by

Jodi Naugle

Did you ever think of vinegar as a product of a biofilm? Acetic acid is produced by two genera of bacteria, Acetobacter and Gluconobacter. These acetogenic bacteria oxidize ethanol to acetic acid.

This exercise utilizes the "trickle method" of vinegar production. In this, an alcoholic liquid is trickled over beech wood shavings packed into a column. The leachate is collected at the bottom and recycled. This exercise employs the bacteria Acetobacter aceti to produce vinegar from hard cider or "spiked" apple cider.

Procedure:

• A culture of the acetogenic bacterium is grown in 50 ml of nutrient broth.
• Two sterile columns, glass or plastic and approximately 50 cm in length are packed loosely with beech wood shavings. These will form the substrate on which the acetogenic biofilm will form. A double-necked receiving flask is attached to the bottom of each column. Glass coverslips may be placed into the column along with the wood shavings to collect biofilms, which can be stained and examined later.
• The 50 ml bacterial suspension is poured into one column over the wood shavings. It is collected at the bottom and discarded. The other column is a control.
• Apple cider containing 5% of ethanol (non-denatured) is the source of the alcohol. For each column, 120 ml of the alcoholic liquid is needed. The solution is poured into the top of the column and collected in the receiving flask at the bottom.
• A peristaltic or diaphragmatic pump is attached to the secondary neck of the receiving flask and to the top of the column to maintain a constant trickle of the alcoholic liquid through the beech wood shavings.
• A biofilm will develop on the surface of the wood-shavings (and the coverslips) in the column inoculated with the acetogenic bacteria. The alcoholic liquid will undergo oxidation by the bacteria and will start to become acidic (acetic acid) and acquire a vinegar odor. The experiment was performed twice, each "trickling" for 12 days. The second trial used the wood-shavings from the first because the acetogenic biofilm was already in place on the wood shavings.

Analysis:

• The coverslips may be stained with a gram's stain.
• The pH should be tested at intervals during the experiment.
• At the end of the experiment, the percentage of acetic acid may be determined by a titration using 0.1M NaOH into 10 ml of acetic acid with 20 ml of distilled water while measuring the pH. When the data is graphed (pH vs. Volume of Titrated NaOH) an equivalence point is found. A calculation using the equivalence point volume is performed to determine the present acetic acid.

Calculation:

Using the Equivalence Point Volume and the dilution of 10 ml of Acetic Acid to 20 ml of distilled water, the following calculation may be used to find the percent of the Acetic Acid in the leachate.

Percent Analysis of Acetic Acid adapted from Campbell, T., 1997. Chemistry 15, Penn State Altoona College.