DEHALOCOCCOIDES AND REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS - Bioaugmentation for Groundwater Remediation - page 48

et al., 2004 ). Strain CBDB1 appears to be specialized to dechlorinate chlorinated aromatic
compounds and no chlorinated aliphatic compound has been found that supports its growth.
Since all Dhc strains contain multiple predicted reductive dehalogenase (RDase) genes on their
genomes (see below), the true substrate spectrum is likely to exceed the range of chlorinated
electron acceptors currently described. For example, the DCE- and VC-dechlorinating Dhc
isolate strain BAV1 has been implicated in debromination of polybrominated diphenyl ethers
(He et al., 2006 )

2.2 ISOLATION

AND CULTIVATION STRATEGIES
2.2.1 General Considerations

DEHALOCOCCOIDES

Dhc are slow growing, strict anaerobes that require anoxia and reducing conditions for
growth. Even brief exposure to air can be detrimental and kill Dhc (Adrian et al., 2000b ;
Amos et al., 2008a ). Stringent anoxic techniques are essential and required reducing condi-
tions are established by the addition of chemical reductants to the medium (see below). Dhc in
mixed cultures, where the presence of oxygen-consuming microbes affords some protection
against oxygen, are more robust than pure cultures but still require faithful application of
anoxic techniques. Moreover, it is commonly observed that Dhc populations grow more
slowly and become more fastidious as they are purified from other organisms; only a few
laboratories worldwide have succeeded in obtaining and maintaining Dhc isolates (Adrian
et al., 2000b ;Heetal., 2003b , 2005 ;Maym ´ -Gatell et al., 1997 ;M¨ller et al., 2004 ;Sungetal.,
2006b ). Specific techniques for Dhc cultivation and isolation are available in the aforemen-
tioned publications, and a summary by L¨ffler et al. ( 2005 ) provides detailed guidance and
protocols. The following section provides an overview of the general principles involved
in Dhc cultivation.
An enrichment culture for Dhc or other dechlorinators is typically initiated by transferring
an inoculum (1-10%, vol/vol) from a microcosm that exhibits the desired reductive dehalo-
genation reaction(s). The mineral composition of the growth medium used should be as similar
as possible to that of the original habitat, particularly with regard to dominant salts. Concen-
trations of the important mineral nutrients ammonium and phosphate need to be higher than
present in situ , but their concentrations should be maintained below 1 mM. To provide trace
metals, chloride salts of minerals should be used rather than sulfate salts because sulfate serves
as an electron acceptor for sulfate-reducing bacteria. The described Dhc grow best around pH 7
and the pH of the enrichment should be buffered to near neutrality. Commonly, a carbon
dioxide/bicarbonate (CO 2 /HCO 3 ) buffer system is used. Oxygen removal is essential and the
medium must be chemically reduced prior to inoculation with Dhc . Culture vessels should
be sealed with thick butyl rubber stoppers. Teflon ® -coated septa also will work, but require
more experience with anoxic cultivation techniques because leakage of gases out of or into
the culture vessel is more difficult to control. Ideally, slight positive pressure is maintained in
the culture vessels to avoid oxygen contamination during sampling events. Hydrophobic
chlorinated compounds, such as PCE and TCE, are notorious for “disappearing” due to
sorptive losses. Teflon ® -lined rubbers stoppers help to minimize sorptive losses but are more
prone to leakage of volatile compounds out of, and oxygen into, the culture vessels. Hence,
thick rubber stoppers are more appropriate to cope with the oxygen sensitivity of Dhc . In any
event, appropriate control vessels are needed, and the measurement of daughter product
formation is always a more reliable indicator of dechlorination activity than is substrate (e.g.,
PCE, TCE) disappearance.