Toxicity of organic extraction reagents to anaerobic bacteria

Various forms of liquid-liquid extraction systems are being developed to separate products, such as ethanol and volatile fatty acids (VFA), from fermentation liquids, since distillation is energetically expensive. Continuous extraction is advantageous, as product inhibition of the fermentation is minimized. However, some extraction solvents may be toxic or inhibitory to microorganisms.Thirty organic chemicals were examined by means of a small scale (60 mL) batch fermentation bioassay procedure for their toxicity to a commercial inoculum (Methanobac, W.B.E. Ltd.), which was a mixed culture of facultatively anaerobic, acid-producing bacteria. Gas production, pH change of medium, and the concentrations of ethanol, VFA, and lactic acid were measured after 75 h growth. The optimum experimental conditions for toxicity testing were alfalfa as substrate (2 g), a buffered nutrient medium (pH 6.8), "Methanobac" inoculum (10 mL), and test chemicals at levels between 10 and 100 muL/mL.Thirteen chemicals were nontoxic, and included the paraffins (C(6)-C(12)), phthalates, organophosphorus compounds, Freon 113 (1,1,2-trichloro-1,2,2-trifluoro ethane), Aliquat 336 (tricaprylylmethyl ammonium chloride), di-isoamyl ether, and trioctylamine. Other amine extractants were partially toxic. Alcohols (C(5)-C(12)), ketones (C(5)-C(8)), benzene derivatives, isoamyl acetate, and di-isopropyl ether were toxic. Generally, the chemicals were not toxic unless present at levels in excess of that expected to be required to saturate the aqueous phase.Total gas production was a good indicator of toxicity even within 24 h, but the presence of homofermentative (nongas producing) lactic acid bacteria complicated interpretation."Methanobac" inoculum was compared with an inoculum derived from a rumen culture for four test chemicals. The results were essentially the same. However, the toxicity of a chemical to bacteria is likely to vary considerably between bacterial species.