Biomedical Engineering Reference
In-Depth Information
2.2.6 Incubation Conditions
Enrichments typically are incubated at room temperature (20-25
C) under stationary
conditions. Agitation has not been shown to improve growth unless hydrogen is provided as
electron donor and mass transfer of the electron donor from the gas to the aqueous phase limits
dechlorination. Incubation at 30
C also may increase rates, but this should be determined only
after enrichments have been established.
2.2.7 Isolation
After successful enrichment of reductive dehalogenators is achieved, isolation can be
considered. Specific isolation techniques for
Dhc
have been described by L¨ffler et al.
(
2005
). Isolation is essentially a numbers game, in which the chances of success increase as
the organisms of interest begin to outnumber other organisms. Thus, the enrichment conditions
should be optimized to maximize growth of the organohalide respirers and minimize growth of
other “contaminating” microorganisms.
2.3
PURE CULTURES
2.3.1 Isolation of
Dhc mccartyi
Strain 195
DHC
In the case of the first isolation of
Dhc mccartyi
strain 195, several properties of the
dechlorinator facilitated isolation. The ability of strain 195 to withstand high concentrations of
PCE that were inhibitory to methanogens allowed the elimination of methanogenic archaea
(DiStefano et al.,
1991
). This was particularly fortuitous, since 2-bromoethane sulfonate, often
used as an inhibitor of methanogenesis, also can inhibit reductive dehalogenation of chlorinated
ethenes (DiStefano et al.,
1992
;L¨ffler et al.,
1997b
). A feature of
Dhc
that is particularly
useful for isolation is the lack of a peptidoglycan cell wall typically found in bacteria. This
feature makes
Dhc
resistant to antibiotics that interfere with peptidoglycan biosynthesis, like
vancomycin (DiStefano et al.,
1992
) and ß-lactam antibiotics such as ampicillin (He et al.,
2003b
; Maym ´ -Gatell et al.,
1997
). Treatment with these antibiotics helps eliminate many
bacterial contaminants, including acetogens, a group that can convert methanol or hydrogen
plus carbon dioxide to acetate, and rapidly outgrow
Dhc
. A final useful attribute of
Dhc
is its
small cell size;
Dhc
cells can pass through 0.45-micrometer (
m
m) membrane filters that retain
many other bacteria.
In the original studies leading to the isolation of strain 195, enrichment cultures were grown
in medium supplemented with hydrogen, PCE, acetate, vitamins (including high levels of
vitamin B
12
), filter-sterilized extract of anaerobic digestor sludge, and sulfide as a reducing
agent (Maym
´
-Gatell et al.,
1995
). These cultures dechlorinated PCE when transferred to
medium with 1 gram per liter (g/L) ampicillin, an antibiotic that inhibited growth of all
contaminating bacteria, but subsequent transfers showed significantly reduced dechlorination
activity. Therefore, transfer cultures were supplemented with filter-sterilized extracts from the
original mixed culture containing active
Dhc
, which allowed reductive dechlorination of PCE
and growth in transfer cultures. This culture underwent serial dilutions in liquid medium and the
culture obtained from the 10
7
dilution tube only contained tiny, flattened cocci. This disc-
shaped morphology was maintained in medium lacking ampicillin, and this pure culture of strain
195 could ultimately be transferred repeatedly in defined mineral salts medium amended with
the PCE, acetate, hydrogen and the Wolin vitamin mixture (He et al.,
2007
; Wolin et al.,
1963
).
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