Submitted to: Corn Utilization Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: June 9, 2004
Publication Date: June 9, 2004
Citation: Skinner, K.A., Leathers, T.D. 2004. Comparison of identification methods for lactobacillus. In: Proceedings of Corn Utilization Technology Conference. June 7-9, 2004, Indianapolis, Indiana. CDROM. Technical Abstract: Bacterial contamination is an ongoing problem for commercial fuel ethanol production facilities. Both chronic and acute infections are of concern, due to the fact that bacteria compete with the ethanol-producing yeast for sugar substrates and micronutrients. Lactic acid levels often rise during bouts of contamination, suggesting that the most common contaminants are lactic acid bacteria. However, quantitative surveys on the natural occurrence of bacteria in commercial corn-based fuel ethanol production are lacking. Consequently, a survey of representative facilities was performed in an effort to better define the problem, and two methods for identification of Lactobacillus were used simultaneously and results compared. Initially, different incubation conditions were tested using seven control strains chosen from the ARS Culture Collection at the National Center for Agricultural Utilization Research. The bacteria were plated onto solid lactobacilli deMan-Rogosa-Sharpe (MRS) medium and incubated anaerobically, microaerobically, or aerobically at either 28ºC, 30ºC, or 37ºC. In all cases, best growth was obtained under anaerobic conditions at 37ºC. The API 50 CHL and Biolog identification test kits were also tested preliminarily on these seven known strains. Both assays gave the most consistent and accurate results when incubated at 37ºC. For the API test kit, bacteria were harvested into the API 50 CHL medium and distributed into each of 50 wells on the API test strips and incubated at 37ºC for 48-72 h. Color change was then graded and data entered into the APILab Plus computer program. For the Biolog system, cells were harvested into Biolog inoculating fluid and dispersed into wells of AN microplates. The microplates were held under aerobic conditions for 10-15 min and then incubated under anaerobic conditions at 37ºC for 20-24 h. Optical densities of the wells were read on a dual channel plate reader at 590 and 750 nm and analyzed using the Biolog MicroLog 3 program and AN database software. When the Biolog system was used, the most accurate results were obtained when organisms were subcultured twice on blood agar plates before cell harvesting, as opposed to one time only or being tested directly off of MRS plates. For the survey, samples were collected from one wet mill and two dry grind fuel ethanol facilities over a nine month period. Appropriate dilutions were counted for total colony forming units, and random colonies were single-colony isolated three times before being tested with the identification kits. It became apparent that it was necessary to use both methods of identification because they differed in their data base sets. For instance, the Biolog system identified some species that were not present in the API data base, such as Weisella confusa and L. amylovorus. On the other hand, the API assay provided subspecies identification of certain species, such as L. brevis and L. acidophilus. When identifications based on two test methods did not agree, the identification with the higher confidence value was chosen. Overall, the API assay was used solely for 33% of the identifications, the Biolog assay solely for 41%, and the tests agreed in 25% of the cases. A random set of isolates were picked and 16S DNA identification done. In all cases, the DNA identification agreed with that based on the test kits. Lactobacillus species were indeed found to be the most abundant isolates from all three plants, averaging 51%, 38%, and 77% of total isolates from the wet mill and the first and second dry grind facilities, respectively.