3 Results and discussion
3.1 Reactor performance
analysis
3.1.1 Decolorization
performance
The decolorization performance of the three reactors was evaluated over
a period of nearly 100 days, as shown in Figure 2. All three reactors
showed high decolorization efficiency for RB5, but the MBBR reactor
exhibited better decolorization stability. One-way ANOVA results
indicated significant differences (p <0.05) in RB5
decolorization rates among the three reactors, which was probably due to
the fluctuation observed in the biochar and PPC reactors. At around day
21, samples of the carrier material were taken and preserved, and almond
shell biochar was added to the biochar reactor to supplement the
insufficient amount of almond shell biochar. Despite a slight decline
due to the lack of biochar, the biochar reactor’s discoloration rate for
RB5 remained stable above 80%. Around day 70, partial detachment of the
biofilm in the PPC reactor caused fluctuations in its treatment
performance for RB5. Overall, all the reactors’ treatment performance
was relatively stable, but the partial detachment of the biofilm caused
fluctuations in the biochar and PPC reactors. Obvious fluctuation in the
decolorization rate was observed in the biochar and PPC reactors.
3.1.2 Analysis of RB5 degradation
pathways
(1) UV-Vis analysis
The high concentration of dye in the influent exceeded the upper limit
of the UV spectrophotometer; therefore, a four-fold dilution was
necessary for both the influent and effluent samples. Figure 3 presents
the full spectrum scan results of the diluted samples. The
characteristic peaks of RB5 at 310 nm and 595 nm, which were caused by
the auxochrome and chromophore of RB5, were significantly reduced
(Droguett et al., 2020). The biological decolorization of the dye could
have resulted from adsorption onto biochar or biodegradation. Biochar
adsorption caused all peaks in the full spectrum to decrease
proportionally, while biodegradation was accompanied by the
disappearance of absorption peaks and the formation of new peaks (Chen
et al., 2003). The UV-Vis absorbance of the biochar reactor effluent was
slightly lower than that of the MBBR and PPC reactors in the 260-700 nm
range, which could be attributed to the adsorption effect of biochar.
The characteristic peaks of RB5 in
the effluent at 310 nm and 595 nm gradually decreased and tended to be
flat after passing through the biochar, MBBR, and PPC reactors,
indicating that the auxochrome and chromophore of RB5 were destroyed,
and the reactors had a biodegradation effect on RB5. A new
characteristic peak was formed at around 250 nm and 380 nm in the
effluent of the biochar, MBBR, and PPC reactors, which might have been
due to the absorption peak of bacterial metabolic products or colorless
intermediate products (Daneshvar et al., 2007). According to the
location of the new absorption peak (200-400 nm), it could be
preliminarily determined that they were likely to be monocyclic
compounds, and it was speculated that substances such as amine might
have been produced (Tan et al., 2016). It is speculated that there might
be some similarity in the effluent products between the biochar reactor
and the MBBR/PPC reactors.
(2) FTIR analysis
The FTIR analysis (Figure 4) revealed changes in the positions and
intensities of certain functional groups in the effluent of biochar,
MBBR, and PPC reactors. In the high wavenumber region (4000-2500
cm-1), stretching vibrations of O-H, N-H, C-H, and S-H
bonds were typically observed. The peak at 3386.7
cm-1, which might have corresponded to the stretching
vibration of -NH2 bonds, appeared as a broad peak. The
variation in peak area indicated differences in the content of
functional groups. The range of stretching vibration of C-H bonds was
2850-2710 cm-1, with the peak at 2829.67
cm-1 corresponding to the stretching vibration of
-CH2 bonds. The peak at 2829.67 cm-1in the effluent of biochar, MBBR, and PPC reactors was smaller than that
in the influent, indicating replacement of some -CH2bonds.
In the wavenumber region below 1900 cm-1, stretching
vibrations of -C=C-, -C=N-, -C=O bonds, and skeletal vibrations of
aromatic rings were observed, indicating the presence of benzene rings.
The peak at 1589.77 cm-1 might have corresponded to
the stretching vibration of -N=N- bonds, which became weaker after
decolorization, indicating that -N=N- bonds might have been destroyed
during the degradation process. The stretching vibration of the
R-SO2-R group was observed in the range of 1370-1290
cm-1 (Martorell et al., 2017). Additionally, a weaker
peak at 1117.43 cm-1 might have corresponded to the
bending vibration of -OH bonds. The characteristic peak at 835.20
cm-1 in the effluent of biochar, MBBR, and PPC
reactors suggested the presence of polycyclic aromatic hydrocarbons, and
the appearance of a small peak at 696.62 cm-1indicated the occurrence of out-of-plane bending vibrations of the N-H
bonds of amine compounds. The effluent of biochar, MBBR, and PPC
reactors might have generated similar compounds.
(3) LC-MS analysis
Figure 5 displays the liquid chromatography profiles of the influent,
biochar, MBBR, and PPC reactors. The chromatograms illustrate
significant variations between the effluents of the biochar reactor and
the MBBR and PPC reactors. In the MBBR and PPC reactor effluents, a
prominent peak was observed around 9.86 min, which was absent in the
influent chromatogram. This indicates that the production of organic
compounds with higher concentrations occurred in the MBBR and PPC
effluents that were not present in the biochar reactor effluent. It
should be noted that the soluble nature of RB5 in water prevented its
extraction in the organic phase, and therefore it was not detected in
the influent liquid chromatography.
Based on the findings in Figure 6, it can be concluded that there was a
significant increase in the ion peak at m/z 399 in both the MBBR and PPC
reactor effluent at 9.86 min. The molecular ion peak
[M-H+] + at m/z 399 suggested a
relative molecular mass of 400 for this substance, indicating that it
was only present in the effluent of the MBBR and PPC reactors. It is
tentatively suggested that this substance might be a benzene-containing
structure formed by further ring-opening of the naphthalene ring
structure in the active black 5 molecule after the double azo bond was
opened. The maximum absorption peak at around 250 nm in the
characteristic absorption of the benzene ring supports this conclusion.
However, the results also indicate that the MBBR and PPC reactors were
unable to completely mineralize the dye, and additional reaction systems
may be required to achieve thorough degradation.
The UV-Vis and FTIR analysis results indicated that the effluent from
the biochar reactor shared similarities with that of the MBBR and PPC
reactors. This suggests that these reactors may have produced similar
substances during the RB5 degradation process. However, the LC-MS
analysis showed significant differences between the biochar reactor and
the MBBR and PPC reactors. While parallel sample repeatability was good,
the degradation pathways of RB5 in the biochar and polymer carrier
reactors differed to some extent. High-efficiency degradation bacterial
isolation results showed that the microbial species responsible for the
RB5 degradation in the polymer carrier MBBR and PPC reactors were
similar. Therefore, it is possible that the differences and overlaps in
the degradation mechanism can be attributed to the presence of
high-efficiency degradation bacteria. The carrier material could have an
impact on the high-efficiency degradation bacterial community in the
reactor, which may affect the RB5 degradation mechanism.
3.2 Analysis of microbial community structure in
reactors
3.2.1 Analysis of microbial community structure on the
carrier
(1) Statistical analysis of diversity indices
Based on Table 1, the ACE and Chao1 indices indicated that P1 had the
highest richness, while Simpson and Shannon indices showed that P1 had
good evenness. M2 and P1 had similar richness, as shown by the ACE and
Chao1 results. A1 had good evenness according to the Simpson index,
while P1 had good evenness according to the Shannon index. Overall, the
richness of microorganisms on the biochar carrier was lower than that on
MBBR and PPC, possibly due to the smaller pores of the almond shell
biochar carrier, which could be easily covered by a dense biofilm,
limiting dissolved oxygen diffusion into the inner layer of the carrier,
and leading to decreased overall richness. Simpson and Shannon
observations showed that the evenness of microorganisms on MBBR carriers
was slightly lower than that on biochar and PPC carriers. The
PD_whole_tree results indicated that the abundance of microorganisms
on MBBR and PPC was higher than that on biochar, suggesting differences
in microbial diversity among different carriers.
(2) Species distribution histogram
Based on the results presented in Figure 7a, the dominant phyla on the
three different carriers were Proteobacteria , Firmicutes ,
and Bacteroidetes . Specifically, the biochar carrier had a higher
proportion of Firmicutes (53.3±2.0%),while the MBBR and PPC
carriers had relatively abundant Proteobacteria andBacteroidetes . Previous studies had shown thatProteobacteria (Wang et al., 2008a) and Firmicutes(Ramalho et al., 2007a; You and Teng, 2009) were effective in degrading
RB5, and a mixed community of Bacteroidetes could also
effectively degrade RB5 (Forss et al., 2013). Synergistetes were
more abundant on the biochar carrier, while Chloroflexi bacteria
were more commonly found on the MBBR and PPC carriers.
At the family level (Figure 7b), the dominant families on the biochar
carrier were f-Family_XIV , f-Peptococcaceae , andf-s74e-6049 , while on the MBBR carrier, the dominant genera wereParacoccus , Flavobacterium , and Taibaiella , and on
the PPC carrier, the dominant genera were Paracoccus ,f-Peptococcaceae , Desulfuribacillus , andDesulfitibacter . Paracoccus had been shown to effectively
degrade dyes (Bheemaraddi et al., 2014). The results suggested that the
carrier played an important role in the growth and enrichment of
microorganisms. Different carriers had different microbial functional
structures, depending on the characteristics of the carrier material,
porosity, surface area, and other factors (Wang et al., 2014). In
addition, there were very low levels of Shewanella on all three
reactor carriers, with only 0.01%±0.004%, 0.01%±0.01%, and
0.004%±0.006%, respectively, which might have been due to competition
between different genera (Hibbing et al., 2010; Zhu et al., 2019).
The microbial community structure on the carriers in this study was
complex, and the efficient degrading bacteria for RB5 was likely to be a
microbial community. Previous studies had shown that microbial
communities could effectively degrade RB5. For example, the natural
microbial community DDMZ1, composed of Burkholderia ,Achromobacter , Stenotrophomonas , Pseudomonas , andEnterobacter , showed stable decolorization efficiency for RB5
(Zhang et al., 2019). Another study isolated a microbial community
consisting of A. hydrophila, R. mucilaginosa , G.
pseudocandidum , and E. coli from dye wastewater and found that
this community achieved a decolorization rate of over 95% for RB5
within 24 hours, with a more stable decolorization effect than a single
strain (Flórez Restrepo et al., 2018). Compared to single strains, mixed
microbial communities had stronger advantages in completely mineralizing
azo dyes due to the synergistic effect between different degrading
microbial communities, which could better degrade azo dyes (Xie et al.,
2020; Saratale et al., 2010).
3.2.2 Isolation of high-efficiency degradation bacterial
community
The effluent from the reactors was acclimatized with increasing
concentrations of RB5 (Figure 8), starting from 100mg/L. After 8 hours,
no significant discoloration effect was observed, and the
OD600 did not show a significant increase. However,
gradual discoloration began to appear in the effluent of all three
reactors after 8 hours. At a concentration of 200 mg/L, discoloration
was observed in the effluent of all three reactors at 4 and 8 hours. At
a concentration of 300 mg/L, discoloration first appeared in the
MBBR reactor group at 4 hours,
with MBBR reactor group having a slightly higher OD600 than biochar
reactor group and PPC reactor group. This early discoloration may be
attributed to the effect of acclimatization and the accumulation of a
large number of degrading bacteria. At 24 hours, all three reactors had
completed discoloration, with MBBR reactor group having the highest
OD600. At 48 hours, the discoloration rate of all three reactors was
nearly 97%, with MBBR reactor group showing the highest
OD600, indicating the completion of acclimatization.
Subsequently, the enriched liquid after acclimatization was diluted and
spread plated five times, resulting in the isolation of highly efficient
RB5-degrading bacterial communities from the effluent of the three
reactors.
3.2.3 Analysis of the RB5 efficient degradation bacterial
community
structure
(1) Statistical analysis of diversity indices
Table 2 shows that the ACE and Chao1 indices were highest in M1 and M2
in the MBBR reactor, indicating a higher abundance of the microbial
community with high RB5 degradation efficiency in the MBBR reactor.
Additionally, the Simpson and Shannon indices were also highest in M1
and M2, suggesting greater diversity in the MBBR reactor. In contrast,
A1 and A2 had the lowest abundance based on the ACE and Chao1 indices.
Overall, the abundance of the microbial community with high RB5
degradation efficiency was smaller in the biochar reactor than in the
MBBR and PPC reactors based on the ACE and Chao1 indices. However, based
on the Simpson and Shannon indices, the functional bacterial composition
was more complex in the MBBR reactor than in the biochar and PPC
reactors, indicating greater diversity in the MBBR reactor. The
PD_whole_tree analysis showed that the abundance of the three reactors
was similar.
(2) Species distribution histogram
At the phylum level, Figure 9a shows that in the efficient RB5 degrading
bacterial communities of the biochar reactor, Clostridiales andPseudomonadales had the highest proportions, accounting for
nearly 100% of the community. In the MBBR efficient RB5 degrading
bacterial community, Pseudomonadales accounted for approximately
27-30%, Enterobacterales accounted for approximately 23%,Burkholderiales accounted for approximately 25-28%, andBacillales accounted for approximately 20%. In the PPC reactor’s
efficient RB5 degrading bacterial community, Pseudomonadalesaccounted for approximately 65-67%, and Enterobacteralesaccounted for approximately 31-33%. The biochar reactor had a higher
abundance of Clostridiales than the other reactors, butEnterobacterales were not detected in the biochar reactor.Burkholderiales and Bacillales were present in higher
numbers in the MBBR reactor than in the biochar and PPC reactors.Burkholderia , a member of the Burkholderiales order, can
synergistically degrade RB5 with other bacterial species, which may be
why the MBBR reactor showed the earliest decolorization effect among the
efficient RB5 degrading bacterial communities.
According to Figure 9b, at the genus level, the highly efficient
degrading bacterial communities in the biochar reactor were dominated by
the Pseudomonas genus (approximately 62-63%), followed by theClostridium_sensu_stricto_1 genus (approximately 35-37%). In
the MBBR reactor, the dominant genera were Pseudomonas(approximately 27-30%), Shewanella (approximately 23-24%),Delftia (approximately 25-28%), and Bacillus(approximately 20%). In the PPC reactor, the dominant genera werePseudomonas (approximately 65-67%) and Shewanella(approximately 31-33%). The Shewanella genus was not present in
the biochar reactor but accounted for a large proportion in the MBBR and
PPC reactors, possibly due to the electron transfer efficiency and
affinity between Shewanella and the carrier (Shi et al., 2007).Clostridium sensu stricto 1 was found only in the biochar reactor
and was the dominant species. In the MBBR reactor, a large number ofDelftia and Bacillus species were detected, but they were
almost absent in the biochar and PPC reactors. Pseudomonas , which
was present in all three reactors, had been extensively documented to
have good decolorization effects on RB5 (Wang et al., 2008b; Hussain et
al., 2013). Bacillus and Delftia were also capable of
effectively degrading RB5 and solubilizing phosphate in the substrate,
respectively, indicating their potential synergistic action (Dafale et
al., 2008; Liao et al., 2013). In bottle experiments, the efficient
degradation microbial community in the MBBR reactor showed earlier
decolorization of RB5 than those in the biochar and PPC reactors,
possibly due to the diverse functional bacteria present. The efficient
degradation microbial communities in the MBBR and PPC reactors had high
similarities and had consistent degradation mechanisms.Shewanella , which was artificially inoculated as an RB5-degrading
bacterium during reactor setup, did not become the dominant species on
the reactor carrier, but screening showed that it was a dominant
functional bacterium for RB5 degradation in both MBBR and PPC reactors,
working in conjunction with other functional bacteria to effectively
degrade RB5.
3.3 The impact of carrier physicochemical properties on
reactor
performance
Microbial attachment and fixation on carrier surfaces is an important
aspect of reactor performance, influenced by both the physicochemical
properties of the carriers and the surface characteristics of the
microorganisms. Previous studies have shown that carrier roughness, pH,
and pore size affect the growth and stability of biofilms in packed bed
reactors (Al-Amshawee et al., 2021; Jaroszynski et al., 2011).
Almond shell biochar showed slightly alkaline pH, with a pH value of
8.28±0.20. It is worth noting that most biochar samples reported in the
literature had an alkaline pH value (Greenough et al., 2021). The pH of
the MBBR carrier was 7.41±0.22, which was also slightly alkaline.
Alkaline conditions were conducive to microbial growth and the
development of microbial communities on the surface of the MBBR carrier,
as well as the formation of biofilms and diverse functional bacteria
(Dai et al., 2021). The pH of the PPC carrier was 6.80±0.04.
Surface roughness was an important factor in carrier selection and
reactor design, as it influenced mass transfer efficiency, effective
contact area, and protection of microorganisms (Messing and Oppermann,
1979). As shown in Figure 10, biochar carrier had a highly porous
surface with small pore sizes (0.1 - 0.5 μm), which might limit the
initial growth of microorganisms (Messing and Oppermann, 1979). In the
reactor that effectively degraded bacteria with a length of about 0.6 to
1.5 μm, these pores could not serve as their living place, and
microorganisms attached to the top or nearby areas of the pores. After
the reactor had been in operation, these pores were filled with
microbial metabolites, and the pore structure was no longer obvious,
resulting in the appearance of a large number of spherical substances.
Most of the surface of the biochar carrier was covered with a layer of
attached biofilm growth, and there were rod-shaped bacteria present on
the surface and grooves of the biochar carrier.
In contrast, MBBR carriers had many protrusions, grooves, and
micro-cracks on their surface, and their overall structure had a toothed
shape. The high surface roughness of the carrier resulted in slow growth
of biofilm on it, which could form a biofilm structure that was more
resistant to flushing (Al-Amshawee et al., 2020). There were mainly two
reasons for this. Firstly, compared with smooth surfaces, the rough
surface of the carrier increased the effective contact area between
bacteria and the carrier. Secondly, the rough parts of the carrier’s
surface, such as holes and cracks, protected the attached bacteria
(Al-Amshawee et al., 2021). After the reactor operated, microorganisms
accumulated in large numbers on the carrier surface, and the biofilm
structure became compact, with spherical and rod-shaped bacteria
interweaving and growing together. As the biofilm matured, it became
more stable and less likely to detach, which was beneficial for the
sustained and stable operation of the reactor.
The PPC carriers had an average diameter of 1cm and a relatively smooth
surface, but with many large pores ranging from 0.5 to 1.5 μm in
diameter. These pores could serve as attachment and survival sites for
microorganisms, providing protection against hydraulic shear stress.
However, some studies had shown that the porous structure of biofilm
carriers might have led to low mass transfer efficiency (Cao et al.,
2017). After the reactor operates, these large pores were filled with a
large number of rod-shaped bacteria, which grew abundantly and covered
the pores and their surroundings. The microbial abundance on the surface
of PPC carriers was relatively high, and this carrier could effectively
enrich microorganisms. However, there was a risk of the biofilm being
washed away from the polyurethane carriers (Al-Amshawee et al., 2020).
Initially, all carriers had clean surfaces without any attached
biofilms. After reactor operation, dense biofilms formed on all
carriers. The pore size of the almond shell biochar was too small to
serve as a habitat for microorganisms. As a result, the abundance of
microorganisms on the almond shell biochar carrier was lower than that
on the MBBR and PPC carriers. The surface of the MBBR carrier was
rougher than that of the PPC carrier, and the rough surface reduced the
possibility of biofilm detachment (Huang et al., 2018). The MBBR carrier
could form a robust and impact-resistant biofilm structure. At the same
time, the surface heterogeneity of the MBBR carrier provided a better
living space for microorganisms and increased the utilization rate of
adsorbed organic matter by attached microorganisms (Dutta et al., 2014).
Therefore, microorganisms could more easily utilize adsorbed compounds
and convert them into final products.