6-CHLORINE DIOXIDE AGAINST BACTERIA AND YEASTS FROM THE ALCOHOLIC FERMENTATION

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Brazilian Journal of Microbiology (2008) 39:337-343 ISSN 1517-8382 337 *Corresponding Author. Mailing address: Via Anhanguera, km 174 - CEP: 13600-970. Araras, SP, Brasil. Tel./Fax: (+5519) 3543-2612. E-mail: silvana.meneghin@cca.ufscar.br

 CHLORINE DIOXIDE AGAINST BACTERIA AND YEASTS FROM THE ALCOHOLIC FERMENTATION 

Silvana Perissatto Meneghin1,2*; Fabricia Cristina Reis1 ; Paulo Garcia de Almeida3 ; Sandra Regina Ceccato-Antonini4 1 Departamento de Biotecnologia Vegetal, Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP, Brasil; 2 Universidade Estadual Paulista, Microbiologia Aplicada, Rio Claro, SP, Brasil; 3 Beraca Sabará Químicos e Ingredientes Ltda, Unidade São Paulo, São Paulo, SP, Brasil; 4 Departamento de Tecnologia Agroindustrial e Sócio-Economia Rural, Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP, Brasil Submitted: July 12, 2007; Returned to authors for corrections: October 27, 2007; Approved: April 25, 2008.

ABSTRACT 

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration (MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis (10 ppm) and Leuconostoc mesenteroides (50 ppm) than for Lactobacillus fermentum (75 ppm) and Lactobacillus plantarum (125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran® (recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran® dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies (dispersed cells) seemed to be more sensitive than wrinkled yeast colonies (clustered cells/pseudohyphal growth), both isolated from an alcoholproducing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms. Key words: chlorine dioxide, bacteria, yeast, antibacterial agent, alcohol, fermentation. 

RESULTS AND DISCUSSION 

Initially, chlorine dioxide was used as antibacterial agent in concentrations of 10, 25 and 50 ppm for all bacterial strains. However, MIC was observed to be probably under 10 ppm for B. subtilis. Subsequent tests with lower concentrations confirmed that 10 ppm was the lowest concentration of chlorine dioxide to inhibit this bacterium, with remarkable growth decrease in the range of 1 to 10 ppm. With Kamoran®, the recommended concentration for usage in fermentation tanks (3 ppm) was not efficient to suppress B. subtilis growth (Fig. 1). All control treatments (without inoculum) had OD540 variying from 0.01 to 0.02 after 24 hours of cultivation. With Leuconostoc mesenteroides, 50 ppm of chlorine dioxide was the MIC, with complete absence of growth, showing remarkable decrease of growth in the range of 10 to 50 ppm. Other than B. subtilis, dosage of 3 ppm of Kamoran® caused significant reduction, but not cessation of growth as chlorine dioxide did (Fig. 2). Concerning Lactobacillus plantarum, the experiments were performed at 37ºC, and concentrations ranging from 10, 25 and 50 ppm of chlorine dioxide did not inhibit bacterial growth. Concentration in the range of 100-200 ppm of chlorine dioxide was then efficient to prevent growth, as well as 3 ppm of Kamoran®. Statistical analysis has shown that 125 ppm of chlorine dioxide had similar inhibitory effect to that obtained with 150 and 200 ppm (Fig. 3). Total inhibition of growth was obtained with 100 ppm of chlorine dioxide for Lactobacillus fermentum, similarly to that result for 3 ppm of Kamoran® (Fig. 4), both experiments at 37ºC. However, statistically significant result was obtained using 75 ppm of chlorine dioxide. Previous studies have shown that the genus Lactobacillus is more adapted to the fermentative process conditions (10), and so, more resistant to antimicrobial agents. In order to verify whether chlorine dioxide has inhibitory effect on yeast, which would impair its usage as biocide in alcoholic fermentation, fermented must collected from an alcoholoperating unity at 2006/2007 sugar cane harvest was tested using plating and serial dilution techniques, in concentrations of 50, 100 and 200 ppm of chlorine dioxide. The yeast growth was completely inhibited at 100 ppm, but at 50 ppm of chlorine dioxide, the number of colony forming units was three times lower than in control dishes (with no chlorine dioxide), as in Fig. 5. This reduction may be considered low because of the high numbers of yeast cells present into fermentation tanks (around 108 cells/ ml) and did not reach even one-log order decrease. Two different yeast colonies were observed in culture medium, as smooth, bright colonies; and wrinkled, opaque colonies. They were isolated, purified and tested individually in tubes for MIC detection, similarly to the methodology used for bacterial strains, using chlorine dioxide concentrations ranging from 0 to 200 ppm. Cells from the wrinkled colony were slightly more resistant than the ones from the smooth colonies (Fig. 6), though the difference was not statistically significant For wrinkled yeast colonies, there was a decrease in growth from 150 ppm while for smooth yeast colonies, 100 ppm was enough to cause the same effect. Total growth inhibition was not observed in the chlorine dioxide range studied for both yeasts, as observed when plating technique was used. A probable explanation is the fact that plating technique is a measure of viable cells, which are not distinguished from dead cells using optical density as growth evaluation. Yeasts presenting clustered cells, as pseudohyphae, are very common in alcoholic fermentation process, and though they are related to poor fermentative performance (3), this cell arrangement can confer protection against undesirable environment, in a process called filamentation (5). Further studies are needed to assess the supposed higher resistance to chlorine dioxide by the wrinkled yeast colonies. For the safe usage of chlorine dioxide as antibacterial agent in alcoholic fermentation, it is not advisable to utilize more than 50 ppm in order to avoid harmful effects on the yeast inoculum. However, Lactobacillus bacteria had presented minimum inhibitory concentration for chlorine dioxide above 50 ppm. Further studies are encouraged since activation in pH below 4 brought about more efficiency, demanding lower dosages than those recommended here (unpublished data). Besides, other important characteristics should be considered for chlorine dioxide: approval at USDA Organic for application in organic products; prevention of resistance occurrence in bacterial populations by the use of antibiotics; higher profits with the sales of yeast for feed since antibiotics are not allowed; saving of sulphuric acid, once chlorine dioxide has anti-buffering effect when applied to the inoculum production step; besides antibiotics replacing, chlorine dioxide also eliminated the usage of other antibacterial agents.

Fig 1 to Fig.5  are in full text.

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