Development of an innovative method for analyzing cell viability, flocculation and microbial contamination during alcoholic fermentation

• Grant number: 20/09785-4
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - PIPE
• Start date: April 01, 2021
• End date: June 30, 2022
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Paulo Henrique Rocha Latado
• Grantee: Paulo Henrique Rocha Latado
• Company: Poligene Comércio e Serviços Ltda. - ME
• CNAE: Atividades de apoio à agricultura ; Fabricação de produtos químicos orgânicos não especificados anteriormente
• City: Rio Claro
• Associated researchers: Ana Paula Jacobus ; Fábio Barufaldi De Nadai
• Associated scholarship(s): 21/10077-7 - Development of an innovative method for analyzing cell viability, flocculation and microbial contamination during alcoholic fermentation, BP.TT; 21/04082-8 - Development of an innovative method for analyzing cell viability, flocculation and microbial contamination during alcoholic fermentation, BP.BIOEN.PIPE

Abstract

Despite being considered as one of the main agricultural sectors in the country, the Brazilian sugar-energy sector presents technological barriers. Industrial efficiency can only be improved in ethanol production plants if monitoring the quality of the raw material and alcoholic fermentation is implemented and continuously improved. Therefore, it would be interesting if faster and more accurate methods of monitoring cell viability (yeasts) and microbial contamination in raw (or fermenting) cane juice is developed. The standard method for determining the number of viable microbial cells (yeasts and bacteria) in a sample is by means of plating in a solid medium, but this is a method with long-term results (minimum 24 h.). Thus, methods based on the use of dyes and observation under an optical microscope are the most used to determine cell viability. However, they can also be inaccurate as they depend on subjective assessments by technicians. Flow cytometry is a very modern methodology that can be used to replace traditional methods of assessing cell viability because it has some advantages: being faster, more sensitive, more accurate and more reliable. Various cytometry analyzes can be carried out concurrently [viability (live / dead) and yeast cell vitality, determination of microbial contamination, for example] and in just a few minutes. Due to the speed of execution and precision of the results, these analyzes can be carried out with greater frequency in the ethanol production units, in order to better monitor the processes related to the quality of the raw material (raw cane juice) and the quality of fermentation. Consequently, if implemented, it will allow managers to have greater knowledge and agility for decision making and may assist in increasing industrial efficiency in ethanol production units. The objective of this project is the development and validation of the flow cytometry method to perform analyzes of cell viability, flocculation and microbial contamination, during alcoholic fermentation. The project includes laboratory tests to adjust the methodology during fermentation (1st stage). Then, these tests should be validated using samples of fermented sugar cane juice (or in fermentation phase), obtained from ethanol production plants (2nd stage), always comparing the results of flow cytometry with those obtained through analyzes. Traditional: a) plating and culture in solid medium with subsequent incubation, followed by colony count and b) viable cell count by staining and observation under an optical microscope. At the end of the project, it is expected to be able to offer the developed methodologies, in the form of alcoholic fermentation monitoring services, to ethanol production units, aiming at improving their production efficiency. (AU)