Screening of Lipid Production Capacities of Bacillus sp. Strains Isolated from Soil and Lipid Staining with Different Staining Techniques

Yıl 2023, Cilt: 23 Sayı: 5, 1094 - 1102, 30.10.2023

Elif Demirkan , İrem Yıldırım

https://doi.org/10.35414/akufemubid.1265060

Öz

Microbial lipids have been attracting more and more attention in recent years as promising raw
materials for the production of biodiesel and value-added compounds. In the current economic and
environmental situation, finding new renewable sources of lipids will be crucial. Recent research has
necessitated the search for new efficient microbial producers with lipid production efficiency. For this
purpose,50 Bacillus sp. strains previously isolated from the soil were screened for their lipid production
capacity. As a result of the production using a single-cell oil production medium, only two Bacillus spp.
strains showed growth. These bacteria were named as Bacillus sp. E40 and Bacillus sp. E226. Bacteria
were then characterized in terms of their biomass, lipid yields, and lipid contents. The biomass of
Bacillus sp. E40 and E226 bacterial isolates, were 0,28 and 0,22 g.L-1, respectively. The highest lipid
content was reached by E226 with 54.5%. Bacillus sp. E40 lipid content was determined as 46.4%. In
addition, both intracellular lipid staining and colony staining demonstrated the presence of lipids. With
this study, it was reported for the first time that local Bacillus sp. strains isolated from soil have lipid
producing capacity.

Anahtar Kelimeler

Bacillus, Lipid, Staining, Sudan Black B

Topraktan İzole Edilen Bacillus sp. Suşlarının Lipid Üretim Kapasitelerinin Taranması ve Farklı Boyama Teknikleri ile Lipid Boyama

Öz

Mikrobiyal lipitler, biyodizel ve katma değerli bileşiklerin üretimi için umut verici hammaddeler olarak
son yıllarda giderek daha fazla dikkat çekmektedir. Mevcut ekonomik ve çevresel durumda, yeni
yenilenebilir lipit kaynakları bulmak çok önemli olacaktır. Son araştırmalar, lipit üretim verimliliğine
sahip yeni verimli mikrobiyal üreticilerin araştırılmasını gerekli kılmıştır. Bu amaçla daha önce topraktan
izole edilen 50 Bacillus sp. suşu lipid üretme kapasiteleri açısından taranmıştır. Tek hücreli yağ üretimi
ortamında yapılan üretim sonucunda bu bakterilerden sadece iki Bacillus sp. suşları üreme göstermiştir.
Bu bakterilerden sadece 2 Bacillus sp. suşu tek hücreli yağ üretimi ortamında üremiştir. Bu bakteriler
Bacillus sp.E40 ve Bacillus sp. E226 olarak adlandırıldı. Bakteriler daha sonra biyokütleleri, lipid verimleri
ve lipid içerikleri açısından karakterize edildi. Bacillus sp. E40 ve E226 bakteri izolatlarının biyokütlesi
sırasıyla, 0,28 ve 0,22 g.L-1 idi. En yüksek lipid içeriğine %54.5 ile E226'da ulaşılmıştır. Bacillus sp. E40
lipid içeriği %46.4 olarak belirlendi. Ek olarak, lipid varlığı hem hücre içi lipid boyama hem de koloni
boyama ile gösterilmiştir Bu çalışma ile topraktan izole edilen yerel Bacillus sp. suşlarının lipit üretme
kapasitesine sahip olduğu ilk kez rapor edilmiştir.

Anahtar Kelimeler

Bacillus, Lipid, Boyama, ,Sudan Siyahı B

Kaynakça

• Akın, D., 2017. Biochemical and genetic assessment of high value lipid production from local thermo-resistant green microalgae strain. Master thesis, Middle East Technical University The Graduate School of Natural and Applied Sciences, Ankara, 132.
• Ali, T.H. and El-Ghonemy, D.H., 2014. Optimization of culture conditions for the highest lipid production from some oleaginous fungi for biodiesel preparation. Asian Journal of Applied Sciences, 2, 2321–2893.
• Andeden, E.E., 2021. Stres koşullarının bazı mikroalg türlerinde lipit verimine ve triaçilgliserol (tag) içeriğine etkisinin gen ekspresyon düzeyinde ortaya konulması ve yağ asidi profili ile ilişkili biyodizel kalitesinin araştırılması. Doktora Tezi, Gazi Üniversitesi Fen Bilimleri Enstitüsü, Ankara, 107.
• Arda, M., 2000. Temel Mikrobiyoloji. Medisan Yayıncılık, 285-298.
• Bellou, S., Triantaphyllidou, I.E., Aggeli, D., Elazzazy, A. M., Baeshen, M.N. and Aggelis, G., 2016. Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content. Current Opinion in Biotechnology, 37, 24–35.
• Bento, H.B.S., Carvalho, A.K.F., Reis, C.E.R. and De Castro, H.F., 2020. Single-cell oil production and modification for fuel and food applications: assessing the potential of sugarcane molasses as culture medium for filamentous fungus. Industrial Crops and Products, 145, 3–6.
• Bettencourt, S., Miranda, C., Pozdniakova, T.A., Sampaio P., Franco-Duarte, R. and Pais, C., 2020. Single cell oil production by oleaginous yeasts grown in synthetic and waste-derived volatile fatty acids. Microorganisms, 8, 1–18.
• Boswell, K., Koskelo, E.K., Carl, L., Glaza, S., Hensen, D.J., Williams, K.D. and Kyle, D.J., 1996. Preclinical evaluation of single-cell oils that are highly enriched with arachidonic acid and docosahexaenoic acid. Food and Chemical Toxicology, 34, 585–593.
• Chan, L.G., Dias, F.F.G., Saarni, A., Cohen, J., Block, D., Taha A.Y. and Bell, J.M., 2020. Scaling up the bioconversion of cheese whey permeate into fungal oil by Mucor circinelloides. Journal of the American Oil Chemists Society, 97, 703–716.
• Demirkan, E., Kut, D., Sevgi, T., Dogan, M. and Baygın, E., 2020. Investigation of effects of protease enzyme produced by Bacillus subtilis 168 E6-5 and commercial enzyme on physical properties of woolen fabric. The Journal of The Textile Institute, 111, 26–35.
• Denli, Y. and Tekin A., 2000. Oil production and microorganisms. Gıda, 25, 265–270.
• Dzurendova, S., Zimmermann, B., Tafintseva, V., Kohler, A., Ekeberg, D. and Shapaval, V., 2020. The influence of phosphorus source and the nature of nitrogen substrate on the biomass production and lipid accumulation in oleaginous Mucoromycota fungi. Applied Microbiology and Biotechnology. 104, 8065–8076.
• Gogte, S., Polasa, K. and Rukmini, C., 1989. Screening for oleaginous yeasts using replica printing technique coupled with densitometric scanning. Current Science, 58, 1406–1408.
• Gufrana, T., Islam, H., Khare, S., Pandey, A. and Radha, P., 2022. In-situ transesterification of single-cell oil for biodiesel production: a review. Preparative Biochemistry and Biotechnology, 2, 1–16.
• Hartman, T.L., 1940. The use of Sudan Black B as a bacterial fat stain. Stain Technology, 15, 23–28.
• Kyle, D.J. and Ratledge, C., 1992. Industrial application of single cell oils. AOCS Publishing, 245–250.
• Li, Y., Horsman, M., Wu, N., Lan, C.Q. and Dubois-Calero, N., 2008. Biofuels from microalgae. Biotechnology Progress, 24, 815–820.
• Liu, G.Q., Lin, Q.L., Jin, X.C., Wang, X.L. and Zhao, Y., 2010. Screening and fermentation optimization of microbial lipid-producing molds from forest soils. African Journal of. Microbiology Research, 4, 1462–1468. Llamas, M., Dourou, M., González-Fernández, C., Aggelis, G. and Tomás-Pejó, E., 2020. Screening of oleaginous yeasts for lipid production using volatile fatty acids as substrate. Biomass Bioenergy, 138, 2–8.
• Maina, S., Pateraki, C., Kopsahelis, N., Paramithiotis, S., Drosinos, E.H., Papanikolaou, S. and Koutinas, A., 2017. Microbial oil production from various carbon sources by newly isolated oleaginous yeasts. Engineering in Life Sciences, 17, 333–344.
• Masurkar, A.A.K., Gone, G.B., Patil, K.V. and Pandey, K.R., 2023. Production of single cell oils from bacterial sources. Journal of Microbiology and Biotechnology, 8, 2–9.
• Madani, M., Enshaeiehb, M. and Abdoli, A., 2017. Single cell oil and its application for biodiesel production. Process Safety and Environmental Protection, 111, 747–756. Mironov, A.A., Nemashkalov, V.A., Stepanova, N.N., Kamzolova, S.V., Rymowicz, W. and Morgunov, I.G., 2018. The effect of pH and temperature on arachidonic acid production by glycerol-Grown Mortierella alpina NRRLA-10995. Fermentation, 4, 1–17.
• Mhlongo S.I., Ezeokoli O.T., Roopnarain, A., Ndaba, B., Sekoai, P.T., Habimana, O. and Pohl, C.H., 2021. Derived from filamentous fungi as alternative feedstock sources for biodiesel production. Frontiers in Microbiology, 12, 2–3.
• Moi, I.M., Leow, A.T., Ali, M.S., Rahman, R.N., Salleh, A.B. and Sabri, S., 2018. Polyunsaturated fatty acids in marine bacteria and strategies to enhance their production. Applied Microbiology and Biotechnology, 102, 5811–5826.
• Neema, P.M. and Kumari, A., 2013. Isolation of lipid producing yeast and fungi from secondary sewage sludge and soil. Australian Journal of Basic and Applied Sciences, 7, 283–288.
• Pan, L.X., Yang, D.F., Shao, L., Li, W., Chen, G.G. and Liang, Z.Q., 2009. Isolation of the oleaginous yeasts from the soil and studies of their lipid-producing capacities. Food Technology Biotechnology, 47, 215–220.
• Patel, A., Karageorgou, D., Rova, E., Katapodis, P., Rova, U., Christakopoulos, P. and Matsakas, L., 2020. An overview of potential oleaginous microorganisms and their role in biodiesel and omega-3 fatty acid-based industries. Microorganisms, 8, 2–40.
• Ratledge, C., 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86, 807–815.
• Steen, E.J., Kang, Y., Bokinsky, G., Hu, Z., Schirmer, A., McClure, A., del Cardayre, S.B. and Keasling, J.D., 2010. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature, 463, 559–562.
• Wang, B., Rezenom, Y.H., Cho, K.C., Tran, J.L., Lee, D.G., Russell, D.H., Gill, J.J., Young, R. and Chu, K.H., 2014. Cultivation of lipid-producing bacteria with lignocellulosic biomass: Effects of inhibitory compounds of lignocellulosic hydrolysates. Bioresource Technology, 161, 162–170.
• Wynn, J.P., Hamid, A.A., Li Y. and Ratledge, C., 2001. Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiology, 147, 2857–2864.
• Wynn, J.P. and Ratledge, C., 2005. Food Biotechnology. CRCPress, 443–472.
• Zainuddin, M.F., Fai, C.K., Mohamed M.S., Abdul Rahman, N.A. and Halim, M., 2022. Production of single cell oil by Yarrowia lipolytica JCM 2320 using detoxified desiccated coconut residue hydrolysate. PeerJ, 10, e12833.