DEHALOCOCCOIDES AND REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS - Bioaugmentation for Groundwater Remediation - page 49

2.2.2 Electron Acceptor
The electron acceptor provided for Dhc enrichment is the target halogenated organic
compound (e.g., chlorinated ethenes). Ideally, the concentration of the target compound should
be as high as possible to maximize growth of dehalogenating organisms while avoiding toxicity.
Since toxicity cannot be predicted a priori , a good preliminary approach is to add the
chlorinated compound at the minimum concentration that is analytically tractable and supply
more electron acceptor to the cultures as it is consumed. The low solubility and high toxicity of
many chlorinated compounds often indicates that their concentrations will be well below the
millimolar concentrations typically used for enrichment and cultivation of microbes using more
common electron acceptors such as sulfate or nitrate.
One potentially useful strategy to provide larger amounts of toxic, lipophilic CAHs, is to
dissolve them in an inert hydrophobic “carrier” phase. For example, dissolving PCE in hexade-
cane, which is metabolized slowly, if at all, by most anaerobic cultures, allows addition of larger
amounts of chlorinated electron acceptor while keeping the aqueous concentration low (Holli-
ger et al., 1993 ; Krumholz et al., 1996 ;L¨ffler et al., 2005 ). Although this procedure maintains
constant, low chlorinated electron acceptor concentrations in the aqueous phase, utilizing a
carrier compound has drawbacks. The addition of a separate organic phase complicates
analysis of hydrophobic compounds due to partitioning, the organic phase may sequester
aqueous phase micronutrients (e.g., lipophilic vitamins) retarding Dhc growth, and the separate
phase may interfere with downstream procedures such as biomass collection and deoxyribo-
nucleic acid (DNA) extraction.
Whether provided with an organic carrier phase or directly (i.e., undiluted) into the growth
medium, aqueous PCE concentrations above 0.54 mM, about half its solubility limit, inhibit dechlo-
rination and growth of pure cultures (Amos et al., 2007 ). Although PCE-to-ethene-dechlorinating
consortia may tolerate slightly higher PCE concentrations (Amos et al., 2007 , 2009 ), growth of
dechlorinators at saturated aqueous phase PCE concentrations has not been demonstrated. Thus,
sustained growth in batch cultures without a carrier phase will require repeated feedings when
the chlorinated electron acceptor has been consumed. A prerequisite, of course, is that no
inhibitory products accumulate. Maintaining elevated concentrations of PCE that are tolerated
by the dechlorinators has been shown to inhibit methanogenic archaea, and thus can eliminate
certain microorganisms (DiStefano et al., 1991 ). The L¨ffler laboratory web site (University of
Tennessee, Knoxville, Department of Microbiology) offers an Excel spreadsheet that conve-
niently calculates the amounts of chlorinated solvent added to the mediumundiluted or dissolved
in a carrier phase to achieve a target aqueous phase electron acceptor concentration.
2.2.3 Electron Donor
Pure Dhc cultures use only hydrogen as an electron donor. Hydrogen also supports growth
of methanogens and acetogenic bacteria and hydrogen consumption may result in negative
pressure in the culture vessels. Therefore, hydrogen may not be the best electron donor for
enrichment. In initial studies of the PCE-dehalogenating enrichment culture from which strain
195 was isolated, methanol appeared to be a suitable electron donor (Freedman and Gossett,
1989 ). In retrospect, this was probably because methanol-utilizing methanogens and acetogens
were supplying Dhc with vitamin B 12 , a required growth factor (see below) and reducing
equivalents, presumably as hydrogen. Typically, an electron donor that generates hydrogen
upon fermentation, such as lactate, butyrate or benzoate is used. The latter two compounds
have an advantage in that their fermentation proceeds slowly under anoxic conditions due to