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Landfill leachate treatment through the combination of genetically engineered bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane filtration processes
Journal Pre-proof Landfill leachate treatment through the combination of genetically engineered bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane filtration processes Fuliang Bai, Hui Tian, Jun Ma PII:
S0269-7491(19)36728-4
DOI:
https://doi.org/10.1016/j.envpol.2020.114061
Reference:
ENPO 114061
To appear in:
Environmental Pollution
Received Date: 12 November 2019 Revised Date:
20 January 2020
Accepted Date: 22 January 2020
Please cite this article as: Bai, F., Tian, H., Ma, J., Landfill leachate treatment through the combination of genetically engineered bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane filtration processes, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2020.114061. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Landfill leachate treatment through the combination of genetically engineered
2
bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane
3
filtration processes
4 5
Fuliang Bai1, 2, Hui Tian3, Jun Ma1*
6
1
7
Technology, Harbin, Heilongjiang, 150090, People's Republic of China
8
2
9
Republic of China
State Key Laboratory of Urban Water and Environment, Harbin Institute of
Lubin Environmental Protection Equipment (Shanghai) Co., Ltd, Shanghai, People's
10
3
11
Republic of China
12
Corresponding Author: Jun Ma. Address: No 73, School of Environment, Huanghe
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Road, Nangang Area, Harbin, 150090, China. Tel/fax: +86 451 86283010. 12
14
Email: [email protected]; [email protected].
School of Life Science, Harbin Institute of Technology, Harbin, 150001, People's
15 16
Abstract
17
This study developed a process of genetically engineered bacteria Rhodococcus
18
erythropolis expressing Nirs and AMO combined with membrane bioreactor (MBR),
19
nanofiltration (NF) and reverse osmosis (RO) membrane (pRho-NA-MNR) for
20
advanced treatment of landfill leachate. Results demonstrated that pRho-NA-MNR
21
presented higher removal rate of chemical oxygen demand (COD), biological oxygen
22
demand (BOD), ammonia nitrogen (N-NH4), total nitrogen (TN) and total organic
23
carbon (TOC) than activated sludge (AS-MNR) system. Administration of pRho-NA
24
increased nitrification by converting N-NH4 to nitrite (N-NO2) and Nitrate (N-NO3),
25
and promoting denitrification by converting N-NO2 to nitrogen (N2) in the landfill
26
leachate treatment, promoted the pH control, increased sludge activity and effluent
27
yield, shortened phase length adaptation under alternating aerobic-anoxic conditions.
28
pRho-NA increased the nitration and denitrifying rate in the aerobic and anaerobic
29
stage in the system by increasing Cyt cd1 and Cyt c expression in the activated sludge.
30
Nitrogen removal by nitrification and denitrification was positively correlated to the
31
concentration of Nirs and AMO expression. Treatment with pRho-NA promoted
32
pollutant removal efficiency of membrane bioreactor, nanofiltration and reverse
33
osmosis membrane processes in landfill leachate. In conclusion, data suggest that
34
pRho-NA-MNR facilitates the formation of granular sludge and enhances comparable
35
removal of nitrogen and organic compounds, indicating the practice of this process
36
should be considered in landfill leachate treatment system.
37
Keywords: Landfill leachate; Rhodococcus erythropolis; Nirs; AMO; pRho-NA-MNR
38 39
1. Introduction
40
Sanitary landfills are the most widely employed method of municipal waste treatment
41
(Navia and Ross, 2009). Landfill leachate is generated from sanitary landfills and
42
much concern with respect to the pollution potential, which is caused by the
43
degradation of solid waste (Azzouz et al., 2018; Jiang et al., 2018; Warwick et al.,
44
2018). Landfill leachate is composed of various compositions with high
45
contamination, such as high contents of organic matter, inorganic substances and
46
other pollutants (Iskander et al., 2018). Thus, it requires appropriate and efficient
47
technology to remove the toxic compositions before discharge for high contents of
48
COD, N-NH4 and disorder of carbon-nitrogen ratio (Calli et al., 2006). However,
49
landfill leachate is extremely difficult to treat and effluent without qualified treatment
50
processes impacts quality of ecological environment and human health (Dia et al.,
51
2018; Ishak et al., 2018; Silveira et al., 2018).
52
Conventional sewage processes are not efficient for the treatment of landfill leachate
53
due to an inadequate C to N ratio (Fudala-Ksiazek et al., 2014). Currently, many
54
integrated processes have been developed for the treatment of landfill leachate
55
(Zolfaghari et al., 2018). Generally, the membrane bioreactor (MBR) technology is
56
regarded as a prominent process in the treatment of landfill leachate deriving from the
57
decomposition of waste in landfills (Hashisho et al., 2016). MBR combining with
58
sequencing batch reactor (SBR) technologies are introduced in treating high-strength
59
landfill leachate (El-Fadel and Hashisho, 2014). The combination of single reactor for
60
high activity ammonia removal over nitrite (SHARON)-anammox processes is an
61
effective method in removing high organic content of young leachate (Akgul et al.,
62
2013). Integrated MBR-nanofiltration (NF) system effectively removes the high
63
concentrations of COD and N-NH4, which improves the quality and increases reuse of
64
the effluent in landfill leachate (Silva et al., 2018). The reverse osmosis (RO) also is
65
applied to treat landfill leachate and provides high quality effluent by reducing the
66
effluent COD from MBR (Hasar et al., 2009). Thus, we assumed that integrating
67
MBR-NF-RO system may enhance current discharge standards and achieve the
68
effluent feasible for reuse in treatment of municipal landfill leachate.
69
Landfill leachate contains high concentrations of N-NH4 and needs to remove nitrate
70
nitrogen, nitrite nitrogen, N-NH4 and TN using activated sludge biological system
71
(Wang et al., 2018). Autotrophic and heterotrophic nitrifying bacteria play crucial role
72
in achieving nitrogen removal via nitrite pathway from landfill leachate (Sun et al.,
73
2013). Introducing microorganisms with high biodegradation ability and resistance to
74
N-NH4, and TN may be a promising solution in the treatment of landfill leachate
75
(Dadrasnia et al., 2017). A study has achieved the startup of anaerobic ammonium
76
oxidation using the conventional activated sludge integrated processes in a MBR with
77
a maximum nitrogen removal rate (Wang et al., 2009). In addition, another study
78
(Fudala-Ksiazek et al., 2014) demonstrates that nitrogen removal via the nitrite
79
pathway is beneficial for carbon-limited and highly ammonia-loaded mixtures, which
80
can lead to a reduction in the external carbon source needed to support denitrification
81
in landfill leachate. Furthermore, a higher denitrification rate and lower surplus sludge
82
production contributes to achieve shortcut nitrification, the activity of nitrite-oxidising
83
bacteria and nitrogen removal (Zhou et al., 2011). In response to characteristics of
84
landfill leachate, novel and efficient technologies that allow for the reduction of
85
pollutants must be developed, optimized and implemented.
86
Nevertheless, conventional treatment method of landfill leachates is insufficient to
87
remove the high concentration of nitrogen, organic matter, color, heavy metals and
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toxic substances in landfill leachates. Thus, various processes combined with
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biological treatment with additional nitrobacteria have technological and economic
90
advantages in the treatment of landfill leachates. Our previous study indicates that
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Nirs and ppk gene expressed by nitrifying bacteria promotes biological removal of
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N-NH4 in the sewage treatment system (Bai et al., 2019). In this study, we
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investigated the efficiency of a novel genetically engineered bacteria Rhodococcus
94
erythropolis expressing nitrite reductase gene (Nirs) and ammonia monooxygenase
95
(AMO) (pRho-NA) in landfill leachate. The multiprocessing efficiency of
96
MBR-NF-RO integrated pRho-NA was investigated in an industrial scale landfill
97
leachate treatment.
98 99
2. Materials and methods
100
2.1 MBR-NF-RO (MNR) system
101
Landfill leachates were come from Laogang County Landfill Treatment Plant
102
(Shanghai, China). A subexternal MBR, NF and RO membrane processes were used
103
to treat landfill leachates from AS or pRho-NA reactors. Landfill leachates were
104
extracted from the same source in activated sludge (AS-MNR) and pRho-NA-MNR
105
system. Two systems were arranged in the same condition during the experimental
106
period. Both AS-MNR and pRho-NA-MNR systems included aerobic fluidized
107
reactors, anaerobic reactors, MBR, NF and RO membrane. The identical dimensions
108
of aerobic fluidized reactors and anaerobic reactors were 400 × 400 × 4.5 m (L × W ×
109
H) and 300 × 300 × 4.5 m (L × W × H), respectively. The membrane area of each
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MBR, NF and RO was 38.5 m2, 32.0 m2 and 30.0 m2 respectively. Schematic of the
111
reactors and the types of components are illustrated in Figure 1. pRho-NA (1x105/ml)
112
was added into aerobic fluidized reactors in pRho-NA-MNR system for a total of 15 d.
113
During the adaptive phase, the dissolved oxygen is 0.5-0.8 mg L-1 and pH is arranged
114
from 6.9 to 7.8. The landfill leachate was extracted from the same source in two
115
systems (AS-MNR and pRho-NA-MNR) during the experimental period.
116
2.2 Generation of pRho-NA
117
Autotrophic nitrifying bacteria was isolated from landfill leachate and identified as
118
Rhodococcus
119
monooxygenase (AMO) were synthesized by Invitrogen (Invitrogen, Carlsbad, CA,
120
USA) and cloned into pSIM5.0 vector and then transfected into Rhodococcus
erythropolis.
Nitrite
reductase
gene
(Nirs)
and
ammonia
121
erythropolis using electrotransfection (Bio-Rad) according to the manufacturer’s
122
instrument. A genetically engineered bacterium of expressing Nirs and AMO was
123
named pRho-NA. pRho-NA was amplified using fermentation as described previously
124
(Xu et al., 2019).
125
2.3 Parameter analysis
126
Physico-chemical parameters of water such as dissolved oxygen, total suspended
127
solids (TSS), BOD, COD, TN, and N-NH4 were measured using standard methods as
128
described previously (Karre et al., 2012). N-NO3 and N-NO2 were determined on a
129
Metrohm 761 Compact Ion Chromatograph (Zofingen, Switzerland) equipped with a
130
conductivity detector. The pH was measured with a Consort C532 pH meter
131
(Turnhout, Belgium). TOC was analyzed using Multi N/C3000 TOC (Analytik Jena
132
AG, Germany). The amount of N2 production was measured using gas
133
chromatography-mass spectrometry (Agilent Technologies Inc., Santa Clara, CA,
134
USA) (Matsushita et al., 2017).
135
2.4 Quantitative RT-PCR (qRT-PCR)
136
Total RNA was extracted from activated sludge in aerobic reactors and anaerobic
137
reactors using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the
138
manufacturer’s instrument. Total RNA (1 µg) was reverse transcribed into cDNA
139
using High Capacity cDNA Reverse Transcription Kit (cat no. 4368814, Invitrogen,
140
Shanghai, China) according to the manufacturer’s protocol. All gene primer sequences
141
including Nirs, AMO, Cyt cd1, Cyt c and β-actin are shown in Supplementary material
142
file (Table S1). The expression levels of the mRNAs were performed using SYBR
143
Green reagent (Roche, Indianapolis, IN, USA) equipment with ABI 7900HT Fast
144
Real-time PCR System (Applied Biosystems). Relative gene expression was
145
calculated using 2-∆∆Ct method.
146
2.5 Western blotting
147
Whole cell extracts from aerobic reactors and anaerobic reactors were prepared and
148
extracted using a protein extraction reagent (Roche, Switzerland). Concentration of
149
protein was measured with a BCA protein assay kit (Pierce Biotechnology, USA) and
150
equal amounts of the protein (40 µg) were separated by 12.5% SDS-polyacrylamide
151
gels (SDS-PAGE), transferred onto nitrocellulose membranes and then blocked with
152
5.0% BSA in TBST (Tris-buffered saline containing 0.1% Tween 20) at room
153
temperature for 2 h. Membranes were incubated with primary antibody Nirs, AMO,
154
Cyt cd1, Cyt c and β-actin (1:1,200, ABCAM) at 4 °C overnight. Membranes were
155
subsequently incubated with goat anti-mouse or anti-rabbit IgG at 37 °C for 2 h, all
156
bands were detected using the ECL chemiluminescence kit (Pierce Biotechnology,
157
USA). The protein of bands was normalized to β-actin, and relative protein expression
158
was analyzed by densitometry analysis program, ImageJ Software (National
159
Instituties of Health, USA).
160
2.6 Statistical analysis
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Standard statistical parameters are expressed as mean ± standard deviation (SD).
162
SPSS software version 22.0 (SPSS, Chicago, IL, USA) was used for statistical
163
analyses. Experimental data were also analyzed by Student t test or one-way ANOVA
164
followed by Tukey’s post hoc test. All experiments were performed at least three
165
times. P < 0.05 was defined as statistically significant.
166 167
3 Results
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3.1 Characteristics of landfill leachate and reactor performances
169
The monitored parameters for landfill leachate are shown in Table S2. The diagram of
170
landfill leachate system was shown in Figure 1. Landfill leachate system included
171
aerobic reactor, anaerobic reactor, MBR, NF and RO system during the operational
172
period. The average N-NH4 and TN concentration 1,275 mg L-1 and 1,736 mg L-1,
173
respectively. The average TCOD (COD) and BOD5 was 16,725 (15,720) and
174
1,428 mg L-1, respectively. The features of landfill leachate are high ammoniacal
175
nitrogen content, little biodegradability, high TN/BOD5, and poor BOD5/COD and
176
COD/N-NH4. The landfill leachate in this study demonstrated high chloride
177
concentrations (average, 2,350 mg L-1; range, 2,045-2,438 mg L-1). The concentration
178
of TOC and dissolved organic carbon (DOC) was 1,925 mg L-1 and 1,348 mg L-1,
179
respectively. Concentration of heavy metals including Cu, Zn, Cd, Cr, and Ni were
180
0.041, 0.782, 0.017, 0.184 and 0.086 mg L-1, respectively, in the landfill leachate.
181
The parameters of landfill leachate in aerobic reactors during adoptive operation
182
between pRho-NA-MNR and AS-MNR system are shown in Table S3. In the aerobic
183
and anaerobic reactors, all parameters were monitored using online measurements.
184
The hydraulic retention time (HRT) was 118 h and 88 h in aerobic reactors during
185
adoptive operation in AS-MNR and pRho-NA-MNR system, respectively (p < 0.01).
186
The sludge retention time (SRT) was 26.8 h and 24.2 h in aerobic reactors during
187
adoptive operation in AS-MNR and pRho-NA-MNR system, respectively (p < 0.01).
188
The obtained results showed that the HRT of landfill leachate treatment in anaerobic
189
was shorter in pRho-NA-MNR than AS-MNR system (mean, 28.5 h vs 24.0 h, p <
190
0.05). TSS parameter values were higher in adopted in MBR operation, aerobic and
191
anaerobic reactors in pRho-NA-MNR than AS-MNR system (p<0.01). In the
192
accommodation with activated sludge with or without PRho-NA followed by aerobic
193
culture, concentrations of COD, BOD5, DOC, TN, Cl−, N-NH4, N-NO3 and N-NO2
194
were compared in two systems. Concentrations of O2 were > 0.8 mg L-1 and <
195
0.05 mg L-1 in aerobic and anaerobic reactors, respectively. A higher nitrification and
196
denitrification rate and COD removal rate was observed in the aerobic reactors in
197
pRho-NA-MNR compared with AS-MNR system. pRho-NA-MNR reactor had higher
198
SV30 and lower concentrations of COD, TOC, TN and N-NH4 compared to AS-MNR
199
reactor during 15 d accommodation. However, accumulation of N-NO3 and N-NO2
200
were higher in aerobic reactor in pRho-NA-MNR system than that in AS-MNR
201
system. There were no significant differences of concentration of Cl-, BOD5, bulk
202
wasting ratio and bulk wasting flow rate in the aerobic reactors between
203
pRho-NA-MNR and AS-MNR system. The results showed that concentration of
204
N-NO3 and N-NO2 was lower, while pH and N2 production was higher in the
205
anaerobic reactors in pRho-NA-MNR than that in AS-MNR system.
206
3.2 Effectiveness of treatment in the pRho-NA-MNR system
207
In the stable portion period, the landfill leachate treatment efficiency including
208
aerobic and anaerobic reactors, MBR, NF and RO effluent was compared between
209
pRho-NA-MNR and AS-MNR system. As shown in Figure 2A-D, pRho-NA-MNR
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system had higher removal rate of TN, N-NH4, BOD5 and COD than AS-MNR
211
system in aerobic reactor. Administration of pRho-NA promoted stability of system
212
determined by the bulk VSS concentration/the COD mass remove (gCODR)
213
(VSS/gCODR) and decreased value of pH by promoting ammoniation in aerobic
214
reactor compared to AS system (Figure 2E-F). Concentrations of TN, N-NH4, BOD5,
215
and COD in MBR effluent were lower in pRho-NA-MNR system than AS-MNR
216
system (Figure 3). The average COD and N-NH4 removal remained above 98.4% and
217
97.2%, respectively, in pRho-NA-MNR system, which were higher than AS-MNR
218
system. TN removal rate was 95.2% and 84.6% in pRho-NA-MNR and AS-MNR
219
system, respectively. Highly effective removing rate of N-NH4 (> 97.8 %), TN (>
220
96.5%), BOD5 (> 80.2%) and COD (> 93.2%%) was observed in NF effluent in
221
pRho-NA-MNR system, whereas the remove rate of N-NH4, TN, BOD5 and COD
222
averaged 84.3%, 72.2%, 70.1% and 80.6% in NF effluent in AS-MNR system,
223
respectively (Figure 4). During the stable operational phase, the averaged
224
concentrations of TN, N-NH4, BOD5 and COD in the RO effluent were 3.5 mg L-1, 3.0
225
mg L-1, 1.0 mg L-1 and 10.6 mg L-1, respectively, in pRho-NA-MNR system. However,
226
a higher averaged concentrations of TN (22.4 mg L-1), N-NH4 (18.8 mg L-1), BOD5
227
(5.6 mg L-1) and COD (62.3 mg L-1) were observed in the RO effluent in AS-MNR
228
system (Figure 5). The averaged TN and DOC concentrations were higher in
229
anaerobic reactors in pRho-NA-MNR system than AS-MNR system (95.6% vs. 87.4%
230
for TN; 98.2% vs. 93.2% for DOC; Figure 6). It showed that concentrations of N-NO3,
231
N-NO2 and production of N2 were higher in anaerobic reactors in pRho-NA-MNR
232
system than that in AS-MNR system (Figure 7). The mean water recovery rates of
233
MBR, NF and RO membrane separation process in pRho-NA-MNR system were
234
15.4%, 36.5% and 47.8%, respectively. However, the mean water recovery rates of
235
MBR, NF and RO membrane separation process in pRho-NA-AS system were 18.6%,
236
41.8% and 54.6%, respectively. Concentrates of MRB returned to the aerobic reactor.
237
Concentrates of NF and RO were discharged and got further treatment. Consequently,
238
the results demonstrated that the pRho-NA-MNR system and AS-MNR system could
239
effectively remove the heavy metals including Cu, Zn, Cd, Cr, and Ni in the landfill
240
leachate (Table S4). There were no significant differences between two treatment
241
systems.
242
3.3 Efficacy of effluent in the pRho-NA-MNR system
243
During the continuous operation, trans-membrane pressure (TMP) in two systems was
244
< 0.35 bar. When TMP of MBR, NF and RO membrane reached 0.40 bar, membrane
245
needed to wash using 0.1% NaClO. As shown in Table S5, the averaged frequency of
246
membrane cleaning MBR, NF and RO was 60 d/time, 35.5 d/time and 30.0 d/time in
247
the pRho-NA-MNR system. The HRT and SRT in pRho-NA-MNR system were 3
248
min and 7 min, while HRT and SRT in AS-MNR system were 7 min and 3 min in a
249
total of 10 min cycle during operation period. However, the averaged frequency of
250
membrane cleaning MBR, NF and RO was 42.5 d/time, 27.0 d/time and 23.5 d/time
251
in the AS-MNR system. To achieve the standard of effluent, the HRT was 72 h and 96
252
h in aerobic reactors in pRho-NA-MNR system and AS-MNR system, respectively (p
253
< 0.01). The SRT was 20.5 h and 13.6 h in aerobic reactors in pRho-NA-MNR system
254
and AS-MNR system, respectively (p < 0.01). The HRT was 16.5 h and 13.0 h in
255
aerobic reactors in pRho-NA-MNR system and AS-MNR system, respectively (p <
256
0.05). The obtained results showed that the SRT of landfill leachate treatment in
257
anaerobic was shorter in pRho-NA-MNR system than that in AS-MNR system (mean,
258
14.5 h vs 17.5 h, p < 0.05). The final effluent approximately remained 650 m3/d and
259
420 m3/d during continuous operation in pRho-NA-MNR system and AS-MNR
260
system, respectively (p < 0.01).
261
3.4 Nitrate and nitrite metabolism in the pRho-NA-MNR system
262
To verify the treatment efficacy of pRho-NA, N-NH4 reduction rate, N-NO3 and
263
N-NO2 generation rate in aerobic reactors, N-NO3 and N-NO2 reduction rate and N2
264
generation rate in anaerobic reactors were analyzed in two systems. As shown in
265
Figure 8A-C, N-NH4 reduction rate, N-NO3 and N-NO2 generation rate in aerobic
266
reactors were higher in pRho-NA-MNR system than AS-MNR system during the
267
stable operation period. During continuous operation, N2 generation rate in anaerobic
268
reactors was recorded and pRho-NA-MNR system had a higher N2 generation rate
269
than AS-MNR system (Figure 8D). Nitrification and denittification rate was also
270
higher in pRho-NA-MNR system than AS-MNR system. The averaged concentrations
271
of N-NO3 and N-NO2 in the effluent of MBR, NF and RO were 0.30, 0.20, 0.08 mg
272
L-1, respectively, in pRho-NA-MNR system, while was higher and averaged 0.13,
273
0.08 and 0.02 mg L-1, respectively, in AS-MNR system.
274
3.5 The functional gene and protein expression levels in the pRho-NA-MNR
275
system
276
During the operation, Nirs and AMO gene and protein expression levels were
277
identified during landfill leachate treatment operation period. As shown in Figure
278
9A-C, a significantly increasing gene and protein expression levels of Nirs and AMO
279
were observed in pRho-NA-MNR system compared to AS-MNR system. To further
280
explain more treatment efficacy of pRho-NA, detailed analysis of associations
281
between nitrogen removal rate and concentration of pRho-NA or Nirs/AMO
282
expression were undertaken. In the aerobic reactors, expression of Cyt cd1 and Cyt c
283
was up-regulated in pRho-NA-MNR system, compared to AS-MNR system, which
284
increased the denitrifying rate in the aerobic stage (Figure 9D). The results showed
285
that nitrogen removal rate by nitrification and denitrification was positively correlated
286
to the concentration of Nirs and AMO expression in aerobic stage (Figure 9E-F). pH
287
is associated with the nitrification and denitrification rate, thus, we investigated the
288
changes of pH in aerobic reactors in two systems. Compared to AS-MNR system,
289
pRho-NA administration promoted the pH control and increased sludge activity
290
(Figure 9G-H). Also, phase length adaptation under alternating aerobic-anoxic
291
conditions was shortened by additional pRho-NA (Figure 9I).
292 293
4. Discussion
294
Previous studies have shown that various bacterial strains have the capacities to
295
degrade the high toxicity values of DOC, N-NH4, BOD5, and COD in landfill leachate
296
under different suitable treatment conditions (Khattabi et al., 2007; Kim et al., 2006;
297
Xiao et al., 2007). Nitrification and denitrification of landfill leachate under different
298
hydraulic retention time in a two-stage anoxic/oxic combined MBR process presents
299
high removal of COD, BOD5, N-NH4 and TN (Liu et al., 2018). In addition, MBR
300
combined with an upflow anaerobic sludge blanket process is an effective method for
301
the treatment of high nitrogen content in leachate (Akgul et al., 2013). Furthermore,
302
MBR and NF membrane can efficiently remove COD, TOC, TKN, and N-NH4 in a
303
full-scale landfill leachate treatment system (Campagna et al., 2013). Moreover, RO
304
membrane filtration is able to achieve satisfactory results in terms of water quality for
305
landfill leachate discharge, process stability and membrane flux (Li et al., 2009).
306
The present study aimed to evaluate the efficiency of a novel genetic engineering
307
bacterial Rhodococcus erythropolis that was able to express Nirs and AMO gene and
308
identified the N-NH4 removal capacity in landfill leachate. In order to achieve current
309
discharge standards, three membranes of MBR, NF and RO cooperated with
310
pRho-NA were employed as safeguards for the treatment of landfill leachate. Here,
311
this study showed that pRho-NA-MNR achieved satisfactory outcomes of effluent
312
water and treatment efficiency for landfill leachate. In this study, administration of
313
pRho-NA not only shorted phase length of adaptation, increased nitrogen removal,
314
but also promoted the pH control, stability of system, increased sludge concentration,
315
activity and effluent yield and quality in the treatment of landfill leachate. Biological
316
co-treatment of pRho-NA with MNR presented higher removal rate of N-NH4, TN,
317
DOC, BOD5, and COD than AS-MNR system, suggesting it may be a reliable method
318
for landfill leachate toxicity reduction. Although our design genetically modified
319
bacteria to treat landfill leachate in an open system and it could easily spread
320
throughout the environment, these bacteria will not cause any problem in the
321
environment due to bacteria themselves come from the environment. As the current
322
study employed pRho-NA to enhance the treatment efficiency in the treatment of
323
landfill leachate, it would be interesting to investigate the biotoxicity and
324
bioavailability of pRho-NA to environment in future studies.
325
A previous study is undertaken to investigate the excellent removal efficiencies for
326
COD (92.67%) and N-NH4 (98.9%) in landfill leachate treatment process based on
327
UASB and submerged MBR (Wang et al., 2016). Integrated MBR-NF system leads to
328
high removal of pollutants and makes the treated effluent feasible for water reuse
329
(Silva et al., 2018). EDTA-Na2Zn as draw solution can effectively eliminate toxicity
330
of landfill leachate using MBR-NF-RO and data indicate that this method is
331
economical and eco-friendly for treatment of different types of landfill leachate (Niu
332
et al., 2016). However, the efficiency of TN, COD, and DOC removal needs to
333
improve during co-treatment in landfill leachate. Bioaugmentation displays the
334
highest effectiveness in removing N-NH4 and COD among all method of landfill
335
leachate treatments (Yu et al., 2014). Thus, this study explored the treatment efficacy
336
of MBR-NF-RO in dislodging TN, COD, and DOC and data found that removal
337
efficiency of TN, COD, and DOC remarkably increased and improved during landfill
338
leachate treatment. Interestingly, decreasing of toxicity and the membrane fouling was
339
observed in pRho-NA-MNR system compared to AS-MNR during stable operatic
340
period. The removal efficiency of DOC fractions and nitrogen from landfill leachate
341
dependents on the concentration and activity of aerobic granular sludge SBR (Wei et
342
al., 2012). It is quite conceivable that the DOC and TN removal rate is the
343
bioaugmentation process in landfill leachate, and our data demonstrated the enhanced
344
efficiency of nitrification and denitrification by using pRho-NA.
345
Generation of much more microbial products increases the values of soluble inert
346
COD to total COD in the leachate of aerobic landfill with leachate recirculation
347
(Bilgili et al., 2008). The syntrophic association has been observed between the
348
ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, which further explains the
349
incomplete nitrification phenomenon (Yusof et al., 2011). This study confirmed the
350
nitrogen and organic matter removal rate of genetic engineering bacteria pRho-NA
351
and found the efficient strategy of biological-ecological combinative technology via
352
using pRho-NA and MBR-NF-RO membrane, which emphasized the importance of
353
modeling, design, and operation of landfill leachate treatment system. The higher TN,
354
N-NH4, COD and BOD removal efficiency in pRho-NA-MNR system may attribute
355
to DO, pH control and stability of system, suggesting it is a high-efficiency process in
356
the treatment of landfill leachate.
357
TN and N-NH4 removal rate by nitrifying bacteria and denitrifying bacteria
358
dependents on a series of biological processes. The Nirs gene is essential for the
359
attachment of the active site haem group of Wolinella succinogenes cytochrome c
360
nitrite reductase (Pisa et al., 2002). The genetic potential for nitrifying bacteria and
361
the corresponding process activity, fine-scale environmentally induced changes in
362
rates of nitrate reduction are likely to be controlled at gene, cellular and protein levels
363
(Dong et al., 2009). Function and diversity of amoA and nirS gene is associated with
364
denitrification in MBR treating landfill leachate, which affects nitrification and
365
ammonia accumulation, causing a severe suppression of Nitrosomonas and an
366
increase in the relative abundance of Nitrosospira (Remmas et al., 2016). In this study,
367
Nirs gene was cloned into the genome of nitrifying bacteria and presented high TN
368
and N-NH4 removal rate in landfill leachate. The sequential activities of AMO and
369
hydroxylamine dehydrogenase (HAO) play crucial role in translation from oxidize
370
ammonia (N-NH4) to nitrite (NO2-) in denitrification (Bennett et al., 2016).
371
Abundance of AMO gene is associated with sewage treatment efficiency in a
372
full-scale municipal wastewater treatment plant (Wu et al., 2013). Communities in
373
N-removal onsite wastewater treatment systems technologies differ slightly in terms
374
of nitrifying and denitrifying bacterial communities (Wigginton et al., 2018). In this
375
study, we observed strong community similarity patterns driven by Nirs and AMO
376
gene expression in the aerobic reactors in the treatment of landfill leachate. Our
377
previous data have indicated that bacillus subtilis expressing Nirs and ppk cooperated
378
with aerobic fluidized MBR system is suitable for domestic wastewater treatment by
379
promoting metabolism of nitrogen and phosphorus (Bai et al., 2019). However, ppk is
380
not suitable for the treatment of landfill leachate for the lacking of phosphorus.
381
Therefore, we selected Rhodococcus erythropolis expressing Nirs and AMO and
382
membrane filtration processes and analyzed the treatment efficacy of pRho-NA in
383
landfill leachate. The most abundant, distributional and containing pRho-NA was
384
associated with pH and DO changes, which was a useful process controlling
385
parameter for the organics and nitrogen removal at high charge input of landfill
386
leachate. Investigation of the expression of AMO and nirS in denitrifying bacteria
387
revealed that gene and protein level of AMO and nirS was successfully expressed and
388
achieved high N-NH4 and COD removal rate. In addition, the qualified effluent of
389
pRho-NA-MNR system was more by introduction of pRho-NA. Furthermore, the
390
pRho-NA-MNR system realized a highly efficient and deep removal of nitrogen and
391
most of the biodegradable organics without any additional carbon source in the
392
treatment of landfill leachate. Moreover, low-level expression of Nirs in the presence
393
of nitrite may provide NO as a trigger for the full expression of denitrification genes
394
(Kuroki et al., 2014). Moreover, pRho-NA increased sludge concentration and activity,
395
decreased N-NO3 and N-NO2 accumulation, and promoted nitrogen removal rate by
396
nitrification and denitrification, and the efficiency of nitrogen removal rate was
397
positively correlated to the concentration of pRho-NA and Nirs/AMO expression.
398
Although a study indicates that MBR-NF-RO system along with aerobic and activated
399
sludge treatment can remove over 99% of COD and N-NH4 in landfill leachate, the
400
removal rate of TN is low (Ramaswami et al., 2018). In this study, pRho-NA
401
promoted removal rate of TN, N-NH4, COD, BOD, Cl- and heavy metal and
402
maintained system stability in the treatment of landfill leachate. The ability of the
403
pRho-NA-MNR to remove pollutants can be advantageous for achieving an effective
404
and integrated system for the treatment of landfill leachate.
405 406
5. Conclusions
407
A clear understanding of the treatment efficacy of the pRho-NA-MNR requires
408
accurate data on organic matter removal in landfill leachate treatment system. Here
409
we have presented a method to generate pRho-NA and explored the treatment efficacy
410
by combing pRho-NA and membrane processes for landfill leachate. Data in this
411
study demonstrate that pRho-NA highly expresses Nirs and AMO, enhances
412
denitrification and nitrification, increases the activated sludge activity and the stability
413
of aerobic and anoxic system, decreases phase length adaptation. The combined
414
treatment process by integrating pRho-NA biological treatment with membrane
415
processes not only achieves higher removal rate of TN, N-NH4, COD, BOD, Cl- and
416
heavy metal, but also improves the water quality and the amount of effluent in a cycle
417
compared to AS-MNR system. Notably, pRho-NA administration promotes the pH
418
control and decreases accumulation of N-NO3 and N-NO2 in landfill leachate
419
treatment system. Taken together, data in the current study indicate that the
420
pRho-NA-MNR disposal technology is an efficient strategy to reduce pollutants for
421
the treatment of landfill leachate.
422 423
Acknowledgement
424
This study is supported by Functional nano-catalytic materials and decontamination
425
technology of National Key Research and Development Program (2017YFA0207203)
426
and China's Post-doctoral Science Fund (2016M601433).
427
Conflicts of Interest
428
The authors declare that they have no conflicts of interest.
429 430
References
431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459
Akgul, D., et al., 2013. Treatment of landfill leachate using UASB-MBR-SHARON-Anammox configuration. Biodegradation. 24, 399-412. Azzouz, L., et al., 2018. Membrane bioreactor performance in treating Algiers' landfill leachate from using indigenous bacteria and inoculating with activated sludge. Waste Manag. 75, 384-390. Bai, F., et al., 2019. Advanced treatment of sewage by membrane bioreactor associate with genetically engineered autotrophic nitrifying bacteria. Bioresour Technol. 288, 121341. Bennett, K., et al., 2016. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea. Appl Environ Microbiol. 82, 2270-2279. Bilgili, M. S., et al., 2008. COD fractions of leachate from aerobic and anaerobic pilot scale landfill reactors. J Hazard Mater. 158, 157-63. Calli, B., et al., 2006. Comparison of long-term performances and final microbial compositions of anaerobic reactors treating landfill leachate. Bioresour Technol. 97, 641-7. Campagna, M., et al., 2013. Molecular weight distribution of a full-scale landfill leachate treatment by membrane bioreactor and nanofiltration membrane. Waste Manag. 33, 866-70. Dadrasnia, A., et al., 2017. Optimal reduction of chemical oxygen demand and NH3-N from landfill leachate using a strongly resistant novel Bacillus salmalaya strain. BMC Biotechnol. 17, 85. Dia, O., et al., 2018. Hybrid process, electrocoagulation-biofiltration for landfill leachate treatment. Waste Manag. 75, 391-399. Dong, L. F., et al., 2009. Changes in benthic denitrification, nitrate ammonification, and anammox process rates and nitrate and nitrite reductase gene abundances along an estuarine nutrient gradient (the Colne estuary, United Kingdom). Appl Environ Microbiol. 75, 3171-9. El-Fadel, M., Hashisho, J., 2014. A comparative examination of MBR and SBR performance for the treatment of high-strength landfill leachate. J Air Waste Manag Assoc. 64, 1073-84. Fudala-Ksiazek, S., et al., 2014. Nitrogen removal via the nitrite pathway during wastewater co-treatment with ammonia-rich landfill leachates in a sequencing batch reactor. Environ Sci Pollut Res Int. 21, 7307-18. Hasar, H., et al., 2009. Stripping/flocculation/membrane bioreactor/reverse osmosis treatment of municipal landfill leachate. J Hazard Mater. 171, 309-17.
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Hashisho, J., et al., 2016. Hollow fiber vs. flat sheet MBR for the treatment of high strength stabilized landfill leachate. Waste Manag. 55, 249-56. Ishak, A. R., et al., 2018. Stabilized landfill leachate treatment by coagulation-flocculation coupled with UV-based sulfate radical oxidation process. Waste Manag. 76, 575-581. Iskander, S. M., et al., 2018. A review of landfill leachate induced ultraviolet quenching substances: Sources, characteristics, and treatment. Water Res. 145, 297-311. Jiang, G., et al., 2018. Impact of landfill density on transport and hydraulic characteristics of recirculated leachate. Environ Technol. 1-7. Karre, A. K., et al., 2012. Parameters affecting HS emissions removal and re-circulating water quality in a pilot-scale sequential biological treatment system at a wastewater lift station in Brownsville, Texas, USA. J Environ Sci Health A Tox Hazard Subst Environ Eng. 47, 979-89. Khattabi, H., et al., 2007. [Temporal and spatial fluctuations in bacterial abundances in four basins of a landfill leachate treatment (Etueffont, France)]. C R Biol. 330, 429-38. Kim, D. J., et al., 2006. Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH. Bioresour Technol. 97, 459-68. Kuroki, M., et al., 2014. Fine-tuned regulation of the dissimilatory nitrite reductase gene by oxygen and nitric oxide in Pseudomonas aeruginosa. Environ Microbiol Rep. 6, 792-801. Li, F., et al., 2009. Treatment of the methanogenic landfill leachate with thin open channel reverse osmosis membrane modules. Waste Manag. 29, 960-4. Liu, J., et al., 2018. Denitrification of landfill leachate under different hydraulic retention time in a two-stage anoxic/oxic combined membrane bioreactor process: Performances and bacterial community. Bioresour Technol. 250, 110-116. Matsushita, T., et al., 2017. Use of gas chromatography-mass spectrometry-olfactometry and a conventional flask test to identify off-flavor compounds generated from phenylalanine during chlorination of drinking water. Water Res. 125, 332-340. Navia, R., Ross, D., 2009. Sanitary landfills, foundation of the waste hierarchy inverted pyramid. Waste Manag Res. 27, 407-8. Niu, A., et al., 2016. Toxicological characterization of a novel wastewater treatment process using EDTA-Na2Zn as draw solution (DS) for the efficient treatment of MBR-treated landfill leachate. Chemosphere. 155, 100-108. Pisa, R., et al., 2002. The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase. Mol Microbiol. 43, 763-70. Ramaswami, S., et al., 2018. Comparison of NF-RO and RO-NF for the Treatment of Mature Landfill Leachates: A Guide for Landfill Operators. Membranes (Basel). 8. Remmas, N., et al., 2016. Effects of high organic load on amoA and nirS gene diversity of an intermittently aerated and fed membrane bioreactor treating landfill leachate. Bioresour Technol. 220, 557-565. Silva, N. C. M., et al., 2018. Evaluation of fouling mechanisms in nanofiltration as a polishing step of yeast MBR-treated landfill leachate. Environ Technol. 1-11. Silveira, J. E., et al., 2018. Landfill leachate treatment by sequential combination of activated persulfate and Fenton oxidation. Waste Manag. 81, 220-225. Sun, H. W., et al., 2013. Achieving nitrogen removal via nitrite pathway from urban landfill leachate using the synergetic inhibition of free ammonia and free nitrous acid on nitrifying bacteria
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activity. Water Sci Technol. 68, 2035-41. Wang, B., et al., 2016. Influence of sludge reflux ratios on biodegradation performance in a coupled landfill leachate treatment process based on UASB and submerged MBR. J Environ Sci Health A Tox Hazard Subst Environ Eng. 51, 701-6. Wang, K., et al., 2018. Treatment of Landfill Leachate Using Activated Sludge Technology: A Review. Archaea. 2018, 1039453. Wang, T., et al., 2009. Start-up of the Anammox process from the conventional activated sludge in a membrane bioreactor. Bioresour Technol. 100, 2501-6. Warwick, S. J., et al., 2018. Altered chemical evolution in landfill leachate post implementation of biodegradable waste diversion. Waste Manag Res. 36, 857-868. Wei, Y., et al., 2012. Organic and nitrogen removal from landfill leachate in aerobic granular sludge sequencing batch reactors. Waste Manag. 32, 448-55. Wigginton, S., et al., 2018. Nitrifying and Denitrifying Bacterial Communities in Advanced Nitrogen-Removal Onsite Wastewater Treatment Systems. J Environ Qual. 47, 1163-1171. Wu, Y. J., et al., 2013. Responses of ammonia-oxidizing archaeal and betaproteobacterial populations to wastewater salinity in a full-scale municipal wastewater treatment plant. J Biosci Bioeng. 115, 424-32. Xiao, Y., et al., 2007. [Bacterial diversity in sequencing batch biofilm reactor (SBBR) for landfill leachate treatment using PCR-DGGE]. Huan Jing Ke Xue. 28, 1095-101. Xu, Q., et al., 2019. Utilization of acid hydrolysate of recovered bacterial cell as a novel organic nitrogen source for L-tryptophan fermentation. Bioengineered. 10, 23-32. Yu, D., et al., 2014. Bioaugmentation treatment of mature landfill leachate by new isolated ammonia nitrogen and humic acid resistant microorganism. J Microbiol Biotechnol. 24, 987-97. Yusof, N., et al., 2011. Nitrification of high-strength ammonium landfill leachate with microbial community analysis using fluorescence in situ hybridization (FISH). Waste Manag Res. 29, 602-11. Zhou, Y., et al., 2011. The role of nitrite and free nitrous acid (FNA) in wastewater treatment plants. Water Res. 45, 4672-82. Zolfaghari, M., et al., 2018. Removal of Pollutants in Different Landfill Leachate Treatment Processes on the Basis of Organic Matter Fractionation. J Environ Qual. 47, 297-305.
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Figure legends
553
Figure 1 Schematic diagram of pRho-NA-MNR in the treatment of landfill leachate
554
Figure 2 Performance of aerobic reactors between pRho-NA-MNR and AS-MNR
555
system in the treatment of landfill leachate
556
(A-F) Concentration of TN (A), N-NH4 (B), BOD5 (C), COD (D) and VSS/gCODR
557
(E) in aerobic reactors. (F) Changes of pH in aerobic reactors. **p < 0.01 vs.
558
AS-MNR.
559
Figure 3 Performance of MBR between pRho-NA-MNR and AS-MNR system in the
560
treatment of landfill leachate
561
(A-D) Concentration of TN (A), N-NH4 (B), BOD5 (C), and COD (D) in MBR
562
effluent. **p < 0.01 vs. AS-MNR.
563
Figure 4 Performance of NF between pRho-NA-MNR and AS-MNR system in the
564
treatment of landfill leachate
565
(A-D) Concentration of TN (A), N-NH4 (B), BOD5 (C), and COD (D) in NF effluent.
566
**p<0.01 vs. AS-MNR.
567
Figure 5 Performance of RO between pRho-NA-MNR and AS-MNR system in the
568
treatment of landfill leachate
569
(A-D) Concentration of TN (A), N-NH4 (B), BOD5 (C), and COD (D) in RO effluent.
570
**p < 0.01 vs. AS-MNR.
571
Figure 6 Concentration of TN and DOC concentrations in anaerobic reactors in
572
pRho-NA-MNR and AS-MNR system
573
(A-B) The averaged TN (A) and DOC (B) concentrations in anaerobic reactors in
574
pRho-NA-MNR and AS-MNR system. **p < 0.01 vs. AS-MNR.
575
Figure 7 concentrations of N-NO3, N-NO2 and production of N2 in anaerobic reactors
576
in pRho-NA-MNR and AS-MNR system
577
(A-B) Concentrations of N-NO3 (A) and N-NO2 (B) in anaerobic reactors in
578
pRho-NA-MNR and AS-MNR system. (C) Generation rate of N2 in anaerobic reactors
579
in PRho-NA-MNR and AS-MNR system. **p < 0.01 vs. AS-MNR.
580
Figure 8 Nitrate and nitrite metabolism in the pRho-NA-MNR and AS-MNR system
581
during the stable operation period
582
(A-C) NH4-N reduction rate (A), N-NO3 (B) and N-NO2 (C) reduction rate in aerobic
583
reactors. (D-E) Nitrification rate in aerobic reactors (D) and denittification rate in
584
anaerobic reactors (E) in the pRho-NA-MNR and AS-MNR system. (F-G)
585
Concentrations of N-NO3 (F) and N-NO2 (G) in the effluent of MBR, NF and RO in
586
the pRho-NA-MNR and AS-MNR system. *p < 0.05, **p < 0.01 vs. AS-MNR.
587
Figure 9 Functional gene and protein expression levels in the PRho-NA-MNR system
588
in the treatment of landfill leachate
589
(A-B) Relative gene expression levels of Nirs (A) and AMO (B) in aerobic reactors in
590
pRho-NA-MNR and AS-MNR system. (C) Relative protein expression levels of Nirs
591
and AMO in aerobic reactors in pRho-NA-MNR and AS-MNR system. (D) Relative
592
protein expression of Cyt cd1 and Cyt c in anaerobic reactors in pRho-NA-MNR and
593
AS-MNR system. (E-F) Corrections of Nirs (E) and AMO (F) expression with
594
nitrification and denitrification rate in aerobic stage in pRho-NA-MNR and AS-MNR
595
system. (G) Comparison of PH in aerobic stage in pRho-NA-MNR and AS-MNR
596
system. (H) Relative sludge activity in pRho-NA-MNR and AS-MNR system. (I)
597
Phase length adaptation of pRho-NA-MNR and AS-MNR system. **p < 0.01 vs.
598
AS-MNR.
Highlights pRho-NA enhances activated sludge activity in aerobic reactor in landfill leachate treatment system. pRho-NA-MNR enhances the stability of aerobic and anoxic system. pRho-NA enhances denitrification and nitrification in landfill leachate treatment system. Administration of pRho-NA increases the quality of effluent by combing membrane processes.
Author statement Hui Tian: Conceptualization, Resources, Methodology, Software, Data curation. Fuliang Bai: Writing-Original draft preparation, Visualization, Investigation, Supervision. Jun Ma: Writing- Reviewing and Editing, Project administration, Funding acquisition.
Conflict of Interest All authors declare that they have no conflicts of interest.
S0269-7491(19)36728-4
DOI:
https://doi.org/10.1016/j.envpol.2020.114061
Reference:
ENPO 114061
To appear in:
Environmental Pollution
Received Date: 12 November 2019 Revised Date:
20 January 2020
Accepted Date: 22 January 2020
Please cite this article as: Bai, F., Tian, H., Ma, J., Landfill leachate treatment through the combination of genetically engineered bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane filtration processes, Environmental Pollution (2020), doi: https://doi.org/10.1016/j.envpol.2020.114061. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Landfill leachate treatment through the combination of genetically engineered
2
bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane
3
filtration processes
4 5
Fuliang Bai1, 2, Hui Tian3, Jun Ma1*
6
1
7
Technology, Harbin, Heilongjiang, 150090, People's Republic of China
8
2
9
Republic of China
State Key Laboratory of Urban Water and Environment, Harbin Institute of
Lubin Environmental Protection Equipment (Shanghai) Co., Ltd, Shanghai, People's
10
3
11
Republic of China
12
Corresponding Author: Jun Ma. Address: No 73, School of Environment, Huanghe
13
Road, Nangang Area, Harbin, 150090, China. Tel/fax: +86 451 86283010. 12
14
Email: [email protected]; [email protected].
School of Life Science, Harbin Institute of Technology, Harbin, 150001, People's
15 16
Abstract
17
This study developed a process of genetically engineered bacteria Rhodococcus
18
erythropolis expressing Nirs and AMO combined with membrane bioreactor (MBR),
19
nanofiltration (NF) and reverse osmosis (RO) membrane (pRho-NA-MNR) for
20
advanced treatment of landfill leachate. Results demonstrated that pRho-NA-MNR
21
presented higher removal rate of chemical oxygen demand (COD), biological oxygen
22
demand (BOD), ammonia nitrogen (N-NH4), total nitrogen (TN) and total organic
23
carbon (TOC) than activated sludge (AS-MNR) system. Administration of pRho-NA
24
increased nitrification by converting N-NH4 to nitrite (N-NO2) and Nitrate (N-NO3),
25
and promoting denitrification by converting N-NO2 to nitrogen (N2) in the landfill
26
leachate treatment, promoted the pH control, increased sludge activity and effluent
27
yield, shortened phase length adaptation under alternating aerobic-anoxic conditions.
28
pRho-NA increased the nitration and denitrifying rate in the aerobic and anaerobic
29
stage in the system by increasing Cyt cd1 and Cyt c expression in the activated sludge.
30
Nitrogen removal by nitrification and denitrification was positively correlated to the
31
concentration of Nirs and AMO expression. Treatment with pRho-NA promoted
32
pollutant removal efficiency of membrane bioreactor, nanofiltration and reverse
33
osmosis membrane processes in landfill leachate. In conclusion, data suggest that
34
pRho-NA-MNR facilitates the formation of granular sludge and enhances comparable
35
removal of nitrogen and organic compounds, indicating the practice of this process
36
should be considered in landfill leachate treatment system.
37
Keywords: Landfill leachate; Rhodococcus erythropolis; Nirs; AMO; pRho-NA-MNR
38 39
1. Introduction
40
Sanitary landfills are the most widely employed method of municipal waste treatment
41
(Navia and Ross, 2009). Landfill leachate is generated from sanitary landfills and
42
much concern with respect to the pollution potential, which is caused by the
43
degradation of solid waste (Azzouz et al., 2018; Jiang et al., 2018; Warwick et al.,
44
2018). Landfill leachate is composed of various compositions with high
45
contamination, such as high contents of organic matter, inorganic substances and
46
other pollutants (Iskander et al., 2018). Thus, it requires appropriate and efficient
47
technology to remove the toxic compositions before discharge for high contents of
48
COD, N-NH4 and disorder of carbon-nitrogen ratio (Calli et al., 2006). However,
49
landfill leachate is extremely difficult to treat and effluent without qualified treatment
50
processes impacts quality of ecological environment and human health (Dia et al.,
51
2018; Ishak et al., 2018; Silveira et al., 2018).
52
Conventional sewage processes are not efficient for the treatment of landfill leachate
53
due to an inadequate C to N ratio (Fudala-Ksiazek et al., 2014). Currently, many
54
integrated processes have been developed for the treatment of landfill leachate
55
(Zolfaghari et al., 2018). Generally, the membrane bioreactor (MBR) technology is
56
regarded as a prominent process in the treatment of landfill leachate deriving from the
57
decomposition of waste in landfills (Hashisho et al., 2016). MBR combining with
58
sequencing batch reactor (SBR) technologies are introduced in treating high-strength
59
landfill leachate (El-Fadel and Hashisho, 2014). The combination of single reactor for
60
high activity ammonia removal over nitrite (SHARON)-anammox processes is an
61
effective method in removing high organic content of young leachate (Akgul et al.,
62
2013). Integrated MBR-nanofiltration (NF) system effectively removes the high
63
concentrations of COD and N-NH4, which improves the quality and increases reuse of
64
the effluent in landfill leachate (Silva et al., 2018). The reverse osmosis (RO) also is
65
applied to treat landfill leachate and provides high quality effluent by reducing the
66
effluent COD from MBR (Hasar et al., 2009). Thus, we assumed that integrating
67
MBR-NF-RO system may enhance current discharge standards and achieve the
68
effluent feasible for reuse in treatment of municipal landfill leachate.
69
Landfill leachate contains high concentrations of N-NH4 and needs to remove nitrate
70
nitrogen, nitrite nitrogen, N-NH4 and TN using activated sludge biological system
71
(Wang et al., 2018). Autotrophic and heterotrophic nitrifying bacteria play crucial role
72
in achieving nitrogen removal via nitrite pathway from landfill leachate (Sun et al.,
73
2013). Introducing microorganisms with high biodegradation ability and resistance to
74
N-NH4, and TN may be a promising solution in the treatment of landfill leachate
75
(Dadrasnia et al., 2017). A study has achieved the startup of anaerobic ammonium
76
oxidation using the conventional activated sludge integrated processes in a MBR with
77
a maximum nitrogen removal rate (Wang et al., 2009). In addition, another study
78
(Fudala-Ksiazek et al., 2014) demonstrates that nitrogen removal via the nitrite
79
pathway is beneficial for carbon-limited and highly ammonia-loaded mixtures, which
80
can lead to a reduction in the external carbon source needed to support denitrification
81
in landfill leachate. Furthermore, a higher denitrification rate and lower surplus sludge
82
production contributes to achieve shortcut nitrification, the activity of nitrite-oxidising
83
bacteria and nitrogen removal (Zhou et al., 2011). In response to characteristics of
84
landfill leachate, novel and efficient technologies that allow for the reduction of
85
pollutants must be developed, optimized and implemented.
86
Nevertheless, conventional treatment method of landfill leachates is insufficient to
87
remove the high concentration of nitrogen, organic matter, color, heavy metals and
88
toxic substances in landfill leachates. Thus, various processes combined with
89
biological treatment with additional nitrobacteria have technological and economic
90
advantages in the treatment of landfill leachates. Our previous study indicates that
91
Nirs and ppk gene expressed by nitrifying bacteria promotes biological removal of
92
N-NH4 in the sewage treatment system (Bai et al., 2019). In this study, we
93
investigated the efficiency of a novel genetically engineered bacteria Rhodococcus
94
erythropolis expressing nitrite reductase gene (Nirs) and ammonia monooxygenase
95
(AMO) (pRho-NA) in landfill leachate. The multiprocessing efficiency of
96
MBR-NF-RO integrated pRho-NA was investigated in an industrial scale landfill
97
leachate treatment.
98 99
2. Materials and methods
100
2.1 MBR-NF-RO (MNR) system
101
Landfill leachates were come from Laogang County Landfill Treatment Plant
102
(Shanghai, China). A subexternal MBR, NF and RO membrane processes were used
103
to treat landfill leachates from AS or pRho-NA reactors. Landfill leachates were
104
extracted from the same source in activated sludge (AS-MNR) and pRho-NA-MNR
105
system. Two systems were arranged in the same condition during the experimental
106
period. Both AS-MNR and pRho-NA-MNR systems included aerobic fluidized
107
reactors, anaerobic reactors, MBR, NF and RO membrane. The identical dimensions
108
of aerobic fluidized reactors and anaerobic reactors were 400 × 400 × 4.5 m (L × W ×
109
H) and 300 × 300 × 4.5 m (L × W × H), respectively. The membrane area of each
110
MBR, NF and RO was 38.5 m2, 32.0 m2 and 30.0 m2 respectively. Schematic of the
111
reactors and the types of components are illustrated in Figure 1. pRho-NA (1x105/ml)
112
was added into aerobic fluidized reactors in pRho-NA-MNR system for a total of 15 d.
113
During the adaptive phase, the dissolved oxygen is 0.5-0.8 mg L-1 and pH is arranged
114
from 6.9 to 7.8. The landfill leachate was extracted from the same source in two
115
systems (AS-MNR and pRho-NA-MNR) during the experimental period.
116
2.2 Generation of pRho-NA
117
Autotrophic nitrifying bacteria was isolated from landfill leachate and identified as
118
Rhodococcus
119
monooxygenase (AMO) were synthesized by Invitrogen (Invitrogen, Carlsbad, CA,
120
USA) and cloned into pSIM5.0 vector and then transfected into Rhodococcus
erythropolis.
Nitrite
reductase
gene
(Nirs)
and
ammonia
121
erythropolis using electrotransfection (Bio-Rad) according to the manufacturer’s
122
instrument. A genetically engineered bacterium of expressing Nirs and AMO was
123
named pRho-NA. pRho-NA was amplified using fermentation as described previously
124
(Xu et al., 2019).
125
2.3 Parameter analysis
126
Physico-chemical parameters of water such as dissolved oxygen, total suspended
127
solids (TSS), BOD, COD, TN, and N-NH4 were measured using standard methods as
128
described previously (Karre et al., 2012). N-NO3 and N-NO2 were determined on a
129
Metrohm 761 Compact Ion Chromatograph (Zofingen, Switzerland) equipped with a
130
conductivity detector. The pH was measured with a Consort C532 pH meter
131
(Turnhout, Belgium). TOC was analyzed using Multi N/C3000 TOC (Analytik Jena
132
AG, Germany). The amount of N2 production was measured using gas
133
chromatography-mass spectrometry (Agilent Technologies Inc., Santa Clara, CA,
134
USA) (Matsushita et al., 2017).
135
2.4 Quantitative RT-PCR (qRT-PCR)
136
Total RNA was extracted from activated sludge in aerobic reactors and anaerobic
137
reactors using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the
138
manufacturer’s instrument. Total RNA (1 µg) was reverse transcribed into cDNA
139
using High Capacity cDNA Reverse Transcription Kit (cat no. 4368814, Invitrogen,
140
Shanghai, China) according to the manufacturer’s protocol. All gene primer sequences
141
including Nirs, AMO, Cyt cd1, Cyt c and β-actin are shown in Supplementary material
142
file (Table S1). The expression levels of the mRNAs were performed using SYBR
143
Green reagent (Roche, Indianapolis, IN, USA) equipment with ABI 7900HT Fast
144
Real-time PCR System (Applied Biosystems). Relative gene expression was
145
calculated using 2-∆∆Ct method.
146
2.5 Western blotting
147
Whole cell extracts from aerobic reactors and anaerobic reactors were prepared and
148
extracted using a protein extraction reagent (Roche, Switzerland). Concentration of
149
protein was measured with a BCA protein assay kit (Pierce Biotechnology, USA) and
150
equal amounts of the protein (40 µg) were separated by 12.5% SDS-polyacrylamide
151
gels (SDS-PAGE), transferred onto nitrocellulose membranes and then blocked with
152
5.0% BSA in TBST (Tris-buffered saline containing 0.1% Tween 20) at room
153
temperature for 2 h. Membranes were incubated with primary antibody Nirs, AMO,
154
Cyt cd1, Cyt c and β-actin (1:1,200, ABCAM) at 4 °C overnight. Membranes were
155
subsequently incubated with goat anti-mouse or anti-rabbit IgG at 37 °C for 2 h, all
156
bands were detected using the ECL chemiluminescence kit (Pierce Biotechnology,
157
USA). The protein of bands was normalized to β-actin, and relative protein expression
158
was analyzed by densitometry analysis program, ImageJ Software (National
159
Instituties of Health, USA).
160
2.6 Statistical analysis
161
Standard statistical parameters are expressed as mean ± standard deviation (SD).
162
SPSS software version 22.0 (SPSS, Chicago, IL, USA) was used for statistical
163
analyses. Experimental data were also analyzed by Student t test or one-way ANOVA
164
followed by Tukey’s post hoc test. All experiments were performed at least three
165
times. P < 0.05 was defined as statistically significant.
166 167
3 Results
168
3.1 Characteristics of landfill leachate and reactor performances
169
The monitored parameters for landfill leachate are shown in Table S2. The diagram of
170
landfill leachate system was shown in Figure 1. Landfill leachate system included
171
aerobic reactor, anaerobic reactor, MBR, NF and RO system during the operational
172
period. The average N-NH4 and TN concentration 1,275 mg L-1 and 1,736 mg L-1,
173
respectively. The average TCOD (COD) and BOD5 was 16,725 (15,720) and
174
1,428 mg L-1, respectively. The features of landfill leachate are high ammoniacal
175
nitrogen content, little biodegradability, high TN/BOD5, and poor BOD5/COD and
176
COD/N-NH4. The landfill leachate in this study demonstrated high chloride
177
concentrations (average, 2,350 mg L-1; range, 2,045-2,438 mg L-1). The concentration
178
of TOC and dissolved organic carbon (DOC) was 1,925 mg L-1 and 1,348 mg L-1,
179
respectively. Concentration of heavy metals including Cu, Zn, Cd, Cr, and Ni were
180
0.041, 0.782, 0.017, 0.184 and 0.086 mg L-1, respectively, in the landfill leachate.
181
The parameters of landfill leachate in aerobic reactors during adoptive operation
182
between pRho-NA-MNR and AS-MNR system are shown in Table S3. In the aerobic
183
and anaerobic reactors, all parameters were monitored using online measurements.
184
The hydraulic retention time (HRT) was 118 h and 88 h in aerobic reactors during
185
adoptive operation in AS-MNR and pRho-NA-MNR system, respectively (p < 0.01).
186
The sludge retention time (SRT) was 26.8 h and 24.2 h in aerobic reactors during
187
adoptive operation in AS-MNR and pRho-NA-MNR system, respectively (p < 0.01).
188
The obtained results showed that the HRT of landfill leachate treatment in anaerobic
189
was shorter in pRho-NA-MNR than AS-MNR system (mean, 28.5 h vs 24.0 h, p <
190
0.05). TSS parameter values were higher in adopted in MBR operation, aerobic and
191
anaerobic reactors in pRho-NA-MNR than AS-MNR system (p<0.01). In the
192
accommodation with activated sludge with or without PRho-NA followed by aerobic
193
culture, concentrations of COD, BOD5, DOC, TN, Cl−, N-NH4, N-NO3 and N-NO2
194
were compared in two systems. Concentrations of O2 were > 0.8 mg L-1 and <
195
0.05 mg L-1 in aerobic and anaerobic reactors, respectively. A higher nitrification and
196
denitrification rate and COD removal rate was observed in the aerobic reactors in
197
pRho-NA-MNR compared with AS-MNR system. pRho-NA-MNR reactor had higher
198
SV30 and lower concentrations of COD, TOC, TN and N-NH4 compared to AS-MNR
199
reactor during 15 d accommodation. However, accumulation of N-NO3 and N-NO2
200
were higher in aerobic reactor in pRho-NA-MNR system than that in AS-MNR
201
system. There were no significant differences of concentration of Cl-, BOD5, bulk
202
wasting ratio and bulk wasting flow rate in the aerobic reactors between
203
pRho-NA-MNR and AS-MNR system. The results showed that concentration of
204
N-NO3 and N-NO2 was lower, while pH and N2 production was higher in the
205
anaerobic reactors in pRho-NA-MNR than that in AS-MNR system.
206
3.2 Effectiveness of treatment in the pRho-NA-MNR system
207
In the stable portion period, the landfill leachate treatment efficiency including
208
aerobic and anaerobic reactors, MBR, NF and RO effluent was compared between
209
pRho-NA-MNR and AS-MNR system. As shown in Figure 2A-D, pRho-NA-MNR
210
system had higher removal rate of TN, N-NH4, BOD5 and COD than AS-MNR
211
system in aerobic reactor. Administration of pRho-NA promoted stability of system
212
determined by the bulk VSS concentration/the COD mass remove (gCODR)
213
(VSS/gCODR) and decreased value of pH by promoting ammoniation in aerobic
214
reactor compared to AS system (Figure 2E-F). Concentrations of TN, N-NH4, BOD5,
215
and COD in MBR effluent were lower in pRho-NA-MNR system than AS-MNR
216
system (Figure 3). The average COD and N-NH4 removal remained above 98.4% and
217
97.2%, respectively, in pRho-NA-MNR system, which were higher than AS-MNR
218
system. TN removal rate was 95.2% and 84.6% in pRho-NA-MNR and AS-MNR
219
system, respectively. Highly effective removing rate of N-NH4 (> 97.8 %), TN (>
220
96.5%), BOD5 (> 80.2%) and COD (> 93.2%%) was observed in NF effluent in
221
pRho-NA-MNR system, whereas the remove rate of N-NH4, TN, BOD5 and COD
222
averaged 84.3%, 72.2%, 70.1% and 80.6% in NF effluent in AS-MNR system,
223
respectively (Figure 4). During the stable operational phase, the averaged
224
concentrations of TN, N-NH4, BOD5 and COD in the RO effluent were 3.5 mg L-1, 3.0
225
mg L-1, 1.0 mg L-1 and 10.6 mg L-1, respectively, in pRho-NA-MNR system. However,
226
a higher averaged concentrations of TN (22.4 mg L-1), N-NH4 (18.8 mg L-1), BOD5
227
(5.6 mg L-1) and COD (62.3 mg L-1) were observed in the RO effluent in AS-MNR
228
system (Figure 5). The averaged TN and DOC concentrations were higher in
229
anaerobic reactors in pRho-NA-MNR system than AS-MNR system (95.6% vs. 87.4%
230
for TN; 98.2% vs. 93.2% for DOC; Figure 6). It showed that concentrations of N-NO3,
231
N-NO2 and production of N2 were higher in anaerobic reactors in pRho-NA-MNR
232
system than that in AS-MNR system (Figure 7). The mean water recovery rates of
233
MBR, NF and RO membrane separation process in pRho-NA-MNR system were
234
15.4%, 36.5% and 47.8%, respectively. However, the mean water recovery rates of
235
MBR, NF and RO membrane separation process in pRho-NA-AS system were 18.6%,
236
41.8% and 54.6%, respectively. Concentrates of MRB returned to the aerobic reactor.
237
Concentrates of NF and RO were discharged and got further treatment. Consequently,
238
the results demonstrated that the pRho-NA-MNR system and AS-MNR system could
239
effectively remove the heavy metals including Cu, Zn, Cd, Cr, and Ni in the landfill
240
leachate (Table S4). There were no significant differences between two treatment
241
systems.
242
3.3 Efficacy of effluent in the pRho-NA-MNR system
243
During the continuous operation, trans-membrane pressure (TMP) in two systems was
244
< 0.35 bar. When TMP of MBR, NF and RO membrane reached 0.40 bar, membrane
245
needed to wash using 0.1% NaClO. As shown in Table S5, the averaged frequency of
246
membrane cleaning MBR, NF and RO was 60 d/time, 35.5 d/time and 30.0 d/time in
247
the pRho-NA-MNR system. The HRT and SRT in pRho-NA-MNR system were 3
248
min and 7 min, while HRT and SRT in AS-MNR system were 7 min and 3 min in a
249
total of 10 min cycle during operation period. However, the averaged frequency of
250
membrane cleaning MBR, NF and RO was 42.5 d/time, 27.0 d/time and 23.5 d/time
251
in the AS-MNR system. To achieve the standard of effluent, the HRT was 72 h and 96
252
h in aerobic reactors in pRho-NA-MNR system and AS-MNR system, respectively (p
253
< 0.01). The SRT was 20.5 h and 13.6 h in aerobic reactors in pRho-NA-MNR system
254
and AS-MNR system, respectively (p < 0.01). The HRT was 16.5 h and 13.0 h in
255
aerobic reactors in pRho-NA-MNR system and AS-MNR system, respectively (p <
256
0.05). The obtained results showed that the SRT of landfill leachate treatment in
257
anaerobic was shorter in pRho-NA-MNR system than that in AS-MNR system (mean,
258
14.5 h vs 17.5 h, p < 0.05). The final effluent approximately remained 650 m3/d and
259
420 m3/d during continuous operation in pRho-NA-MNR system and AS-MNR
260
system, respectively (p < 0.01).
261
3.4 Nitrate and nitrite metabolism in the pRho-NA-MNR system
262
To verify the treatment efficacy of pRho-NA, N-NH4 reduction rate, N-NO3 and
263
N-NO2 generation rate in aerobic reactors, N-NO3 and N-NO2 reduction rate and N2
264
generation rate in anaerobic reactors were analyzed in two systems. As shown in
265
Figure 8A-C, N-NH4 reduction rate, N-NO3 and N-NO2 generation rate in aerobic
266
reactors were higher in pRho-NA-MNR system than AS-MNR system during the
267
stable operation period. During continuous operation, N2 generation rate in anaerobic
268
reactors was recorded and pRho-NA-MNR system had a higher N2 generation rate
269
than AS-MNR system (Figure 8D). Nitrification and denittification rate was also
270
higher in pRho-NA-MNR system than AS-MNR system. The averaged concentrations
271
of N-NO3 and N-NO2 in the effluent of MBR, NF and RO were 0.30, 0.20, 0.08 mg
272
L-1, respectively, in pRho-NA-MNR system, while was higher and averaged 0.13,
273
0.08 and 0.02 mg L-1, respectively, in AS-MNR system.
274
3.5 The functional gene and protein expression levels in the pRho-NA-MNR
275
system
276
During the operation, Nirs and AMO gene and protein expression levels were
277
identified during landfill leachate treatment operation period. As shown in Figure
278
9A-C, a significantly increasing gene and protein expression levels of Nirs and AMO
279
were observed in pRho-NA-MNR system compared to AS-MNR system. To further
280
explain more treatment efficacy of pRho-NA, detailed analysis of associations
281
between nitrogen removal rate and concentration of pRho-NA or Nirs/AMO
282
expression were undertaken. In the aerobic reactors, expression of Cyt cd1 and Cyt c
283
was up-regulated in pRho-NA-MNR system, compared to AS-MNR system, which
284
increased the denitrifying rate in the aerobic stage (Figure 9D). The results showed
285
that nitrogen removal rate by nitrification and denitrification was positively correlated
286
to the concentration of Nirs and AMO expression in aerobic stage (Figure 9E-F). pH
287
is associated with the nitrification and denitrification rate, thus, we investigated the
288
changes of pH in aerobic reactors in two systems. Compared to AS-MNR system,
289
pRho-NA administration promoted the pH control and increased sludge activity
290
(Figure 9G-H). Also, phase length adaptation under alternating aerobic-anoxic
291
conditions was shortened by additional pRho-NA (Figure 9I).
292 293
4. Discussion
294
Previous studies have shown that various bacterial strains have the capacities to
295
degrade the high toxicity values of DOC, N-NH4, BOD5, and COD in landfill leachate
296
under different suitable treatment conditions (Khattabi et al., 2007; Kim et al., 2006;
297
Xiao et al., 2007). Nitrification and denitrification of landfill leachate under different
298
hydraulic retention time in a two-stage anoxic/oxic combined MBR process presents
299
high removal of COD, BOD5, N-NH4 and TN (Liu et al., 2018). In addition, MBR
300
combined with an upflow anaerobic sludge blanket process is an effective method for
301
the treatment of high nitrogen content in leachate (Akgul et al., 2013). Furthermore,
302
MBR and NF membrane can efficiently remove COD, TOC, TKN, and N-NH4 in a
303
full-scale landfill leachate treatment system (Campagna et al., 2013). Moreover, RO
304
membrane filtration is able to achieve satisfactory results in terms of water quality for
305
landfill leachate discharge, process stability and membrane flux (Li et al., 2009).
306
The present study aimed to evaluate the efficiency of a novel genetic engineering
307
bacterial Rhodococcus erythropolis that was able to express Nirs and AMO gene and
308
identified the N-NH4 removal capacity in landfill leachate. In order to achieve current
309
discharge standards, three membranes of MBR, NF and RO cooperated with
310
pRho-NA were employed as safeguards for the treatment of landfill leachate. Here,
311
this study showed that pRho-NA-MNR achieved satisfactory outcomes of effluent
312
water and treatment efficiency for landfill leachate. In this study, administration of
313
pRho-NA not only shorted phase length of adaptation, increased nitrogen removal,
314
but also promoted the pH control, stability of system, increased sludge concentration,
315
activity and effluent yield and quality in the treatment of landfill leachate. Biological
316
co-treatment of pRho-NA with MNR presented higher removal rate of N-NH4, TN,
317
DOC, BOD5, and COD than AS-MNR system, suggesting it may be a reliable method
318
for landfill leachate toxicity reduction. Although our design genetically modified
319
bacteria to treat landfill leachate in an open system and it could easily spread
320
throughout the environment, these bacteria will not cause any problem in the
321
environment due to bacteria themselves come from the environment. As the current
322
study employed pRho-NA to enhance the treatment efficiency in the treatment of
323
landfill leachate, it would be interesting to investigate the biotoxicity and
324
bioavailability of pRho-NA to environment in future studies.
325
A previous study is undertaken to investigate the excellent removal efficiencies for
326
COD (92.67%) and N-NH4 (98.9%) in landfill leachate treatment process based on
327
UASB and submerged MBR (Wang et al., 2016). Integrated MBR-NF system leads to
328
high removal of pollutants and makes the treated effluent feasible for water reuse
329
(Silva et al., 2018). EDTA-Na2Zn as draw solution can effectively eliminate toxicity
330
of landfill leachate using MBR-NF-RO and data indicate that this method is
331
economical and eco-friendly for treatment of different types of landfill leachate (Niu
332
et al., 2016). However, the efficiency of TN, COD, and DOC removal needs to
333
improve during co-treatment in landfill leachate. Bioaugmentation displays the
334
highest effectiveness in removing N-NH4 and COD among all method of landfill
335
leachate treatments (Yu et al., 2014). Thus, this study explored the treatment efficacy
336
of MBR-NF-RO in dislodging TN, COD, and DOC and data found that removal
337
efficiency of TN, COD, and DOC remarkably increased and improved during landfill
338
leachate treatment. Interestingly, decreasing of toxicity and the membrane fouling was
339
observed in pRho-NA-MNR system compared to AS-MNR during stable operatic
340
period. The removal efficiency of DOC fractions and nitrogen from landfill leachate
341
dependents on the concentration and activity of aerobic granular sludge SBR (Wei et
342
al., 2012). It is quite conceivable that the DOC and TN removal rate is the
343
bioaugmentation process in landfill leachate, and our data demonstrated the enhanced
344
efficiency of nitrification and denitrification by using pRho-NA.
345
Generation of much more microbial products increases the values of soluble inert
346
COD to total COD in the leachate of aerobic landfill with leachate recirculation
347
(Bilgili et al., 2008). The syntrophic association has been observed between the
348
ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, which further explains the
349
incomplete nitrification phenomenon (Yusof et al., 2011). This study confirmed the
350
nitrogen and organic matter removal rate of genetic engineering bacteria pRho-NA
351
and found the efficient strategy of biological-ecological combinative technology via
352
using pRho-NA and MBR-NF-RO membrane, which emphasized the importance of
353
modeling, design, and operation of landfill leachate treatment system. The higher TN,
354
N-NH4, COD and BOD removal efficiency in pRho-NA-MNR system may attribute
355
to DO, pH control and stability of system, suggesting it is a high-efficiency process in
356
the treatment of landfill leachate.
357
TN and N-NH4 removal rate by nitrifying bacteria and denitrifying bacteria
358
dependents on a series of biological processes. The Nirs gene is essential for the
359
attachment of the active site haem group of Wolinella succinogenes cytochrome c
360
nitrite reductase (Pisa et al., 2002). The genetic potential for nitrifying bacteria and
361
the corresponding process activity, fine-scale environmentally induced changes in
362
rates of nitrate reduction are likely to be controlled at gene, cellular and protein levels
363
(Dong et al., 2009). Function and diversity of amoA and nirS gene is associated with
364
denitrification in MBR treating landfill leachate, which affects nitrification and
365
ammonia accumulation, causing a severe suppression of Nitrosomonas and an
366
increase in the relative abundance of Nitrosospira (Remmas et al., 2016). In this study,
367
Nirs gene was cloned into the genome of nitrifying bacteria and presented high TN
368
and N-NH4 removal rate in landfill leachate. The sequential activities of AMO and
369
hydroxylamine dehydrogenase (HAO) play crucial role in translation from oxidize
370
ammonia (N-NH4) to nitrite (NO2-) in denitrification (Bennett et al., 2016).
371
Abundance of AMO gene is associated with sewage treatment efficiency in a
372
full-scale municipal wastewater treatment plant (Wu et al., 2013). Communities in
373
N-removal onsite wastewater treatment systems technologies differ slightly in terms
374
of nitrifying and denitrifying bacterial communities (Wigginton et al., 2018). In this
375
study, we observed strong community similarity patterns driven by Nirs and AMO
376
gene expression in the aerobic reactors in the treatment of landfill leachate. Our
377
previous data have indicated that bacillus subtilis expressing Nirs and ppk cooperated
378
with aerobic fluidized MBR system is suitable for domestic wastewater treatment by
379
promoting metabolism of nitrogen and phosphorus (Bai et al., 2019). However, ppk is
380
not suitable for the treatment of landfill leachate for the lacking of phosphorus.
381
Therefore, we selected Rhodococcus erythropolis expressing Nirs and AMO and
382
membrane filtration processes and analyzed the treatment efficacy of pRho-NA in
383
landfill leachate. The most abundant, distributional and containing pRho-NA was
384
associated with pH and DO changes, which was a useful process controlling
385
parameter for the organics and nitrogen removal at high charge input of landfill
386
leachate. Investigation of the expression of AMO and nirS in denitrifying bacteria
387
revealed that gene and protein level of AMO and nirS was successfully expressed and
388
achieved high N-NH4 and COD removal rate. In addition, the qualified effluent of
389
pRho-NA-MNR system was more by introduction of pRho-NA. Furthermore, the
390
pRho-NA-MNR system realized a highly efficient and deep removal of nitrogen and
391
most of the biodegradable organics without any additional carbon source in the
392
treatment of landfill leachate. Moreover, low-level expression of Nirs in the presence
393
of nitrite may provide NO as a trigger for the full expression of denitrification genes
394
(Kuroki et al., 2014). Moreover, pRho-NA increased sludge concentration and activity,
395
decreased N-NO3 and N-NO2 accumulation, and promoted nitrogen removal rate by
396
nitrification and denitrification, and the efficiency of nitrogen removal rate was
397
positively correlated to the concentration of pRho-NA and Nirs/AMO expression.
398
Although a study indicates that MBR-NF-RO system along with aerobic and activated
399
sludge treatment can remove over 99% of COD and N-NH4 in landfill leachate, the
400
removal rate of TN is low (Ramaswami et al., 2018). In this study, pRho-NA
401
promoted removal rate of TN, N-NH4, COD, BOD, Cl- and heavy metal and
402
maintained system stability in the treatment of landfill leachate. The ability of the
403
pRho-NA-MNR to remove pollutants can be advantageous for achieving an effective
404
and integrated system for the treatment of landfill leachate.
405 406
5. Conclusions
407
A clear understanding of the treatment efficacy of the pRho-NA-MNR requires
408
accurate data on organic matter removal in landfill leachate treatment system. Here
409
we have presented a method to generate pRho-NA and explored the treatment efficacy
410
by combing pRho-NA and membrane processes for landfill leachate. Data in this
411
study demonstrate that pRho-NA highly expresses Nirs and AMO, enhances
412
denitrification and nitrification, increases the activated sludge activity and the stability
413
of aerobic and anoxic system, decreases phase length adaptation. The combined
414
treatment process by integrating pRho-NA biological treatment with membrane
415
processes not only achieves higher removal rate of TN, N-NH4, COD, BOD, Cl- and
416
heavy metal, but also improves the water quality and the amount of effluent in a cycle
417
compared to AS-MNR system. Notably, pRho-NA administration promotes the pH
418
control and decreases accumulation of N-NO3 and N-NO2 in landfill leachate
419
treatment system. Taken together, data in the current study indicate that the
420
pRho-NA-MNR disposal technology is an efficient strategy to reduce pollutants for
421
the treatment of landfill leachate.
422 423
Acknowledgement
424
This study is supported by Functional nano-catalytic materials and decontamination
425
technology of National Key Research and Development Program (2017YFA0207203)
426
and China's Post-doctoral Science Fund (2016M601433).
427
Conflicts of Interest
428
The authors declare that they have no conflicts of interest.
429 430
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Figure legends
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Figure 1 Schematic diagram of pRho-NA-MNR in the treatment of landfill leachate
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Figure 2 Performance of aerobic reactors between pRho-NA-MNR and AS-MNR
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system in the treatment of landfill leachate
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(A-F) Concentration of TN (A), N-NH4 (B), BOD5 (C), COD (D) and VSS/gCODR
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(E) in aerobic reactors. (F) Changes of pH in aerobic reactors. **p < 0.01 vs.
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AS-MNR.
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Figure 3 Performance of MBR between pRho-NA-MNR and AS-MNR system in the
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treatment of landfill leachate
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(A-D) Concentration of TN (A), N-NH4 (B), BOD5 (C), and COD (D) in MBR
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effluent. **p < 0.01 vs. AS-MNR.
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Figure 4 Performance of NF between pRho-NA-MNR and AS-MNR system in the
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treatment of landfill leachate
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(A-D) Concentration of TN (A), N-NH4 (B), BOD5 (C), and COD (D) in NF effluent.
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**p<0.01 vs. AS-MNR.
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Figure 5 Performance of RO between pRho-NA-MNR and AS-MNR system in the
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treatment of landfill leachate
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(A-D) Concentration of TN (A), N-NH4 (B), BOD5 (C), and COD (D) in RO effluent.
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**p < 0.01 vs. AS-MNR.
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Figure 6 Concentration of TN and DOC concentrations in anaerobic reactors in
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pRho-NA-MNR and AS-MNR system
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(A-B) The averaged TN (A) and DOC (B) concentrations in anaerobic reactors in
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pRho-NA-MNR and AS-MNR system. **p < 0.01 vs. AS-MNR.
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Figure 7 concentrations of N-NO3, N-NO2 and production of N2 in anaerobic reactors
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in pRho-NA-MNR and AS-MNR system
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(A-B) Concentrations of N-NO3 (A) and N-NO2 (B) in anaerobic reactors in
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pRho-NA-MNR and AS-MNR system. (C) Generation rate of N2 in anaerobic reactors
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in PRho-NA-MNR and AS-MNR system. **p < 0.01 vs. AS-MNR.
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Figure 8 Nitrate and nitrite metabolism in the pRho-NA-MNR and AS-MNR system
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during the stable operation period
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(A-C) NH4-N reduction rate (A), N-NO3 (B) and N-NO2 (C) reduction rate in aerobic
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reactors. (D-E) Nitrification rate in aerobic reactors (D) and denittification rate in
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anaerobic reactors (E) in the pRho-NA-MNR and AS-MNR system. (F-G)
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Concentrations of N-NO3 (F) and N-NO2 (G) in the effluent of MBR, NF and RO in
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the pRho-NA-MNR and AS-MNR system. *p < 0.05, **p < 0.01 vs. AS-MNR.
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Figure 9 Functional gene and protein expression levels in the PRho-NA-MNR system
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in the treatment of landfill leachate
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(A-B) Relative gene expression levels of Nirs (A) and AMO (B) in aerobic reactors in
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pRho-NA-MNR and AS-MNR system. (C) Relative protein expression levels of Nirs
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and AMO in aerobic reactors in pRho-NA-MNR and AS-MNR system. (D) Relative
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protein expression of Cyt cd1 and Cyt c in anaerobic reactors in pRho-NA-MNR and
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AS-MNR system. (E-F) Corrections of Nirs (E) and AMO (F) expression with
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nitrification and denitrification rate in aerobic stage in pRho-NA-MNR and AS-MNR
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system. (G) Comparison of PH in aerobic stage in pRho-NA-MNR and AS-MNR
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system. (H) Relative sludge activity in pRho-NA-MNR and AS-MNR system. (I)
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Phase length adaptation of pRho-NA-MNR and AS-MNR system. **p < 0.01 vs.
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AS-MNR.
Highlights pRho-NA enhances activated sludge activity in aerobic reactor in landfill leachate treatment system. pRho-NA-MNR enhances the stability of aerobic and anoxic system. pRho-NA enhances denitrification and nitrification in landfill leachate treatment system. Administration of pRho-NA increases the quality of effluent by combing membrane processes.
Author statement Hui Tian: Conceptualization, Resources, Methodology, Software, Data curation. Fuliang Bai: Writing-Original draft preparation, Visualization, Investigation, Supervision. Jun Ma: Writing- Reviewing and Editing, Project administration, Funding acquisition.
Conflict of Interest All authors declare that they have no conflicts of interest.