3.4 The washout of microbial communities at a very short HRT
In Table 4, the microbial (bacteria and archaea) richness and diversity
were obviously higher at a 5d-HRT and this gradually decreased with HRT
shortening. To be specific, OTUs of Chao1 estimations decreased by 26%
and 33% in bacteria and 23% and 29% in archaea when accompanied by a
decrease in the HRT from 5 to 3 and 1.5d, respectively. The same trend
was observed with Shannon and Simpson values although the reduction of
archaea diversity was greater than bacteria. However, all richness and
diversity scores observed in this study during the shortest HRT (1.5d)
were quite similar to data observed with different substrate (cow
manure) at a longer HRT (25 d) while bacteria richness in the current
study was obviously higher (Sun et al., 2015).
qPCR was performed during the reducing of HRTs. Genes of total bacteria
sharply decreased from 2.17×106 copies
μL-1 at HRT of 5d down to 1.55×104
copies μL-1 at HRT of 1.5d. This data was much lower
than that previously reported for an AD process dealing with food waste
at HRT of 20d and OLR of 8.21 g-COD·L-1, i.e.
1.2×107 copies μL-1 (Jang et al.,
2016). The trend of the three most abundant archaeal populations during
the HRT decreasing showed a pattern close to that of the
bacterial/archaeal ratio (Fig. 4), i.e. for HRT of 5, 3 and 1.5d, the
total archaeal population sharply decreased from
3.47×105 to 3.4×104 and then to
0.9×104 copies μL-1.
Correspondingly, the bacterial/archaeal ratio showed its steepest
decline when the HRT dropped from 3d to 1.5d, accompanied by the decline
of the methane yield from 223 to 51 mL-1
g-1 VS. This indicated that the methanogenic
population had declined significantly and was unable to support a
remarkable methane production at HRT between 3d to 1.5d. Moreover, the
significant decrease of VS removal efficiency occurred (from 39.4% to
5.2%, Fig. 3d) together with the accumulation of the VFA level (Fig.
3c). This thus indicated that HRT of 1.5d was too short and led to a
fast-microbial washout which reduced the process performance (Table 2).
Three major methanogenic orders at HRTs of 5, 3 and 1.5 d were also
quantified. The order Methanobacteriales significantly decreased
from 4.76×104 to 0.84×104 copies
μL-1 when passing from HRT of 5d to 1.5d. OrdersMethanosarcinales and Methanemicrobiales were
2.49×104 and 2.75×105 copies
μL-1 at 3d HRT, and both decreased to
0.05×104 copies μL-1 when the HRT
was shortened to 1.5d (Fig. 4). The results indicated that a very short
HRT cannot sustain the thermophilic AD operation although the
thermophilic bacterial had a faster growth rate.
3.5 Microbial community
under suboptimal and disturbed states targeting HRT
In the second stage (231-270 days), the bacterial genera were mainly
consisted of S1 (Thermotogae phyla),Cellulosibacter (Firmicutes phyla) and Clostridium(Firmicutes phyla) (Fig.5 a-c). These results corresponded with
those reported previously in similar substrates, i.e. food waste and/or
rain tree leaf with a longer HRT (20 days) (Ratanatamskul and Manpetch
2016; Zamanzadeh et al., 2017). It was previously shown that the genusS1 was probably SAOB (Li et al., 2018b) but it has not yet been
identified. In the present study, possible SAOB (generaTepidanaerobacter and Clostridium ) (Tachaapaikoon et al.,
2012) were detected at HRTs of 5, 3, and 1.5 day (Table 3). The total
percentage of potential SAOB increased from 6% to 11% as the HRT was
shortened from 5d to 1.5d. The abundance of genusTepidanaerobacter was 0.8% at HRT of 5d (Fig.5a), which
significantly increased to 2.9% and 4.2% when the HRT decreased to 3
and 1.5d, respectively (Fig.5b and 5c). A species within genusTepidanaerobacter , i.e. Tepidanaerobacter acetatoxydans ,
has previously been proved as a SAO bacterium (Westerholm et al., 2011)
and Tepidanaerobacter syntrophicus was isolated from thermophilic
(55°C) AD fed with either municipal solid waste or sewage sludge
(Sekiguchi et al., 2006). SAOB genus Clostridium did not change
significantly with the decrease in the HRT from 5d to 1.5d (Fig. 5a-c).
The detected increase of SAOB indicated the presence of a SAO-HM
pathway, which was promoted with the stepwise decrease of the HRT.
The archaeal community contained only hydrogenotrophic methanogens and
strongly differed between the different HRTs (Fig. 5 a-c). When the HRT
was decreased from 5d to 3d and then to 1.5d, the community richness and
diversity decreased as depicted in former section (Table 4). GenusMethanoculleus dominated with 57% at an HRT of 5d (Fig.5a), and
when the HRT was shortened to 3d, the genusMethanothermobacter became
the most dominate archaea (93.8%) (Fig.5b), which accompanied by an
increase in its co-culture SAOB Tepidanaerobacter syntrophicus .Methanosarcia gradually decreased 1.7- and 2.4- fold along with
shortening the HRT from 5 to 3 and 1.5 day, respectively. The doubling
time of Methanothermobacterium sp. was around 1.8h (Huber et al.,
1982), which was much shorter than the doubling time ofMethanoculleus sp. (12h) (Seely and Fahrney 1983). In
addition, the doubling time of genus Methanosarcina andMethanosaeta were reported to be at least 36h (Westerholm et al.,
2011). During short HRT stages (5 to 1.5d), the faster growing
hydrogenotrophic methanogens tend to dominate and revealed its
functionally redundant properties which may syntrophically metabolize
acetate with SAOB. In this study, the methane formation under the short
HRT condition was therefore possibly through the SAO-HM pathway.