N-167. Monitoring of the Microbial Succession of Fermented Alfalfa Haylage under Various Conditions

S. J. Zinkel, C. Wehnes, A. H. Smith, T. G. Rehberger;
Agtech Products Inc., Waukesha, WI.

The quality of fermented forage is very important in the diary industry, with 40-60% of the diet consisting of ensiled field crops. The objective of this study was to monitor the bacterial communities in alfalfa haylage under ideal and less favorable conditions over the course of fermentation. Under ideal conditions the moisture content at ensiling is 45-55%. Cut alfalfa was wilted in windrows for 24 hrs (control, 48% moisture), 48 hrs (dry, 31% moisture) and 48 hrs with a half inch of simulated rain after the first 24 hrs (rain, 31% moisture). Duplicate control and treatment (dry and rain) samples were collected from two laboratory scale mini-silos, air tight 5 gallon buckets containing approximately 31 pounds of packed haylage, on days 0, 1, 2, 6, 8, 10, 14, 35 and 59 post ensiling. Silos were unpacked after day 59 and the aerobic stability was monitored for twelve days. Aerobic stability is the length of time it takes for haylage to spoil after exposure to oxygen. Genomic DNA was extracted from samples and bacterial 16s ribosomal DNA was amplified by PCR using eubacterial primers, targeting the V3 region of the 16s rDNA. PCR products were separated by denaturing gradient gel electrophoresis (DGGE) to analyze bacterial communities. Volatile fatty acids (VFAs) and pH were measured at each time point, as well as the enumeration of lactic acid bacteria and spoilage (coliforms, yeasts, molds) on selective media. Unique DGGE profiles at day 0 for the control and treated samples were obtained, with the rain samples having the most bands. Within each treatment, an observable microbial succession is apparent. Analysis of the pH and VFA indicate the treated samples fermented poorly, the pH of the treated samples never going below 5.6 and lactic acid less than 0.1% whereas the control was below pH 4.8 and lactic acid greater than 3.5%. Treated samples also spoiled faster under aerobic conditions, with yeast reaching 2.0E+6 cfu/g after 2 days, where the yeast in the control samples stayed below 2.0E+6 cfu/g until day twelve. A better understanding of the microbial succession in both well and poorly fermented alfalfa will allow us to manipulate the bacterial communities with silage inoculants to ensure production of high quality haylage.