Effects of Initial Rice Bran Concentration and Inoculum's Ratio on Microbial Growth of Co-culture Fermentation


  • Jack Mink Tan Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, Johor, Malaysia.
  • Roslina Rashid Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, Johor, Malaysia.
  • Siti Marsilawati Mohamed Esivan Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, Johor, Malaysia.
  • Nor Athirah Zaharudin Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, Johor, Malaysia.




Co-culture fermentation, Microbial growth, Yield coefficient, Lactobacillus casei, Propionibacterium jensenii


Co-culture fermentation is widely applied for its synergistic effects. The synergistic effects of lactic acid bacteria (LAB) and propionic acid bacteria (PAB) are reported to improve the ruminant feed efficiency through the supplementations of probiotics. However, although co-culture fermentation of LAB and PAB has been recently demonstrated, the effects of carbon source and inoculum’s ratio on the microbial growth in co-fermentation are still not well-explored. Thus, this study was carried out to investigate the effect of rice bran concentration, as carbon source and inoculum’s ratio on the growth of L. casei and P. jensenii in co-culture fermentation. Reducing sugar content was extracted from rice bran through autoclave at 121℃ for 15 minutes. Co-culture fermentation was carried out in 2 stages: rice bran extract concentration’s variation and inoculum’s ratio variation. Co-culture in 20% w/v of RBE concentration showed the highest yield coefficient of YX/S of 0.265 g biomass/g substrate and YP/S of 0.715 g propionic acid/g substrate. Therefore, 20% w/v RBE concentration was used for the study of inoculum’s ratio. The YX/S (0.254 g biomass/g substrate) and YP/S (0.653 g propionic acid/g substrate) of ratio 1:4 was slightly lower than ratio 1:8, but the viability of L. casei (8.934 log10 CFU/mL) and P. jensenii (9.420 log10 CFU/mL) was the highest in ratio 1:4. Although increase of PAB ratio can increase biomass produced, but ratio 1:4 can achieve higher microbes’ viability which is important in the development of probiotics products.

Author Biography

Roslina Rashid, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, Johor, Malaysia.

Associate Professor Dr Roslina Rashid

Department of Bioprocess Engineering,

School of Chemical and Energy Engineering, Faculty of Engineering, 

Universiti Teknologi Malaysia, Johor Bahru, Johor. 


Ahmadi, N., Khosravi-Darani, K., Mohammad Mortazavian, A. and Mashayekh, S.M., (2016) ‘Effects of process variables on fed-batch production of propionic acid’, Journal of Food Processing and Preservation, 41(2), e12853. doi:10.1111/jfpp.12853.

Akoetey, W. (2015). Direct fermentation of sweet potato starch into lactic acid by Lactobacillus amylovorus: The prospect of an adaptation process’, Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/41.

Alazzeh, A. Y., Smith, A. H., Beauchemin, K. A., Meale, S. J. and McAllister, T. A. (2014) ‘Supplementing Propionibacterium acidipropionici P169 does not affect methane production or volatile fatty acid profiles of different diets in in vitro rumen cultures from heifers', Acta Agriculturae Scandinavica, Section A - Animal Science, 64(3), 170-177. doi: 10.1080/09064702.2014.988746.

Bader, J., Mast-Gerlach, E., Popović, M. K., Bajpai, R. and Stahl, U. (2010) ‘Relevance of microbial coculture fermentations in biotechnology’, Journal of Applied Microbiology, 109, 371-387. doi:10.1111/j.1365-2672.2009.04659.x.

Campaniello, D., Bevilacqua, A., Sinigaglia, M. and Altieri, C. (2015) ‘Screening of Propionibacterium spp. for potential probiotics properties’, Anaerobe, 34, 169-173. doi: 10.1016/j.anaerobe.2015.06.003.

Canon, F., Nidelet, T., Guédon, E., Thierry, A. and Gagnaire, V. (2020) ‘Understanding the mechanisms of positive microbial interactions that benefit lactic acid bacteria co-cultures’, Frontiers in Microbiology, 11, 2088. doi: 10.3389/fmicb.2020.02088.

Chen, X., Huang, H., Khanna, M., & Önal, H. (2011). Meeting the mandate for biofuels: implications for land use, food, and fuel prices. In The intended and unintended effects of US agricultural and biotechnology policies (pp. 223-267). University of Chicago Press.

Das, P. K., Rani, J., Rawat, S. and Kumar, S. (2021) ‘Microalgal co-cultivation for biofuel production and bioremediation: Current status and benefits,’ BioEnergy Research, 15, 1-26. doi: 10.1007/s12155-021-10254-8.

Derev, S. A., Peeva, Tz., Radulova, P., Stancheva, N., Staykova, G., Beev, G., Todorova, P., & Tchobanova, S. (2007) ‘Yeast cultures in ruminant nutrition’, Bulgarian Journal of Agricultural Science, 13, 357-374.

Elghandour, M. M. Y., Salem, A. Z. M., Castñeda, J. S. M., Camacho, L. M., Kholif, A. E. and Chagoyán, J. C. V. (2015) ‘Direct-fed microbes: A tool for improving the utilization of low-quality roughages in ruminants’, Journal of Integrative Agriculture, 14(3), 526-533. doi: 10.1016/S2095-3119(14)60834-0.

Farhadi, S., Khosravi-Darani, K., Mashayekh, M., Mortazavian, A. M., Mohammadi, A. and Shahraz, F. (2013) ‘Production of propionic acid in a fermented dairy beverage’, International Journal of Dairy Technology, 6(1), 127-134. doi: 10.1111/1471-0307.12004.

Guyot, J. P., Calderon, M., & Morlon-Guyot, J. (2001). Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T’, Journal of Applied Microbiology, 88(1), 176–182. doi: 10.1046/j.1365-2672.2000.00953.x.

Hamza, F., Kumar, A. R., Zinjarde, S. (2018) ‘Coculture induced improved production of biosurfactant by Staphylococcus lentus SZ2: Role in protecting Artemia salina against Vibrio harveyi,’ Enzyme and Microbial Technology, 114, 33-39. doi: 10.1016/j.enzmictec.2018.03.008.

Hu, H., Catchmark, J. M. and Demirci, A. (2021) ‘Co-culture fermentation on the production of bacterial cellulose nanocomposite produced by Komagataeibacter hansenii,’ Carbohydrate Polymer Technologies and Applications, 2, 1-9. doi: 10.1016/j.carpta.2020.100028.

Hugenschmidt, S., Schwenninger, S. M., & Lacroix, C. (2011). Concurrent high production of natural folate and vitamin B12 using a co culture process with Lactobacillus plantarum SM39 and Propionibacterium freudenreichii DF13. Process Biochemistry, 46(5), 1063-1070.

Kim, D-H., Yun, H-S., Kim, Y-S. and Kim, J-G. (2020) ‘Effects of co-culture on improved productivity and bioresource for microalgal biomass using the floc-forming bacteria Melaminivora jejuensis,’ Frontiers in Bioengineering and Biotechnology, 8, Article 588210. doi: 10.3389/fbioe.2020.588210.

Klongklaew, A., Unban, K., Kanpiengjai, A., Wongputtisin, P., Pamueangmun, P., Shetty, K. and Khanongnuch, C. (2021) ‘Improvement of enantiomeric L-lactic acid production from mixed hexose-pentose sugars by coculture of Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9’, Fermentation, 7, 95. doi: 10.3390/fermentation7020095.

Lee, I. H., Fredrickson, A. G. and Tsuchiya, H. M. (1974) ‘Diauxic growth of Propionibacterium shermanii’, Applied and Environmental Microbiology, 28(5), 831-835.

Liu, J. A. P. and Moon, N. J. (1982) ‘Commensalistic interaction between Lactobacillus acidophilus and Propionibacterium shermanii’, Applied and Environmental Microbiology, 44(3), 715-722.

Maier, R. N. and Pepper, I A. (2015) ‘Chapter 3 – Bacterial Growth’, In Pepper, I. L., Gerba, C. P. and Gentry, T. J. (eds.) Environmental Microbiology (Third Edition). Academic Press, pp. 37-56. doi: 10.1016/B978-0-12-394626-3.00003-X.

Mohamed Esivan, S. M., Rashid, R., Jati, A. and Zaharudin, N. A. (2021) ‘Growth of Lactobacillus casei and Propionibacterium jensenii in different glucose concentration and incubation temperature’, In: Abdul Karim, S.A., Abd Shukur, M.F., Fai Kait, C., Soleimani, H., Sakidin, H. (eds) Proceedings of the 6th International Conference on Fundamental and Applied Sciences. Springer Proceedings in

Complexity. Springer, Singapore. doi: 10.1007/978-981-16-4513-6_9.

Ranadheera, C. S., Evans, C. A., Adams, M., Baines, S. K. (2016) ‘Co-culturing of probiotics influences the microbial and physicochemical properties but not sensory quality of fermented dairy drink made from goat’s milk’, Small Ruminant Research, 136, 104-108. doi: 10.1016/j.smallrumres.2016.01.016.

Seo, J. K., Kim, S. W., Kim, M. H., Upadhaya, S. D., Kam, D. K. and Ha, J. K. (2010) ‘Direct-fed microbial for ruminant animals’, Asian-Aust. J. Anim. Sci., 23(12), 1657-1667.

Wu, Q. Q., You, H. J., Ahn, H. J., Kwon, B. and Ji, G. E. (2012) ‘Changes in growth and survival of Bifidobacterium

by coculture with Propionibacterium in soy milk, cow’s milk, and modified MRS medium’, International Journal of Food Microbiology, 157(1), 65-72. doi: 10.1016/j.ijfoodmicro.2012.04.013.

Xie, C., Coda, R., Chamlagain, B., Varmanen, Piironen, V., Katina, K. (2019) ‘Co-fermentation of Propionibacterium freudenreichii and Lactobacillus brevis in Wheat Bran for in situ Production of Vitamin B12’, Frontiers in Microbiology, 10, Article 1541. doi: 10.3389/fmicb.2019.01541.