Anaerobe Clostridium acetobutylicum and Biodiesel Glycerol Byproduct in Undergraduate Research

Graves, James, and Vincent Vuljaj

Acetone-butanol-ethanol (ABE) fermentation by anaerobe Clostridium acetobutylicum is a source of biofuel.  Growth of the organism in the presence of biodiesel glycerol byproduct was examined in this investigation.  The Oxyrase For Broth (Oxyrase, Inc.) enzyme system was used to assist in creating anaerobic conditions.  C. acetobutylicum grew well on Schaedler blood agar Oxyplates, brain heart infusion (BHI) agar with Oxyrase, in fluid thioglycollate medium with or without Oxyrase and in phenol red broth with 0.2% agar and Oxyrase.  The organism did not grow well in minimal broth or phenol red broth, with Oxyrase. Tube cultures had closed caps and anaerobic plate cultures were incubated in zip-lock bags.  In thioglycollate medium with Oxyrase growth was assessed by measuring culture optical density (OD) by use of a Klett-Summerson colorimeter.  In cultures containing added glucose or a mixture of glucose and byproduct growth was increased while that in cultures containing byproduct or pure glycerol was similar to that in the base medium.  In phenol red broth that contained 0.2% agar and Oxyrase cultures that contained added glucose or a mixture of glucose and byproduct exhibited a positive fermentation reaction in contrast to those containing byproduct or pure glycerol.  Test paper (Hydrion) revealed the pH of cultures containing glucose dropped from 8.0 to 5.0.  A glucose oxidase and peroxidase assay (Precision Labs, Inc.) showed the concentration of glucose decreased to an undetectable level.  An alcohol oxidase and peroxidase assay (AlcoScreen) indicated the presence of alcohol in all cultures.  Experiments with glycerol byproduct made by catalyst column or transesterification produced similar results.  It could be profitable to find a use in industry for the waste glycerol byproduct from biodiesel plants.