activity, thus significantly improving the nZVI performance. Results from the
monitoring process reflecting the effect of nZVI on microbial communities are
therefore an important criterion for successfully evaluating in situ remediation.
The presence, quantity, and/or diversity of microorganisms can be monitored by
either simple microbiological methods such as cultivation techniques or using more
advanced methods such as epifluorescence microscopy or molecular genetics.
Researchers and remediation engineers should thoroughly consider various specific
aspects of these methods to choose the most appropriate one. The key criteria are
time, cost effectiveness, and the diagnostic value. Here, we summarize the most
commonly utilized microbiological methods for monitoring the effect of nZVI on
microorganisms.
34.2 Cultivation
Cultivation methods are used to estimate the number of microorganisms in the given
sample volume which are able to grow on the given medium. It is important to know
that only a small portion of microorganisms can be cultivated (0.1–2%). The most
commonly used methods are colony-forming unit (CFU) counting (spread-plate
technique and the membrane filter cultivation on agar plates) and the end-point
dilution method (most probable number—MPN). The end-point dilution method
presumes that the distribution of cells in the liquid medium is uniform, and the cells
do not cluster together, which is not typical for environmental samples from polluted
sites. Therefore only the CFU method is described here.
The CFU method is based on the assumption that a single colony develops from
each cell. However, cells can sometimes clump and a single colony may grow from
several cells clustered together. The viable count could be thus invariably lower than
the real total cell number present in a sample. A suspension must be prepared from
soil or water samples and then diluted. Usually a ten-fold dilution series is applied.
There is a wide range of cultivation media available supporting the growth of
different functional groups of microorganisms. Since the application of nZVI
might favor iron-reducing or iron-oxidizing bacteria depending on environmental
conditions (Emerson et al. 2010; Weber et al. 2006) selective media might be used in
addition to more general media in order to focus on these specific groups of
microorganisms (APHA 2005). Particularly, Citrate Agar (HiMedia) was proved
to be suitable and highly selective for iron-oxidizing bacteria in our laboratory
(Fig. 34.1).
In brief, a known volume (e.g., 0.1 mL) of the dilutions is plated onto the
surface of a suitable growth medium pre-prepared in Petri dishes. The incubation
is held under defined conditions (temperature), and after 3–7 days the average
CFU on the plates is determined. Plates containing between 20–200 colonies are
optimal for counting. The number of CFU per mL (or g) of the initial sample can
be calculated from the average CFU on parallel plates and the known dilution
factor (CFU/mL or g).
646
A. Ševců et al.
monitoring process reflecting the effect of nZVI on microbial communities are
therefore an important criterion for successfully evaluating in situ remediation.
The presence, quantity, and/or diversity of microorganisms can be monitored by
either simple microbiological methods such as cultivation techniques or using more
advanced methods such as epifluorescence microscopy or molecular genetics.
Researchers and remediation engineers should thoroughly consider various specific
aspects of these methods to choose the most appropriate one. The key criteria are
time, cost effectiveness, and the diagnostic value. Here, we summarize the most
commonly utilized microbiological methods for monitoring the effect of nZVI on
microorganisms.
34.2 Cultivation
Cultivation methods are used to estimate the number of microorganisms in the given
sample volume which are able to grow on the given medium. It is important to know
that only a small portion of microorganisms can be cultivated (0.1–2%). The most
commonly used methods are colony-forming unit (CFU) counting (spread-plate
technique and the membrane filter cultivation on agar plates) and the end-point
dilution method (most probable number—MPN). The end-point dilution method
presumes that the distribution of cells in the liquid medium is uniform, and the cells
do not cluster together, which is not typical for environmental samples from polluted
sites. Therefore only the CFU method is described here.
The CFU method is based on the assumption that a single colony develops from
each cell. However, cells can sometimes clump and a single colony may grow from
several cells clustered together. The viable count could be thus invariably lower than
the real total cell number present in a sample. A suspension must be prepared from
soil or water samples and then diluted. Usually a ten-fold dilution series is applied.
There is a wide range of cultivation media available supporting the growth of
different functional groups of microorganisms. Since the application of nZVI
might favor iron-reducing or iron-oxidizing bacteria depending on environmental
conditions (Emerson et al. 2010; Weber et al. 2006) selective media might be used in
addition to more general media in order to focus on these specific groups of
microorganisms (APHA 2005). Particularly, Citrate Agar (HiMedia) was proved
to be suitable and highly selective for iron-oxidizing bacteria in our laboratory
(Fig. 34.1).
In brief, a known volume (e.g., 0.1 mL) of the dilutions is plated onto the
surface of a suitable growth medium pre-prepared in Petri dishes. The incubation
is held under defined conditions (temperature), and after 3–7 days the average
CFU on the plates is determined. Plates containing between 20–200 colonies are
optimal for counting. The number of CFU per mL (or g) of the initial sample can
be calculated from the average CFU on parallel plates and the known dilution
factor (CFU/mL or g).
646
A. Ševců et al.