As a result, the tricarboxylic acid cycle is blocked at isocitrate which is subsequently converted to citrate, transported to the cytosol and metabolized to acetyl Co-A. Simultaneously, the generation of NADPH occurs by conversion of malate to pyruvate by the malic enzyme and/or NADPH regeneration by the pentose-phosphate pathway. When there is a continuous supply of acetyl Co-A and NADPH, reverse beta-oxidation occurs and that advances to lipid synthesis. These lipids are converted to triacylglycerols and stored in the microbial cell (Ochsenreither et al., 2016).
The ability of oleaginous fungi to produce hydrolytic enzymes enables them to grow on unmanageable wastes (Khot et al., 2018). A. niger produces several extracellular enzymes such as pectinases, amylases and cellulases to name a few. The release of these enzymes makes the organism grow on culture medium derived from inexpensive and inedible waste products such as cellulosic biomass, industrial wastewater and waste molasses (Chuppa-Tostain et al., 2018; Kanti and Sudiana 2017; Veana et al., 2014).
Onion or Allium cepa is the most widely grown and consumed vegetable worldwide as well as it is the routinely used ingredient in Indian cuisine. As the world’s second-most consumed horticultural crop and the exceeding demand for processed onions, its production interventions generate an ample amount of waste (Celano et al., 2021). The waste is mainly in the form of onion peels and the disposal of which, if not managed properly, can have damaging effects on the environment such as release of carbon monoxide and greenhouse gases (Maji et al., 2020). Most of the agro waste management includes conversion of the crop residue to animal feed, but onion peels cannot be used for this purpose because of their strong aroma (Kumar et al., 2022). Considering the nature of onion peels as one of the habitats for Aspergillus niger and its unique composition of carbohydrates (88.56%) and nitrogen (15%) which can enhance lipid synthesis and storage in an oleaginous microbe (Shabir et al., 2022; Ochsenreither et al., 2016), we developed a growth medium from onion peels to culture A. niger.
2.1 Isolation of A. niger:
Black mould found on onion bulbs was used as the source of Aspergillus niger (Ko et al., 2002). The mould was suspended in 5ml of sterile distilled water and the absorbance of the suspension was taken at 600nm (Cortesão et al., 2020). The suspension was inoculated in 100ml Potato-dextrose broth (PDB) and was kept at 37°C for a 4-day incubation period (Passamani et al., 2014).
The Potato-dextrose broth was composed of (in g/L): 200g potatoes and 20g dextrose. The broth was autoclaved at 15psi at 121°C for 20 minutes before use.
2.2 Identification of A. niger
Apart from colony characteristics, the cultured A. niger was identified by preparing two different mounts: one using 0.3% Sudan black stain (0.3g in 100 ml of 70% alcohol) and Safranin as the counterstain, and other using Lactophenol Cotton Blue stain. The mounts were observed under a light microscope.
2.2.1 Steps for Sudan Black B staining:
1. Take a clean microscope slide.
2. Place a drop of sterile-distilled water on it.
3. Transfer a loopful of A. niger culture onto the slide, tweeze the culture and spread it evenly over an oval area up to 2 cm in length.
4. Air-dry the smear and heat-fix it by passing the slide through the flame a few times.
5. Flood the smear with 0.3% Sudan Black B stain and keep for 15 mins.
6. Keep the slide in the xylene for 15-20 seconds and let it air dry.
7. Counterstain with Safranin.
8. After 2 minutes, drain off the excess stain and wash with sterile distilled water.
9. Air-dry the slide and observe under an oil-immersion lens.
2.2 2 Steps for Lactophenol Cotton Blue staining:
1. Take a clean microscope slide.
2. Place a drop or two of ethanol onto the slide.
3. Transfer a loopful of A. niger culture over ethanol, tweeze the culture and spread it evenly over an oval area up to 2 cm in length.
4. Heat-fix the smear by passing the slide over the flame a few times.
5. Add a few drops of the stain and let it stay for 15 mins.
6. Drain off the excess stain and place a coverslip on the smeared area.
7. Observe the mount under a 45x objective lens.
2.3 Biomass weight calculation:
The biomass developed in PDB was transferred to a pre-weighed petri plate and was dried by keeping the petri plates at 105°C for 1 hour (Xie et al., 2007).
2.4 Extraction of microbial oil:
A significant amount of dried and powdered A. niger biomass was used for lipid extraction. The sample was packed in a pre-weighed thimble made from Whatman filter paper No. 2. Microbial oil was extracted using the Soxhlet apparatus with n-hexane as the solvent of choice (Li et al., 2014). 12-14 cycles were sufficient for the complete extraction of the microbial oil. The extracted oil was stored in an airtight vial at 4°C until further analysis.
2.5 Biochemical analyses:
Microbial oil extracted from the A. niger grown in PDB was analyzed for its acid value and peroxide value. The estimation methods were referred from the book—Biochemical Methods by S. Sadasivam (1996). The acid and peroxide values were compared to the values obtained from soybean oil (Refined soybean oil by Fortune Foods was used for the purpose).
2.6 Antimicrobial activity of A. niger oil:
The antimicrobial activity of A. niger oil against Staphylococcus aureus was checked by the disc-diffusion method. The paper discs were made out of Whatman paper No.2; the test sample was applied along with a positive control (1% Ampicillin) and a negative control (Saline—0.9% NaCl) (Peechakara et al., 2022). The antimicrobial susceptibility was checked by measuring the zone of inhibition.
2.7 Using onion-peel waste as the culture medium:
Onion peels were used to prepare a sustainable culture medium for A. niger. The culture medium is hereafter referred to as Onion-peel broth (OPB) and comprised (in g/L): 100g dried and powdered onion peels plus 20g dextrose. The onion peels were sun-dried, ground into a fine powder, and boiled in 1000 ml of distilled water for 30 minutes. The infusion was cooled and strained through cheesecloth. 20 g of dextrose was added to the infusion and the volume was made up to 1000 mL with distilled water. The broth was sterilised by autoclaving at 15 psi at 121°C for 20 minutes. The growth characteristics, culture morphology and biomass yield of A. niger grown in OPB were compared to that of grown in PDB.
2.8 Growth curve of A. niger cultured in Onion-peel broth
A growth-curve analysis of Aspergillus niger cultured on Onion-peel broth was performed over ten days by gravimetric method. Ten flasks, each containing 100 ml of Onion-peel broth, were inoculated with A. niger culture. The absorbance of the inoculum was kept between 0.5-0.6 to ensure a similar inoculum size in all the flasks. The flasks were numbered 1-10: corresponding to the day on which the biomass yield from that particular flask will be recorded. The yield from each flask was separated and dried and the dry-cell weight was determined. A plot of the time in days versus the dry-cell weight of A. niger in grams was plotted.
3.1. Microbial oil:
As shown in Fig. 1, when Aspergillus niger was stained with 0.3% Sudan black B, the sterigmata, vesicle and conidiophore took up the stain indicating the presence of lipids in those parts, whereas only the sterigmata and vesicles were stained with Lactophenol Cotton Blue stain. Thus, the staining methods confirmed the presence of A. niger and lipids in the fungus.
The oil yield from Aspergillus niger grown in a PDB was found to be 11.8%. The results of the biochemical analyses of the A. niger oil and soybean oil are given in the Tables 1 and 2.
|
Constant burette readings (mL) |
A. niger oil |
Soybean oil |
|---|---|---|
|
Acid value |
3.1 |
0.1 |
|
Peroxide value |
18..3 |
4.0 |
The following formulas (Sadasivam 1996) were used to calculate the acid value and peroxide value for A. niger oil and soybean oil from the constant brunette readings (Table 1) obtained by the titrimetric methods.
|
Sample |
Acid Value (mg KOH/g oil) |
Peroxide Value (mEq of peroxide/kg oil) |
|---|---|---|
|
A. niger oil |
49.13 |
48.03 |
|
Soybean oil |
0.47 |
7.86 |
As seen from Table 2, the acid value and peroxide value of microbial oil are greater than those of soybean oil. Higher acid value could be attributed to the autoxidation of fatty acid or due to exposure to elevated temperature conditions during the extraction process. Higher acid and peroxide values could also be a result of high levels of unsaturated fatty acids present in the oil as they are unstable at elevated temperatures and undergo autoxidation readily (Mhlongo et al., 2021); also the concentration of unsaturated fatty acids is positively correlated with oil rancidity (Feiner 2016, Negash et al., 2019).
The extracted A. niger oil showed antimicrobial activity against Staphylococcus aureus with a zone of inhibition measuring 8mm (Fig. 2). Compared to ampicillin, the clear zone achieved by A. niger oil was extremely less. The minimum zone of inhibition could be a consequence of less diffusion of oil in the agar medium due to differences in polarity. The susceptibility of A. niger oil against other pathogens such as E. coli or Salmonella species could be further assessed to know its spectrum of activity.
3.2. Onion-peel broth (OPB)
Figure 3 depicts the before and after images of Onion-peel broth inoculated with A. niger. For comparison of the growth of A. niger cultured in OPB with that cultured in PDB, the picture was taken on the fourth day, as the PDB culture showed maximum growth after 4-day incubation.
The A. niger culture from OPB had slightly different characteristics from that cultured in PDB. The differences are pointed out in Table 3. These differences will help in the screening of Aspergillus niger when cultured in OPB.
|
Sr. No |
Culture Characteristics |
Onion-peel Broth |
Potato-dextrose Broth |
|---|---|---|---|
|
1 |
Colour of the upper side |
Black |
Black |
|
2 |
Appearance of the upper side |
Cottony |
Cottony |
|
3 |
Colour of the reverse side |
Pinkish |
Yellowish |
|
4 |
Appearance of the reverse side |
Flat |
Uneven |
To know when the optimum growth of A. niger is seen when cultured in Onion-peel broth, a growth curve was plotted (Fig. 4). We recorded that maximum growth of A. niger was seen on the sixth day after inoculation. After the sixth day, due to nutrient deficiency in the medium, the fungus attained a death phase showing a decrease in the dry-cell yield. From this result, a comparison was made between the dry-cell yield of A. niger cultured in PDB and that cultured in OPB (Table 4). After a 6-day incubation period, the culture grown in Onion-peel broth was separated and dried at 105°C for 1 hour and the dry-cell weight of the culture was noted. The cell biomass from PDB was separated on the fourth day as the optimum growth of the culture in PDB was observed on that day. From the result, it was seen that the weight of culture from OPB was less than that
|
Culture medium |
Dry-cell weight (g/100mL) |
|---|---|
|
OPB (after 6-day incubation) |
0.308 ± 0.01 |
|
PDB (after 4-day incubation) |
0.672 ± 0.02 |
obtained from PDB. Therefore, it would be more beneficial if OPB is used for screening or other purposes where the yield of the culture is not an important factor. Nevertheless, OPB is more profitable as inferred from the cost-benefit analysis (Table 5) and can be used as an alternative to PDB.
3.3. Cost-benefit analysis of Onion-peel broth
To understand the cost-benefit of choosing OPB over PDB as a culture medium for A. niger, all the materials that go into the making of the two media were listed and their respective costs were calculated. It was found that 24.04% can be saved if OPB is used instead of PDB.
|
Culture medium |
Requirements (Per litre) |
Source |
Cost (Rupees) |
Total cost (Rupees) |
|---|---|---|---|---|
|
Potato-dextrose Broth (PDB) |
Potatoes: 200g |
Local market |
5.0 |
25.8 |
|
Dextrose:20g |
Commerciala |
20.8 |
||
|
Distilled water: 1L |
Lab prepared |
0.0 |
||
|
Onion-peel Broth (OPB) |
Onion peels: 150g |
Field waste |
0.0 |
20.8 |
|
Dextrose:20g |
Commerciala |
20.8 |
||
|
Distilled water:1L |
Lab prepared |
0.0 |
||
|
Cost-benefit from using OPB as an alternative to PDB = 24.04% |
||||
| aSDFCL S D Fine-Chem Limited, Mumbai, India | ||||
Thus, the Onion-peel broth is not just suitable to culture Aspergillus niger, it is also easy to prepare and is more profitable when compared to Potato-dextrose broth. Using OPB will also play a role in the sustainable management of agro waste and would be an inexpensive choice over PDB.
[1] Nutrient Agar by HIMEDIA M001, Mumbai, India
Aspergillus niger was successfully isolated from onion peels as well as the oil extracted from A. niger cultured on Potato-dextrose broth showed a substantial yield. When the biochemical analyses of A. niger oil were compared to that of soybean oil, the difference in the values was fairly high. The high acid value and peroxide value of A. niger oil could be attributed to the auto-oxidation of fatty acids, exposure to high-temperature conditions during the extraction process and/or the presence of high amounts of unsaturated fatty acids. Accordingly, the addition of certain antioxidants in the oil or using other extraction processes could prevent oxidation and generation of free radicals. A. niger oil possesses anti-microbial activity against Staphylococcus aureus. However, the antimicrobial components, the spectrum of activity and toxicity levels of A. niger oil need to be further assessed. The production of microbial oil can be made more profitable and sustainable by using onion peels to prepare the growth medium for A. niger. Using onion peels as a growth medium agrees with the nutrient conditions required for lipid synthesis and storage in the microbial cell. Furthermore, compared to Potato-dextrose broth, Onion-peel broth is more cost-effective and will aid in reducing the upstream processing costs in microbial oil production. On the whole, with further modifications, A. niger oil can be considered as an alternative to plant-based oils and fats, playing a part in improving the energy crisis and preventing the depletion of these resources. Moreover, culturing A. niger in Onion-peel broth will not only reduce the upstream processing cost of microbial oil production but will also help in the sustainable management of this agro waste.
The extracted microbial oil needs to be further assessed using sophisticated techniques, such as HPTLC or HPLC, to understand the lipid profile concerning the unsaturated and saturated fatty acid content. If unsaturated fatty acid content is higher than the saturated fatty acid content, the former (if therapeutically important) can be isolated and incorporated into nutraceuticals. If the saturated fatty acid content is higher, the microbial oil can be suitable for biodiesel production. The biochemical parameters of microbial oil from A. niger cultured in Onion-peel broth can also be determined to figure out the possible applications of the oil. Other than A. niger, Onion-peel broth can be used to culture other oleaginous microorganisms as well.
Funding: No funding was received for conducting this study.
Conflict of interest: The authors declare no conflict of interest. The authors have no relevant financial or non-financial interests to disclose.
Author contributions: The idea was conceived by Sneha Salgaonkar. Both the authors have contributed to the design of the study. Experiments were performed and analysed by Saloni Malekar. The first draft of the manuscript was written by Saloni Malekar and reviewed by Sneha Salgaonkar. Both the authors read and approved the final manuscript.
- Alrubayae, I. M., & Kadhim, K. F. (2020). Mycodiesel production from some isolates of oleaginous fungi isolated from oil-rich soils in Basrah. International Journal of Pharmaceutical Research, 12(4). https://doi.org/10.31838/ijpr/2020.12.04.640
- André, A., Diamantopoulou, P., Philippoussis, A., Sarris, D., Komaitis, M., & Papanikolaou, S. (2010). Biotechnological conversions of bio-diesel derived waste glycerol into added-value compounds by higher fungi: Production of biomass, single cell oil and oxalic acid. Industrial Crops and Products, 31(2), 407–416. https://doi.org/10.1016/j.indcrop.2009.12.011
- Celano, R., Docimo, T., Piccinelli, A. L., Gazzerro, P., Tucci, M., Di Sanzo, R., Carabetta, S., Campone, L., Russo, M., & Rastrelli, L. (2021). Onion peel: Turning a food waste into a resource. Antioxidants, 10(2), 304. https://doi.org/10.3390/antiox10020304
- Chuppa-Tostain, G., Hoarau, J., Watson, M., Adelard, L., Shum Cheong Sing, A., Caro, Y., Grondin, I., Bourven, I., Francois, J. M., Girbal-Neuhauser, E., & Petit, T. (2018). Production of Aspergillus niger biomass on sugarcane distillery wastewater: Physiological aspects and potential for biodiesel production. Fungal Biology and Biotechnology, 5(1), 1. https://doi.org/10.1186/s40694-018-0045-6
- Cortesão, M., De Haas, A., Unterbusch, R., Fujimori, A., Schütze, T., Meyer, V., & Moeller, R. (2020). Aspergillus niger spores are highly resistant to space radiation. Frontiers in Microbiology, 11, 560. https://doi.org/10.3389/fmicb.2020.00560
- Feiner, G. (2016). Meat and Fat. Salami, 3–30. https://doi.org/10.1016/B978-0-12-809598-0.00001-9
- Kanti, A., & Sudiana, I. M. (2017). C0-CULTURE OF AMYLOLYTIC FUNGI Aspergillus niger AND OLEAGINOUS YEAST Candida orthopsilosis ON CASSAVA WASTE FOR LIPID ACCUMULATION. Berita Biologi, 16(2), 111–119. http://doi.org/10.14203/beritabiologi.v16i2.2207
- Khot, M., Katre, G., Zinjarde, S., & RaviKumar, A. (2018). Single Cell Oils (SCOs) of oleaginous filamentous fungi as a renewable feedstock: A biodiesel biorefinery approach. In F. Biorefineries (Ed.). Fungal Biorefineries (pp. 145–183). Springer. https://doi.org/10.1007/978-3-319-90379-8_8
- Ko, S. S., Huang, J. W., Wang, J. F., Shanmugasundaram, S., & Chang, W. N. (2002). Evaluation of onion cultivars for resistance to Aspergillus niger, the causal agent of black mold. Journal of the American Society for Horticultural Science, 127(4), 697–702. https://doi.org/10.21273/JASHS.127.4.697
- Kumar, M., Barbhai, M. D., Hasan, M., Punia, S., Dhumal, S., Radha, N., Rais, N., Chandran, D., Pandiselvam, R., Kothakota, A., Tomar, M., Satankar, V., Senapathy, M., Anitha, T., Dey, A., Sayed, A. A. S., Gadallah, F. M., Amarowicz, R., & Mekhemar, M. (2022). Onion (Allium cepa L.) peels: A review on bioactive compounds and biomedical activities. Biomedicine and Pharmacotherapy, 146, 112498. https://doi.org/10.1016/j.biopha.2021.112498
- Li, S. L., Feng, S. L., Li, Z. T., Xu, H., Yu, Y. P., Qiao, D. R., & Cao, Y. (2011). Isolation, identification and characterization of oleaginous fungi from the soil of Qinghai Plateau that utilize D-xylose. African Journal of Microbiology Research, 5, 2075–2081.
- Li, Y., Ghasemi Naghdi, F., Garg, S., Adarme-Vega, T. C., Thurecht, K. J., Ghafor, W. A., Tannock, S., & Schenk, P. M. (2014). A comparative study: The impact of different lipid extraction methods on current microalgal lipid research. Microbial Cell Factories, 13(1), 14. https://doi.org/10.1186/1475-2859-13-14
- Mhlongo, S. I., Ezeokoli, O. T., Roopnarain, A., Ndaba, B., Sekoai, P. T., Habimana, O., & Pohl, C. H. (2021). The potential of single-cell oils derived from filamentous fungi as alternative feedstock sources for biodiesel production. Frontiers in Microbiology, 12, 637381. https://doi.org/10.3389/fmicb.2021.637381
- Negash, Y. A., Amare, D. E., Bitew, B. D., & Dagne, H. (2019). Assessment of quality of edible vegetable oils accessed in Gondar City, Northwest Ethiopia. BMC Research Notes, 12(1), 793. https://doi.org/10.1186/s13104-019-4831-x
- Ochsenreither, K., Glück, C., Stressler, T., Fischer, L., & Syldatk, C. (2016). Production strategies and applications of microbial single cell oils. Frontiers in Microbiology, 7, 1539. https://doi.org/10.3389/fmicb.2016.01539
- Passamani, F. R. F., Hernandes, T., Lopes, N. A., Bastos, S. C., Santiago, W. D., Cardoso, M. D. G., & Batista, L. R. (2014). Effect of temperature, water activity, and pH on growth and production of ochratoxin A by Aspergillus niger and Aspergillus carbonarius from Brazilian grapes. Journal of Food Protection, 77(11), 1947–1952. https://doi.org/10.4315/0362-028X.JFP-13-495
- Patel, A., Karageorgou, D., Rova, E., Katapodis, P., Rova, U., Christakopoulos, P., & Matsakas, L. (2020). An overview of potential oleaginous microorganisms and their role in biodiesel and omega-3 fatty acid-based industries. Microorganisms, 8(3), 434. https://doi.org/10.3390/microorganisms8030434
- Peechakara, B. V., Basit, H., & Gupta, M. (2022). Ampicillin. In StatPearls [Internet]. StatPearls Publishing.
- Ratledge, C. (2013). Microbial production of polyunsaturated fatty acids as nutraceuticals. In Microbial production of food ingredients, enzymes and nutraceuticals (pp. 531–558). Woodhead Publishing. https://doi.org/10.1533/9780857093547.2.531
- Sadasivam, S. (1996). Biochemical methods. New age international.
- Schuster, E., Dunn-Coleman, N., Frisvad, J. C., & Van Dijck, P. W. (2002). On the safety of Aspergillus niger–a review. Applied Microbiology and Biotechnology, 59(4–5), 426–435. https://doi.org/10.1007/s00253-002-1032-6
- Shabir, I., Pandey, V. K., Dar, A. H., Pandiselvam, R., Manzoor, S., Mir, S. A., Shams, R., Dash, K. K., Fayaz, U., Khan, S. A., Jeevarathinam, G., Zhang, Y., Rusu, A. V., & Trif, M. (2022). Nutritional profile, phytochemical compounds, biological activities, and utilisation of onion peel for food applications: A review. Sustainability, 14(19), 11958. https://doi.org/10.3390/su141911958
- Veana, F., Martínez-Hernández, J. L., Aguilar, C. N., Rodríguez-Herrera, R., & Michelena, G. (2014). Utilization of molasses and sugar cane bagasse for production of fungal invertase in solid state fermentation using Aspergillus niger GH1. Brazilian Journal of Microbiology. Publication of the Brazilian Society for Microbiology, 45(2), 373–377. https://doi.org/10.1590/s1517-83822014000200002
- Wynn, J. P., & Ratledge, C. (2005). Oils from microorganisms. Bailey’s Industrial Oil and Fat Products. https://doi.org/10.1002/047167849X.bio006
- Xie, G. X., & West, T. P. W. (2007). Citric acid production by Aspergillus niger on condensed corn distillers solubles. Research Journal of Microbiology, 2(5), 481–485. https://doi.org/10.3923/jm.2007.481.485
- Zhu, S., & Bonito, G. (2020). Oleaginous fungi in biorefineries. Reference module in life sciences. https://doi.org/10.1016/B978-0-12-819990-9.00004-4
No competing interests reported.