UPGMA clustering analysis helped visualize clustering in the various treatment
groups at the phylum level (Fig. 4A). The contents and distributions of the nine major
phyla (occupied above 1.0%) in the five experimental groups revealed that the
22
structure of the exposed microbial community deviated from that of the control, and
clustering depended on the aging status of the TiO
2
-NPs instead of their crystalline
phase. That is to say, the sludge communities in the two pristine TiO
2
-NPs groups
(TiO
2
-A and TiO
2
-R) exhibited spiking and were all clearly clustered, whereas the two
aged TiO
2
-NPs groups (aTiO
2
-A and aTiO
2
-R) and the control group exhibited spiking
and were all apparently clustered. The clustering seemed to imply that aging slowed
down the stress and/or inhibition induced by the NPs. Proteobacteria (65.3%),
Chloroflexi (6.8%), and Bacteroidetes (7.4%) were the three main microbial phyla of
the control sample. After exposure, the prevalence of Chloroflexi—the main phylum
responsible for carbohydrate biodegradation (Miura et al., 2007) in waste-activated
sludge (Zhou et al., 2015)—decreased dramatically in all the exposed groups, which
might explain the marked decline in COD removal. Proteobacteria and Bacteroidetes
were the other two dominant phyla in our activated SBR system, and they also exist in
conventional anaerobic digesters (Hao & Wang, 2015) and continuous flow-enhanced
biological phosphorus removal systems (Li et al., 2016). It's seemly unexpected and
irreconcilable that Proteobacteria, to which many of the PAOs that play key roles in P
removal belong (Hu et al., 2017), increased in proportion in the exposed groups as the
efficiency of P removal decreased in this study. Therefore, we need to further compare
Proteobacteria at the genus level to better understand the effects of TiO
2
-NPs on the
PAOs in SBRs.
At the genus level, the percentages of the nine representative bacterial genera
were closely related to dephosphorization, the consumption of carbon sources, and
EPS secretion, as shown in Fig. 4B.
Candidatus
Accumulibacter, which includes the
genus
Burkholderia
, comprises some well-known and predominant bacteria that are
accumulators of poly-P in enhanced biological P removal systems, and its presence
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23
was consistent with the typical phenotype of PAOs under anaerobic/aerobic conditions
(Hu et al., 2017; Mullan et al., 2002). Compared to the control, exposure to the four
TiO
2
-NPs (50 mg/L in each case) significantly (p < 0.05) decreased the abundances of
Burkholderia
and Accumulibacter, which might explain the reduction in the P removal
(intake) rates (Fig. 2B). However, the abundances of Comamonadaceae-unclassified
(affiliated with β-Proteobacteria) and Rhodocyclaceae-uncultured (affiliated with
α-Proteobacteria)—the other two major microbial families involved in P removal (Ge
et al., 2015; Kong et al., 2005; Xu et al., 2016)—increased in the groups exposed to
pristine and aged TiO
2
-NPs with different crystal forms. The differential responses of
the PAOs mentioned above may be attributed to the regional micro-environment
structures of the activated sludge flocs (Li et al., 2019b). That is to say, aerobic
Burkholderia
usually colonizes the outer layers of the sludge floc and would be the
first to bear the brunt when directly exposed to NPs. The facultative anaerobic and
anaerobic genera of Rhodocyclaceae and Comamonadaceae exist in the deeper layers,
and suffer minor effects or even short-term hormonal effects (Xu et al., 2016). The
phenotype of the GAOs has been defined as ‘‘Candidatus_Competibacter phosphatis’’
(Taya et al., 2013), and was present in smaller proportions (3.8–11.2%) than
Accumulibacter (12.5–32.2%) before and after exposure, indicating that the SBRs
were more highly enriched with Accumulibacter than with Competibacter. GAOs are
considered an important factor in the deterioration of biological P removal. The
genera
Acetobacter
and
Acidocella
, which are well-known degraders of carbon
sources (Araujo et al., 2012), also decreased in abundance, which did not benefit COD
degradation (Fig. 2D) in the SBR cycles. Interestingly, the abundances of
Pseudomonas
and
Novosphingobium
, two specific EPS-secreting genera (Lv et al.,
2014; Shrout & Nerenberg, 2012), evidently increased after short-term exposure. This
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24
suggests that greater secretion of EPS triggered by bacterial community quorum
sensing improved the resistance of the sludge to the toxicity of the NPs (Li et al.,
2019b). Therefore, the species aspect reasons of the decline in sludge COD and P
removal rates was due to the reduction of sludge microbial diversity and the severe
suppression of the abundance of specific functional species at the levels of phylum
and genus.
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