Environmental Engineering Reference
SYBR Green I (Invitrogen, USA), can also be used for staining. In this case the
concentration is 20
L (undiluted solution) in 200 mL of distilled water.
DNA Recovery from DGGE Gel and Sequencing of 16S rDNA
Major DGGE bands were recovered from the gel by using the razor or nib of a
Pasteur pipette, and washed with sterile purified water to use as template for re-
amplification of PCR performed using the original protocols except for the forward
primer, B341F (5 -CCTACGGGAGGCAGCAG-3 , E . coli position 341-356). The
partial 16S rDNAs were sequenced by using an appropriate DNA sequencer, and a
homology search was then conducted using the BLASTN database .
About 0.2 g wet wt of samples from the reactors were each suspended in 5 mL of
saline and gently shaken for 10 min at room temperature. Thereafter, 0.1 mL of
the suspension was diluted with 0.9 mL of saline and subsequently filtered through
L-pore-size Anodisc filter (Whatman, UK). One drop of 0.25% of SYBR
Gold (Invitrogen, USA) was placed on the Anodisc filter and kept in the dark for
15 min. The stained Anodisc filter was completely dried and mounted on a glass
slide with a drop of antifade reagent (SlowFade Antifade Kit, Invitrogen, USA). The
enumeration of cells was estimated by direct cell counting using epifluorescence
microscopy . The mean value was obtained from 20 independent experiments.
4 Current Outcome of Technological Implementation
With the progress of molecular biological techniques, microbial analysis meth-
ods have improved. 16S rDNA clone analysis and DGGE method have made
possible to clarify the accurate microbial community structures including VBNC
microorganisms that could not be detected by traditional culture dependent method.
As a case study, we compared the bacterial communities in (1) different con-
ditions (decomposing rate) of the reactors, using (2) different types of bulking
agent (wood chip or polyethylene terephthalate), and in (3) small-scale and large-
scale reactor. Each result showed different communities and presence of the VBNC
microorganisms (Figs. 1, 2, 3, and 4).
As previously reported, the dominant bacteria consisted of members of the order
Bacillales in the typical condition of composting with woodchips as bulking agent
[2-7, 10]. In reactor A, the order Bacillales was also dominant. However, in reactor
B, they decreased to 14%, and anaerobes or facultative anaerobes increased when
the decomposition rate of organic compound dropped following aggregation of the
contents (Figs. 1 and 2). This might be correlated with accumulation of persistent
substance like inorganic salt and decreasing of oxygen concentration . The order
Lactobacillales was not detected from the reactor A and B which using woodchips
as bulking agents, however, the order Lactobacillales were detected from reactor