Bioresource Technology 265 (2018) 17–24

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Cell membrane characteristics and microbial population distribution of T

MBBR and IFAS with different dissolved oxygen concentration
⁎
Si-jia Ma, Li-li Ding, Hai-dong Hu, Hai-jun Ma, Ke Xu, Hui Huang, Jin-ju Geng, Hong-qiang Ren
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, Jiangsu, PR China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: This paper investigated the influences of different dissolved oxygen (DO) concentration (0.71–1.32, 2.13–3.02
Dissolved oxygen level and 4.31–5.16 mg/L) on cell membrane characteristics and microbial population distribution of moving biofilm
Moving bed biofilm reactor reactors. Two representative reactors, i.e., moving bed biofilm reactors and integrated fixed-film activated
Integrated fixed-film activated sludge sludge were operated. Results indicated that both DO concentration of 0.71–1.32 mg/L and 4.31–5.16 mg/L
Cell membrane characteristics
could increase membrane lipid mobile fraction (49.4%-67.4%) of the microbes, however, through prompting the
Microbial population distribution
synthesis of branched fatty acids and unsaturated fatty acids, respectively. For the biofilms, the abundance of
Bacteroidetes decreased and Actinobacteria increased with the increase of DO levels. The lowest EfOM content and
the highest microbial diversities (1.14–1.52) was observed at DO of 2.13–3.02 mg/L. Redundancy analysis
showed that changes of DO levels could alter cell membrane properties and bacterial community structures, and
subsequently significantly influenced effluent organic matter composition of moving biofilm reactors.

1. Introduction providing a large surface for the growth of biofilms (Huang et al.,
2015). Two representative reactors, i.e., moving-bed biofilm reactor
Increasingly stringent limits on wastewater effluent control have (MBBR) and integrated fixed-film activated sludge (IFAS) are increasing
spurred demand for more reliable and better optimization of waste- popular modification of conventional activated sludge (AS). Those re-
water treatment plants (WWTPs). Moving biofilm reactors rely on the actors have higher nitrification rates and organic matter removal effi-
use of moving plastic carriers in which microorganisms form biofilms, ciency (Onnis-Hayden et al., 2011; Hu et al., 2017), greater resistance
keeping slow-growing microbes in the system (Bassin et al., 2011), and to shock loading (Wells et al., 2017), less sludge production (Gu et al.,

⁎
Corresponding author.
E-mail address: [email protected] (H.-q. Ren).

https://doi.org/10.1016/j.biortech.2018.03.111
Received 30 January 2018; Received in revised form 20 March 2018; Accepted 23 March 2018
Available online 30 March 2018
0960-8524/ © 2018 Published by Elsevier Ltd.
S.-j. Ma et al. Bioresource Technology 265 (2018) 17–24

2017) and better micropollutants removal (Luo et al., 2015), compared always accounted for a small part of the total bacteria (Liang et al.,
to AS system. MBBRs and IFAS reactors have been also used to treat 2015). Therefore, it is crucial to investigate the impacts of different DO
industrial wastewater (Huang et al., 2015; Hu et al., 2017). concentration on microbial community structure, since they affect the
Accurate and reliable control of dissolved oxygen (DO) concentra- EfOM composition and stability of biological process.
tion is necessary for the operation of WWTPs, because DO significantly The objectives of this paper were to investigate the impacts of dif-
influences the removal of organics and nitrogen, effluent quality and ferent DO concentration on the PLFA composition and fluidity of cell
operational costs in WWTPs. The impacts of DO concentration on the membrane lipids, and the bacterial community structure in MBBRs and
features of AS have been widely reported (Yadav et al., 2014; Ma et al., IFAS reactors. A better understanding of the impact of DO levels on
2016; Li et al., 2018), but few published research articles are available moving biofilm reactor will be useful to minimizing energy consump-
on the influences of DO levels in moving biofilm reactors. Wang et al. tion and developing reliable and better operating strategies of WWTPs.
(2006) succeeded to decrease the chemical oxygen demand (COD) re-
moval efficiency by 13% in MBBRs by decreasing DO from 2 to 1 mg/L. 2. Materials and methods
Cao et al. (2017) found good removal of COD and nitrogen at a DO of
2.5 mg/L, and increasing the DO level was inhibitory to denitrification 2.1. MBBRs and IFAS reactors operation
in a moving bed sequencing batch reactor. IFAS and MBBR systems
have prominent advantages compared with conventional AS system, Three MBBRs (referred to as M-A, M-B, and M-C) and three IFAS
but IFAS and MBBR systems are complex and insufficiently understood reactors (referred to as I-A, I-B, and I-C) were operated. DO of reactor
(Rosso et al., 2011; Malovanyy et al., 2015; Cao et al., 2017). Although M-A and I-A was 0.71–1.32 mg/L, DO of reactor M-B and I-B was
the COD removal efficiencies were similar, the EfOM underwent a 2.13–3.02 mg/L and, DO reactor of M-C and I-C was 4.31–5.16 mg/L.
transformation that increased its complexity under both IFAS and AS Air was introduced through six distributors on the bottom of the re-
treatment, and bacteria was the most likely reason for the increase in actors from an air pump. The DO level in each reactor was controlled by
the effluent’s molecular diversity (Tseng et al., 2013). Moreover, it has adjusting the aeration fluxes with the help of six rotameters. The
been reported that biofilm or flocs derived from IFAS reactors maybe oxygen levels were detected with an oxygen meter and probe (SG6,
distinctly differed from those originating from comparable moving bed METTLER TOLEDO Inc., USA). Sludge (mixed liquor suspended soilds
reactors or AS reactor (Zhang et al., 2006; Mahendran et al., 2012). was about 3500 mg/L) collected from the aeration tank in Dachang
Therefore, further investigation to optimize the DO level is needed to Municipal WWTP was seeded in the six reactors. After 24 h, the acti-
improve MBBR and IFAS performance and reduce operating costs. vated sludge was discharged from the MBBRs. The synthetic wastewater
The fluidity of cell membrane is indispensable in implementing consisted of glucose (400 mg/L), NH4Cl (120 mg/L), KH2PO4 (19 mg/
many functions of biological membrane, such as the absorption of nu- L), MgSO4·7H2O (25 mg/L), CaCl2·2H2O (11 mg/L). 0.6 mL of trace
trients or contaminants, as well as the transportation or extrusion of elements solution was added to 1 L of synthetic wastewater and the
many organic compounds, such as protein or carbohydrate (Ramos composition of the trace elements solution can be found in Ma et al.
et al., 2002; Laganowshy et al., 2014). Variations of cell membrane (2016). The cyclic time was 12 h, and consisted of filling (15 min), re-
fluidity could be having significant influence on the adsorption or de- action (690 min) and discharging (15 min) for the MBBRs, and filling
gradation of organic matters and the generation of soluble microbial (10 min), reaction (660 min), settling (40 min) and discharging
products (Ma et al., 2016; Ramos et al., 2002). Microbes can change the (10 min) for the IFAS reactors, respectively. The operational HRT and
constitution of membrane lipids to maintain cell membrane fluidity in organic loading rate was 11.5 h and 0.8 kg COD/m3·d. The operation of
responding to different conditions. And accumulation of branched fatty the six reactors was performed at temperature of 25 ± 2 °C and pH of
acids (BFA) or unsaturated fatty acids (UFA) could increase the fluidity 7 ± 0.1. In each cycle, 1 L of treated wastewater was discharged from
of cell membrane (Kaneda, 1991). It has been reported that an in- each reactor and 1 L of fresh wastewater was then added to each re-
creasing of aeration off time could decrease UFA content and increase actor. The mixing intensity was made similar by setting same rotating
BFA content in submerged membrane bioreactor (Chang et al., 2011). speed of the electric mixer in each reactor.
He et al. (2016) found that the increase of UFA in AS reactors can en-
hance the removal of organic matters at low temperature. Recently, Ma 2.2. Water quality analysis
et al. (2016) reported that DO of 0.71–1.32 mg/L and 4.31–5.16 mg/L
can increase the content of BFA in AS systems, and BFA significantly COD was measured according to Standard Methods (APHA, 2005).
influenced the EfOM composition. So far, most researches have focused Influent and effluent COD were measured every three days. To de-
on the impacts of operation parameters on cell membrane fluidity and termine the removal rate of COD, samples were collected at 0, 0.5, 1, 2,
phospholipid fatty acid (PLFA) composition of microorganisms in AS 3, 5, 7, 9 and 11 h at reaction periods, and this process was repeated
systems, little is known about the influences of DO levels on those five times on the 25th, 30th, 35th 40th, 45th and 60th day, respec-
properties of microorganisms in moving biofilm reactors. Analysis of tively. The method illustrated in Huang et al. (2014) was used to detect
shifts of microbial cell membrane properties (PLFA and fluidity) in re- the excitation and emission matrix (EEM) fluorescence of the effluent
sponse to different DO levels and the changes with time under same DO on the 30th and 60th day.
levels would be helpful to enhance treatment efficiency and decrease
SMP content in moving biofilm reactors. 2.3. Adhesion force measurements
Previous studies suggested that changing of DO levels could alter
microbial community structures in AS or membrane bioreactors, and The sludge and biofilm samples on the 60th day dried naturally and
the shifts of microbial population significantly influence the EfOM atomic force microscope (Multimode 8, Bruker Inc., Germany) was used
composition (Menniti et al., 2010; Ma et al., 2013). A high bacteria to detect the adhesion force by force-distance measurements (Ma et al.,
retention time could be maintained in attached biofilm system, thus 2016). Adhesion force and images were analyzed by NanoScopeAna-
increased microbial population diversity (Huang et al., 2015). Un- lysis software. Each sample was tested five times.
fortunately, limited information is known about the microbial ecology
of biological wastewater treatments combining suspended biomass and 2.4. Measurement of cell membrane lipid fluidity
attached biofilms (Reboleiro-Rivas et al., 2016). Recently, it has been
reported that the number of ammonia-oxidizing bacteria (AOB) and Lipid fluidity was determined by measuring the mobile fraction of
nitrite-oxidizing bacteria (NOB) increased with the increase of DO level the BODIPY FL-C12 (a fluorescent lipid probe) tagged plasma membrane
in moving bed sequencing batch reactor. However, AOB and NOB lipid (Mullineaux et al., 2006). Briefly, the biofilms were detached from

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S.-j. Ma et al. Bioresource Technology 265 (2018) 17–24

carriers and converted to suspended sludge with a concentration about Reactor M-A (100 ± 5.7 mg/L·h) and Reactor I-C (360 ± 14.4 mg/
3000 mg/L. The biofilms or sludge was incubated for 30 min with a L·h) > Reactor I-B (253.2 ± 25.32 mg/L·h) > Reactor I-A
final concentration of 1 μmol/L of BODIPY FL-C12. A FV1000 confocal (160 ± 16 mg/L·h). Nogueira et al. (2015) reported that a higher
laser scanning microscope (Olympus Corp., Tokyo, Japan) was used to mixing intensity (higher hydraulic shear strength and DO level) can
detect the fluorescence intensity of the selected area. The mobile frac- improve the external mass transfer coefficient in MBBR for wastewater
tion (Mf) of cell membrane lipid can be determined by treatment and can, therefore, lead to a faster removal of organic ma-
terial or harmful nutrients. The results indicated that the increase of DO
Mf = (Ft−F0)/(Fi−F0) (1)
levels caused by increasing aeration rate facilitated the quick removal
where Ft is the fluorescence intensity of the selected region after re- of COD in MBBRs and IFAS reactors. To better understand the impacts
covery for t min, Fi is the fluorescence intensity of the selected region of DO concentration on COD removal rates, adhesion forces of sludge
before bleaching and F0 is the fluorescence intensity of the selected and biofilms will be studied in Section 3.2.
region just after bleaching. Detailed experimental protocol can be found EEM peaks (Supplementary File), exhibiting peak maxima at Ex/
in Ma et al. (2016). Em = 275–285/305–310 nm and Ex/Em = 220–230/330–340 nm
(protein-like substances), Ex/Em = 340–345/380–390 nm (humic acid-
2.5. Measurements of phospholipid fatty acid (PLFA) like substances) and Ex/Em = 240–250/460–475 nm (fulvic acid-like
substances) were observed in the samples of the six reactors according
PLFA composition of microbes in MBBRs and IFAS reactors was to the method from Hu and Ren (2016). The fluorescence intensity of
analyzed on the 30th and 60th day. The PLFA extraction method was the peaks can be seen in Table 1. Protein-like substances was the major
used following the methodology outlined by Niu et al. (2013). Briefly, substance in all the samples. On day 60, the fluorescence intensity of
after extraction, a solid phase extraction column filled with silicic acid the humic acid-like substance decreased but the fulvic acid-like sub-
used to separate phospholipid. Through saponification and methyla- stance increased in the effluent of MBBRs, and all the fluorescence in-
tion, the PLFA was transformed into fatty acid methyl esters (FAMEs). tensity of peaks decreased in the effluent of IFAS reactors. The fluor-
Detection of the FAMEs was performed by Aglient 7890A GC, and the escence intensity of EfOM was the highest when operating at DO of
MIDI Sherlock Microbial Identification System (MIDI, Newark, DE). The 4.13–5.16 mg/L, which is agreed with the observations that increasing
nomenclature of PLFA was performed as: total carbon atoms number: DO levels or shear force (DO of 6 mg/L) lead to an increase of soluble
double bonds number, following the position ω of the double bond from microbial products in activated sludge or MBR (Menniti et al., 2010; Ma
the methyl end of the molecule. The “iso” and “anteiso” branching are et al., 2016). Particularly, the fluorescence intensity of EfOM of IFAS
indicated by the prefixes “i” and “a”, respectively. reactors was lower than those in MBBRs. The hybrid nature and higher
effective biomass of the IFAS system might explain the superior effluent
2.6. Microbial community structures and data analysis results (Huang et al., 2015). The results indicated that the variation of
DO level obviously influenced the COD removal efficiency and DO of
The samples collected on the 30th and 60th day were used to detect 2.13–3.02 mg/L can lead to the lowest fluorescence intensity of protein-
the population distribution of the six reactors using an Illumina MiSeq like and humic acid-like or fulvic acid-like in effluents of both IFAS
platform (Illumina Inc., USA). DNA of the sludge or biofilm samples was reactors and MBBRs.
extracted using the FastDNA™ Spin Kit for soil (MP Biomedicals, Santa
Ana, CA) according to the manufacturer’s manual. To amplify and se- 3.2. Adhesion force of biofilms and sludge
quence the V1V2 hypervariable region of the 16S rRNA gene, forward
primer (50-AGAGTTTGATYMTGGCTCAG-30) and reverse primer (50- The adhesion forces and Ra roughness of biofilms were the highest
TGCTGCCTCCCGTAGGAGT-30) were selected and different 8-bases at DO of 4.13–5.16 mg/L (Table 2) and an increase of DO concentration
barcodes and a Guanine were linked to the 50 end of each primer. results in an increase of adhesion force for biofilms in IFAS and MBBRs
Detailed experimental protocol can be found in Supplementary File. (Supplementary File). The adhesion forces of biofilms in Reactor M-C
Heatmap was drawn by “R” statistical packages. Redundancy analysis and I-C (DO of 4.13–5.16 mg/L) were 37.9 ± 2.8 nN and 31.2 ± 4.2
(RDA) was performed by CANOCO 4.5 software and SPSS 19.0 software nN. These results are similar to the adhesion force of biofilms found by
was used to calculate the significant correlation (P). Zhu et al. (2015) of 35.1 ± 5.2 nN at DO of 4.0–7.5 mg/L. The adhe-
sion force of sludge in Reactor I-C was the highest but the Ra roughness
3. Results and discussion of sludge was the highest in Reactor I-A.
Pussak et al. (2014) found that increasing of adhesion forces could
3.1. Effluent quality of MBBRs and IFAS reactors promote the adhesion of carbohydrate on specific receptors in biolo-
gical processes. Recently, BinAhmed et al. (2018) reported that hy-
It can be seen that about 10 days and 13 days were used to allow the drophilic polydopamine coatings decrease bacterial (Pseudomonas) ad-
IFAS reactors and MBBRs to achieved stable COD removal efficiencies hesion forces during short bacterium-membrane contact times, and the
(Fig. 1a and 1c), respectively. The HRT (11.61 h), pH (6.7–7.7), DO adhesion forces are weakened by the presence of natural organic
(3.62–6.34 mg/L), OLR (0.22–0.31 kg COD/m3·d, COD (107–149 mg/L) matter. Furthermore, these results are effective as a biofouling control
concentration of seed sludge in the WWTP was different from the HRT strategy. The removal of organics includes of two steps: adsorption and
(23 h), pH (7 ± 0.1), DO (0.71–5.16 mg/L), OLR (0.8 kg COD/m3·d, degradation. As shown in Fig. 1b and 1d, the removal rate of COD was
COD (400 mg/L) concentration of the lab-scale reactors. The changes of the highest at DO concentration of 4.13–5.16 mg/L (Reactor M-C and I-
those operational parameters significantly influenced the microbial C); meanwhile, the adhesion forces were also the highest. Those results
physiological status, thus determining the start-up of the reactors (Cao suggested that an increase of DO concentration might promote the
et al., 2017; Ma et al., 2017; Bassin et al., 2011). This is agreed with the adhesion of glucose on sludge or biofilms surface in IFAS and MBBRs,
findings that a period of time intended for biofilm formation in MBBRs which promotes quick COD removal. This phenomenon is contrary to
(Bassin et al., 2011). The average removal efficiency of COD increased the findings stating that an increased DO concentration results in a
with the increase of DO level. This is consistent with the findings that decrease of adhesion forces in activated sludge (Ma et al., 2016). Li
increasing aeration pressure results in enhanced COD removal effi- et al. (2014) found that physicochemical properties of cell surface sig-
ciencies (Xin et al., 2016). As shown in Fig. 1b and 1d, on 1th hour, the nificantly influence the adhesion of organic matter. BinAhmed et al.
removal rate of COD decreased as follows: Reactor M-C (2018) also presented evidence that the adhesion of Pseudomonas to the
(192.8 ± 11.14 mg/L·h) > Reactor M-B (112.4 ± 4.14 mg/L·h) > UF membrane surface is mediated by cell surface macromolecules. The

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Fig. 1. COD concentration variation in the six reactors, (a) influent and effluent of MBBRs; (b) COD removal efficiency in one cycle; (c) influent and effluent of IFAS
reactors; (d) COD removal efficiency in one cycle.

Table 1 above results suggested that the variation of DO level can influence the
EEM fluorescence intensity of the peaks in the effluent. M-A30 represents the mechanical properties of biofilm and sludge, therefore affecting the
effluent of Reactor M-A on the 30th day. COD removal performance.
Reactor code Protein-like Humic acid-like Fulvic acid-like

M-A30 134.6 144.5 61.4 3.3. Fluidity and PLFAs composition of cell membranes in biofilms and
M-B30 116.7 128.9 53.2 sludge
M-C30 314.6 71.4 122.3
M-A60 179.6 38.7 136.1 Fang et al. (2007) found that microbes can change cell membrane
M-B60 148.9 63.2 182.9
M-C60 247.2 33.4 119.9
fluidity to adapt to different environmental conditions and maintain
I-A30 113.8 132.6 64.1 optimal growth. The fluorescence intensity recovery percentages could
I-B30 97.64 74.32 121.4 be used to represent the fluidity of cell membrane lipids. As shown in
I-C30 143.2 76.21 132.6 Tables 3 and 4, the microbes in Reactor M-B (36.7%) and I-B (44.6% for
I-A60 99.01 92.27 32.7
biofilm and 26.5% for sludge) had the lowest fluidity in cell membrane
I-B60 86.39 66.32 63.4
I-C60 116.7 69.46 73.6 lipids; meanwhile, the fluorescence intensity of effluents of Reactor M-B
and I-B by EEM was the lowest (Supplementary File). This phenomenon
is confirmed by observing a slower mobility rate in cell membrane lipid
Table 2 synchronization with lower effluent protein content in AS (Ma et al.,
Adhesion force and Ra roughness of sludge and biofilms surface on the 60th 2016). Ramos et al. (2002) found that microorganisms can change cell
day. membrane fluidity to regulate the extrusion of compounds to external
Reactor code Adhesion force (nN) Roughness (nN)
Table 3
M-A 16.4 ± 6.4 155 ± 16.2 Changes of fluorescence intensity of selected area on the 60th day in IFAS re-
M-B 25.7 ± 3.9 179 ± 19.5 actors.
M-C 37.9 ± 2.8 254 ± 21.2
IA-A 13.1 ± 2.6 152.1 ± 14.3 Reactor code IM-A IM-B IM-C IA-A IA-B IA-C
IA-B 12.4 ± 2.3 75.6 ± 7.8
IA-C 16.5 ± 1.2 88.9 ± 6.8 Before bleaching 1785.6 1741.7 2360.1 1258.1 669.8 1183.2
IM-A 14.5 ± 1.4 91.7 ± 11.2 After bleaching 1082.8 841.5 1306.7 595.3 360.3 519.1
IM-B 29.7 ± 3.4 157.9 ± 12.1 1 min 1120.8 982.6 1455.2 670.7 377.9 577.9
IM-C 31.2 ± 4.2 223 ± 16.5 3 min 1190.7 1060.5 1619.2 779.9 387.7 666.1
5 min 1334.2 1128.7 1687.8 845.4 393.9 714.7
7 min 1394.4 1228.4 1798.2 877.7 404.8 814.8
9 min 1486.6 1315.8 1973.7 950.5 462.1 872.9
11 min 1556.3 1243.4 1925.1 947.4 442.4 846.1
Mf 67.4% 44.6% 58.7% 53.1% 26.5% 49.2%

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Table 4 increased obviously and UFA content changed slightly at DO of

Changes of fluorescence intensity of selected area on the 60th day in MBBR 0.71–1.32 mg/L and 2.13–3.02 mg/L, while UFA increased obviously
reactors. and BFA content changed slightly at DO of 4.31–5.16 mg/L. For IFAS
Reactor code M-A M-B M-C reactors, the content of SFA of biofilms and sludge changed slightly
from the 30th day to the 60th day. UFA also increased with the increase
Before bleaching 1876.2 3061.7 2463.1 of DO levels and anteiso-fatty acids (AFA) was dominant in sludge at
After bleaching 1041.6 1964.9 1531.1
DO of 0.71–1.32 mg/L.
1 min 1117.5 1982.5 1588.5
3 min 1305.3 2050.1 1663.3 It has been reported that the increase of UFA can cause the fall of
5 min 1398.2 2121.4 1727.4 the lipid melting point of a membrane to maintain good mobility and
7 min 1471.4 2287.6 1815.6 enhance the mass transfer of microbes at low temperature (Niu et al.,
9 min 1521.8 2309.1 1913.7
2013). BFA are necessary for the optimal growth of two B. subtilis
11 min 1587.7 2367.3 1991.7
Mf 65.4% 36.7% 49.4%
mutants (Kaneda, 1991). The changes of environmental conditions
could result in different PLFA compositions, thus lead to the changes of
BFA or UFA content (Fang et al., 2007). In this study, BFA were more
media. It has been reported that an increase of cell membrane fluidity of abundant than UFA at low DO level, and UFA were more abundant than
sludge can enhance the mass transfer efficiency at low temperature (Niu BFA at high DO levels. C14:0 iso, C15:0 anteiso, C16:0 iso and C18:0 iso
et al., 2012). Microbes in MBBR (65.4%) and IFAS (67.4% for biofilm were the major BFA in MBBRs and IFAS reactors. 18:1w7c, 17:1w8c
and 53.1% for sludge) operating at DO of 0.71–1.32 mg/L showed the and 16:1w7c were the dominant UFA in MBBRs, and 18:1w9c, 18:1w7c
highest Mf, but the EfOM content was not the highest. This phenom- and 16:1w7c were the dominant UFA in IFAS reactors. The abundance
enon might be due to the fact that membrane protein can also affect cell of C15:0 anteiso was the highest at DO of 0.71–1.32 mg/L. Kaneda
membrane fluidity. (1991) found that C15:0 anteiso has a phase transition temperature of
As shown in Fig. 2, for MBBRs, the abundance of saturated fatty 23 °C, which is lower than other BFA. Consequently, microorganisms
acids (SFA) decreased in all samples from the 30th day to 60th day. SFA can efficient maintain the fluidity of cell membrane. These results in-
were dominant at DO of 4.31–5.16 mg/L and 2.13–3.02 mg/L with a dicate that microbes of sludge or biofilms in MBBRs and IFAS could
content of 58.6%–60.2%. BFA content decreased and UFA content in- increase the synthesis of BFA (especially the content of AFA) to increase
creased with the increase of DO levels. On the 60th day, BFA content the cell membrane lipid fluidity at DO concentration of 0.71–1.32 mg/

Fig. 2. PLFA composition of the biofilms and sludge in MBBR and IFAS reactors. M-A30 represents the biofilms samples of M-A on the 30th day. IM-A30 and IA-A30
represents the biofilms and sludge samples of I-A on the 30th day, respectively.

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Fig. 3. The relative abundance of different phylum and heat map of genera in MBBR and IFAS reactors. (a) and (b), MBBRs in phylum and genera level; (c) and (d)
IFAS reactors in phylum and genera level on the 30th day; (e) and (f) IFAS reactors in phylum and genera level on the 60th day, respectively.

L, and mainly increases the synthesis of UFA to increase the cell (30.64%). It has been reported that Proteobacteria was the most domi-
membrane fluidity at DO of 4.31–5.16 mg/L. The above results re- nant phyla during the start-up period in MBBRs and these bacteria are
flected the effects of DO levels on cell membrane properties. easy to attach to the surface of bio-carriers (Zhu et al., 2015; Atabek
and Camesano, 2007). Yadav et al. (2014) reported that the changes of
DO levels (1–4 mg/L) obviously influenced the abundance of Proteo-
3.4. Evolution of bacterial community
bacteria in industrial wastewater treatment by activated sludge. At DO
of 0.71–1.32 mg/L, Actinobacteria has the highest abundance and in-
The bacterial community at phylum level dominantly consist of
creases from 36.05% to 45.96% from the 30th day to 60th day. On the
Proteobacteria, Bacteroidetes and Actinobacteria throughout the whole
60th day, the content of Bacteroidetes increased and Actinobacteria de-
operation of the six reactors (Fig. 3), which agrees with the findings by
creased with the increase of DO levels. The content of Candidatus Sac-
previous studies (Zhu et al., 2015; Ma et al., 2017). And the changes of
charibacteria was 20.33% in Reactor M-A at the 30th day and decreased
DO levels obviously influenced the relative abundance of different
to 0.57% at the 60th day, while its abundance increased slightly in
phyla in the reactors. In MBBRs, Proteobacteria was the most abundant
Reactor M-B and M-C from the 30th day to the 60th day. For IFAS
phylum at DO of 2.13–3.02 mg/L and 4.31–5.16 mg/L. On the 30th day,
reactors, the content of Proteobacteria decreased with the increase of DO
Proteobacteria increased with the increase of the DO level, and the
level in biofilms and increased with the increase of DO levels in sludge
content of Proteobacteria at DO of 4.31–5.16 mg/L (66.01%) was more
on day 30. TM7 was more abundant at DO of 0.71–1.32 mg/L
than twice that at DO of 0.71–1.32 mg/L (28.41%) and 2.13–3.02 mg/L

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Fig. 4. RDA analysis of bacterial community with PLFA and EfOM composition, (a) MBBR; (b) IFAS, red represent sludge and purple represent biofilms. Peak p
(protein-like), peak f (fulvic acid-like) and peak h (humic acid-like). (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

(4.91%–6.27%) and 2.13–3.02 mg/L (1.65%–6.43%) than at DO of polysaccharides in their cells (Begum and Batista, 2012; Ma et al.,
4.31–5.16 mg/L (0.42% to 0.57%). It has been reported that TM7 are 2016). The content of Flavobacterium in Reactor I-C was lower than that
filamentous bacteria and increased at the period of biofilm formation in Reactor M-C, but the COD removal efficiency was faster in Reactor I-
(Zhu et al., 2015). On the 60th day, Bacteroidetes was the most abun- C than that in Reactor M-C, which means the fast removal of COD may
dant phyla with DO of 0.71–1.32 mg/L and Actinobacteria was the most depend on other bacteria. The above results showed that the variation
abundant phyla at DO of 2.13–3.02 mg/L and 4.31–5.16 mg/L. Bacter- of DO levels can change the microbial community, therefore influen-
oidetes plays a key role in degrading high molecular organic matters, cing the COD removal performance and the effluent quality of MBBRs
such as protein and chitin (Ma et al., 2013). In this study, the reactors and IFAS reactors. Shannon-Wiener index of microbial diversity
(M-A and I-C) had the highest Bacteroidetes content, but the content of (Supplementary File) were higher in both MBBR and IFAS reactors
EfOM was not the lowest. The results of this study agree with the operating at DO of 2.13–3.02 mg/L. Tocchi et al. (2012) reported that a
findings that the removal of protein may rely on other bacteria (Ma high biodiversity in the activated sludge was prerequisite for high
et al., 2016). The structure and adaptability of Actinobacteria are more performances in dairy wastewater treatment.
stable and higher than others; thus, microbes can better maintain bio-
film structures (Zhu et al., 2015).
3.5. RDA analysis of bacterial population distribution with PLFA and EfOM
For MBBRs, operating at DO of 0.71–1.32 mg/L, the microbes at
composition
genus level were dominated by Saccharibacteria_genera_incertae_sedis
(20.33%) and Nakamurella (12.02%) on day 30. The abundance of the
As shown in Fig. 4a, for MBBRs, the redundancy analyses (Fig. 4a),
two genera decreased, and the abundance of Sphingomonas and
RDA1 and RDA2, provided 65.7% and 25.8% of the total variance,
Enterobacter increased on day 60. On the 30th day, Nakamurella
respectively. Pseudomonas, Paludibacter and Acidisoma were related to
(3.26%) and Microbacterium (3.49%) were dominating at DO of
protein-like substances (p < 0.05). Nakamurella was related to humic
2.13–3.02 mg/L, while Arcobacter (26.88%) and Acinetobacter (5.11%)
acid-like and fulvic acid-like substances (p < 0.05). SFA correlated to
were abundant at DO of 4.31–5.16 mg/L. The content of
protein-like substances, and AFA was correlated with Pseudomonas,
Saccharibacteria_genera_incertae_sedis and Nakamurella increased on the
Mucilaginibacte and Paludibacter (p < 0.05). For IFAS reactors, the
60th day in Reactor M-B and M-C. Furthermore, in Reactor M-C, the
RDA1 and RDA2, provided 78.7% and 13.9% of the total variance
content of Flavobacterium increased to 3.41% on the 60th day. It has
(Fig. 4b). In sludge, Flavobacterium was related to protein-like sun-
been found that an increase of aeration intensity could lead to an in-
stances (p < 0.05); Ferruginibacter, Nakamurella, TM7_gener-
crease of Flavobacterium in activated sludge, and Flavobacterium plays
a_incertae_sedis and Geothrix were correlated with humic acid-like sub-
an important role in the rapid degradation of organics (Xin et al., 2016).
stances (p < 0.05); Thauera and Dechloromonas were related to fulvic
The observations of this paper verified this phenomenon by finding that
acid-like substances (p < 0.05); AFA was correlated with Ottowia
the fastest COD removal efficiency in Reactor M-C had the largest
(p < 0.05). In biofilms, Nakamurella and Microbacterium were related
amount of Flavobacterium. Nitrospira affiliated to the nitrite oxidizing
to humic acid-like substances (p < 0.05); IFA was correlated with
bacteria (NOB) were found to be more abundant in Reactor M-C
Nakamurella, TM7_genera_incertae_sedis and Tetrasphaera (p < 0.05). It
(0.44%) compared to Reactor M-A where the NOB was 0.04% and
has been widely reported that these PLFAs and genera can significantly
compared to Reactor M-B where the NOB was 0.03%. It has been re-
affect the removal of organic matters and EfOM (Ma et al., 2013; Niu
ported that moving plastic carriers can keep slow-growing microbes in
et al., 2013; Young et al., 2017). These results show that a change of DO
the system and attached biomass had higher nitrification activity than
concentration, promotes the changes of bacterial communities and the
suspended biomass (Bassin et al., 2011). For IFAS reactors, operating at
cell membrane fluidity and PLFA composition, which influences the
DO of 0.71–1.32 mg/L, Nakamurella was the most abundant genus in
EfOM in MBBRs and IFAS system.
both biofilms (50.84%) and sludge (51.58%) on the 30th day. At DO of
2.13–3.02 mg/L, Micropruina was the most dominated genus in sludge.
Notably, due to the variation in DO levels, the bacterial community 4. Conclusions
varied greatly. Nakamurella and Micropruina can store a lot of
The results suggested that an increase of DO concentration lead to

23
S.-j. Ma et al. Bioresource Technology 265 (2018) 17–24

an increasing removal rate of COD and increasing surface adhesion 171–178.

force in MBBR and IFAS systems. Both Do concentration of Liang, W., Yu, C., Ren, H., Geng, J., Ding, L., Xu, K., 2015. Minimization of nitrous oxide
emission from CASS process treating low carbon source domestic wastewater: effect
0.71–1.32 mg/L and 4.31–5.16 mg/L can increase membrane lipid of feeding strategy and aeration rate. Bioresour. Technol. 198, 172–180.
mobile fraction (49.4%-67.4%) of the microbes, however, through Luo, Y., Jiang, Q., Ngo, H.H., Nghiem, L.D., Hai, F.I., Price, W.E., Wang, J., Guo, W.,
prompting the synthesis of BFA and UFA, respectively. Microbial di- 2015. Evaluation of micropollutant removal and fouling reduction in a hybrid
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versities were the highest and EfOM content was the lowest operating at 355–359.
DO of 2.13–3.02 mg/L. PLFA compositions were significantly correlated Ma, J., Wang, Z., Yang, Y., Mei, X., Wu, Z., 2013. Correlating microbial community
with some genera, such as Nakamurella, Pseudomonas, and Ottowia. structure and composition with aeration intensity in submerged membrane bior-
eactors by 454 high-throughput pyrosequencing. Water Res. 47 (2), 859–869.
Ma, S.J., Ding, L.L., Huang, H., Geng, J.J., Xu, K., Zhang, Y., Ren, H.Q., 2016. Effects of
Acknowledgement DO levels on surface force, cell membrane properties and microbial community dy-
namics of activated sludge. Bioresour. Technol. 214, 645–652.
Ma, W., Han, Y., Ma, W., Han, H., Zhu, H., Xu, C., Wang, D., 2017. Enhanced nitrogen
This work was supported by the National Natural Science
removal from coal gasification wastewater by simultaneous nitrification and deni-
Foundation of China (51608254), Natural Science Foundation of trification (SND) in an oxygen-limited aeration sequencing batch biofilm reactor.
Jiangsu Province (BK20160655), National Science and Technology Bioresour. Technol. 244, 84–91.
Major Project (2017ZX07204001), Jiangsu Science and Technology Mahendran, B., Lishman, L., Liss, S.N., 2012. Structural, physicochemical and microbial
properties and biofilms in integrated fixed-film activated sludge systems. Water Res.
Projects (BE2017632, BA2016012). 46, 5085–5101.
Malovanyy, A., Trela, J., Plaza, E., 2015. Mainstream wastewater treatment in integrated
Appendix A. Supplementary data fixed film activated sludge (IFAS) reactor by partial nitritation/anammox process.
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