Identification of the release and effects of AHLs in anammox culture for bacteria communication

Accepted Manuscript Identification of the release and effects of AHLs in anammox culture for bacteria communication Xi Tang, Sitong Liu, Zuotao Zhang, Guoqiang Zhuang PII: DOI: Reference:

S1385-8947(15)00361-7 http://dx.doi.org/10.1016/j.cej.2015.03.045 CEJ 13396

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

29 November 2014 3 March 2015 10 March 2015

Please cite this article as: X. Tang, S. Liu, Z. Zhang, G. Zhuang, Identification of the release and effects of AHLs in anammox culture for bacteria communication, Chemical Engineering Journal (2015), doi: http://dx.doi.org/ 10.1016/j.cej.2015.03.045

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1

Identification of the release and effects of AHLs in anammox

2

culture for bacteria communication

3

Xi Tanga,b, Sitong Liu a,b, Zuotao Zhang a,b,Guoqiang Zhuangc

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a, Department of Environmental Engineering, Peking University, Beijing 100871, China

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b, Key Laboratory of Water and Sediment Sciences, Ministry of Education of China,

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Beijing 100871, China

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c, Research Center for Eco-Environmental Sciences, Chinese Academy of

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Sciences, Beijing 100085, China

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*Corresponding author:

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Sitong Liu

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E-mail: [email protected]

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Tel/Fax: 0086-10-62754290

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Address: College of Environmental Science and Engineering, Peking University,

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Beijing 100871, China

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1

1 2

Abstract Quorum sensing (QS) is a microorganism communication system. This study verified

3

the potential existence of the QS system among ANaerobic AMMonium OXidation

4

(anammox) bacteria. The release of three Acyl Homoserine Lactones (AHLs; C6-HSL,

5

C8-HSL, C12-HSL) by anammox culture was confirmed. By conducting series of batch

6

test, the introduction of exogenous C6-HSL and C8-HSL could significantly increase

7

activity of anammox bacteria, and meanwhile, C6-HSL significant increased the growth

8

rate, finally resulting in the increased ammonium nitrogen removal performance of 35%

9

and 20%, respectively, whereas the exogenous C12-HSL apparently promoted the

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growth of heterotrophic bacteria in the community. To further identify how exogenous

11

signal molecular affects anammox culture, analyses of anammox culture supernatant

12

and biomass extraction revealed that these additional signal molecules activated bacteria

13

by inducing the production of endogenous signal molecular by anammox culture. This

14

study might open a new window to overcome the disadvantage of the long-start-up

15

period of anammox reactor restricting the development of current anammox process.

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Graphical abstract

17 2

1

Keywords

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ANAMMOX; quorum sensing; nitrogen removal; growth rate

3

1. Introduction

4

Anammox is a process that combines ammonium and nitrite to form nitrogen gas. It

5

requires no Chemical Oxygen Demand (COD), reduces oxygen demand and produces

6

low quantities of sludge compared with the traditional method [1]. The nitrogen

7

removal rate as high as 5 kg N m−3 day−1 of the anammox system [2], in addition to the

8

advantages mentioned above, demonstrate that nitrogen removal in wastewater

9

treatment processes will save energy and provide lots of benefits [3]. However, because

10

anammox bacteria grow slowly (doubling time is 10–14 days) and are greatly affected

11

by several environmental conditions, such as temperature and organics in surroundings

12

[4-6], anammox processes often require long start-up periods. Numerous solutions have

13

been presented to improve the activity of anammox bacteria and shorten the reactor

14

start-up period [7], such as modifying the carrier to provide a better growing

15

environment for the bacteria and choosing the suitable bioreactor type. However, these

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solutions are unable to essentially regulate the bacterial metabolism. To improve the

17

activity, growth rate and competitive strength of anammox bacteria, the promotions of

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the expression of several key genes in the anammox bacteria are very important.

19

A milestone observation of bacterial ecology is that bacteria can communicate with

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one another by a system called Quorum sensing (QS) [8], which regulates gene

21

expression in response to fluctuations in the cell population density [9]. The bacteria

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which have QS system produce quorum signaling molecules and then release them to 3

1

their surroundings. When the cell population density increased to a certain value, the

2

signals concentration reached a threshold value, and the binding of signal molecules and

3

regulatory proteins activates quorum sensing-regulated genes to activate relevant

4

phenotypes. Generally, signal molecules of Gram-negative bacteria are AHLs [9].

5

AHLs-based QS-regulated phenotypes, such as symbiosis, competence, conjugation,

6

antibiotic production and virulence, have been studied in medicine and toxicology [10].

7

Recently, the biofilm formation, activity and growth rates of bacteria for the wastewater

8

treatment have been investigated because they provide information that is required to

9

solve problems related to water treatment [11]. Previous studies have been verified the

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community activity (phenol degradation) could be improved under conditions that 2 µM

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AHL were added to a sludge sample from an industrial wastewater treatment plant [12].

12

N-Octanoyl-L-Homoserine lactone (C8-HSL) was reported to be closely associated with

13

membrane biofouling of the membrane bioreactor, while the formation of membrane

14

biofilms can be prevented by quenching AHLs [13]. Moreover, De Clippeleir et al.

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demonstrated that the oxidation rate of Oxygen Limited Autotrophic Nitrification

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Denitrification (OLAND) biomass at low concentrations could be significantly

17

increased with the addition of exogenous AHLs [14].

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Although the question about whether anammox bacteria owns QS systems has not

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been well answered, some indirect evidences about the existence of some key

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characteristics about the QS systems in anammox bacteria have been confirmed. First,

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the activity of anammox bacteria has an apparent density-dependent phenomenon. It has

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been discovered that anammox bacteria do not take on activity until the cell 4

1

concentration is higher than 10 10–1011 cell mL-1 [15]. This is similar to Vibrio fischeri,

2

the bioluminescence of which could be inspired when the population density reaches a

3

threshold concentration. Vibrio fischeri are typical bacteria regulated by QS system [16].

4

Moreover, the aggregation of anammox bacteria is similar to the biofilm formation.

5

AHLs-based QS system has been shown to influent biofilm formation of Gram-negative

6

bacteria in water and wastewater systems [17, 18]. During anammox reactor operation,

7

the anammox bacteria were frequently found to form biofilm or attach to the container

8

wall. In the activated sludge reactor, high activity anammox bacteria are easy to

9

aggregate as red granules settling in the bottom of the reactor [19]. In addition, the

10

existence of the genes coding for the quorum sensing compounds has been evidenced by

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Strous et al. [20], where the meta-genome of anammox bacteria was shown. It has been

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confirmed that four gene clusters encode fatty acid biosynthesis of Kuenenia

13

stuttgartiensis, two of which have functions to encode fatty acid biosynthesis and

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S-adenosyl methionine (SAM) radical enzymes (significant biosynthetic pathway for

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AHLs signals) [8, 21]. Even though increasing evidences have indicated that anammox

16

bacteria might have QS regulation systems, the direct evidences for the anammox

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bacteria communication by QS systems are remained to be explored. Therefore, the

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potential existence of QS system in anammox bacteria and its effects are very urgent to

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be investigated, which might be useful to regulate the growth rate and activity of

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anammox bacteria with the final aim to realize the fast start-up of anammox process .

21 22

In this study, three different AHLs were identified in the anammox culture supernatant and culture extraction by applying AHLs biosensor and High-performance 5

1

liquid chromatography (HPLC). To estimate the potential effects of C6-HSL, C8-HSL

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and C12-HSL on the regulation of anammox bacteria behavior, batch tests were

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conducted by adding exogenous AHLs. Moreover, the probable working mechanism of

4

exogenous signals on anammox culture was proposed by real-time analyzing the AHLs

5

concentration after its addition to the system.

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2. Material and Methods

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2.1 Inoculum bacteria and chemical analysis

8 9

The anammox culture used in this study was mainly composed of Candidatus brocadia fulgida, which accounted for around 80% of the total population [22].

10

According to standard methods set out by the American Publish Health Association [23],

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the concentrations of NH4+-N were measured by preliminary distillation step and

12

titrimetric method, NO3--N were determined by Ultraviolet Spectrophotometric

13

Screening Method, NO2--N were measured by Colorimetric Method, and Volatile

14

Suspended Solids (VSS) were defined under the conditions that suspended solids were

15

dried at 103-105 °C for 12 h and volatile solids were ignited at 550 °C for 2 h. pH was

16

obtained by pH meter (Mettler, Switzerland).

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2.2 Reactor operation

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Initial experiment was conducted in a 3.0 L bio-fermentor inoculated with anammox

19

bacteria. Cultivation medium, as described previously [24], were added to the fermentor

20

to keep certain concentrations suitable for bacteria activation (the initial concentration

21

of NH4+-N and NO2--N were kept among 80-100 mg L-1). The solutions were purged

22

with a gas mixture of N2-CO2 (95/5%) to exclude the dissolved oxygen (DO) and keep 6

1

the anaerobic conditions for anammox bacteria. pH, DO and temperature in the

2

fermentor were measured online and controlled at 7.8-8.2, 0-0.05 mg L-1 and 37±1 °C,

3

respectively. The stirring speed of standard six-blade disk impeller was set at 100 rpm to

4

produce shear force and improve gas-liquid transfer. The initial solids concentration for

5

the reactor operation was 0.58 g VSS/L. The experiment was conducted in a 3.0 L

6

bio-fermentor with bulk volume of 2.5 L. HRT was shortened from 48 h (0－340 day of

7

the operation) to 24 h (340－400 day of the operation) with improvement of bacterial

8

activity during these days. When the concentration of NO2--N was under 20 mg L-1, the

9

used medium was discarded, new medium was added and one cultivation cycle was

10

finished, which indicted the operations were continuous. During the 400 days’ operation,

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the nitrogen removal performance, activity and VSS were recorded every 30 day. Every

12

data was measured for three times to get the average value.

13

2.3 AHLs standard

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AHLs structures all have the homoserine lactone ring moiety and acyl side-chain of

15

different length, degree of substitution, and saturation. The acyl side-chain length (n) is

16

typically 4−14 carbons in length. AHLs standards (C6-HSL, C8-HSL, C12-HSL) were

17

purchased from Sigma Aldrich.

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2.4 Extraction of AHLs

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The culture supernatant (300 mL) was harvested, filtered through a 0.22 µm cellulose

20

filter to remove bacteria and extracted with an equal volume of ethyl acetate and 0.1%

21

(v/v) formic acid [25]. The solvent extracts were evaporated using a rotary evaporator

22

(Zhengjie, RE-52AA) at 40 °C, and reconstituted in 1 mL ethyl acetate, stored at 7

1

–20 °C.

2

AHLs extraction from the anammox bacteria was described below. The biomass was

3

homogenized in a 100 mL serum bottle, and 1 mL mixture was extracted using a syringe.

4

The supernatant was removed by centrifugation at 4000 rpm for 2 min, and the bacteria

5

were obtained and dispersed in methanol. The biomass was completely disrupted using

6

sonication at 300 W for 12 min and filtered through a 0.22 µm syringe filter. The solvent

7

extracts were dried under a nitrogen flow at 40 °C, reconstituted in 400 µL of methanol

8

for concentration and stored at -20 °C.

9

2.5 AHLs reporter plate assays

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Agrobacterium tumefaciens A136 (pCF218) (pCF372) is a biosensor strain used in

11

agar plate-based bioassays because it hydrolyzes

12

5-Bromo-4-chloro-3-indolyl-beta-D-galactopy-ranoside (X-gal) into a blue pigment in

13

the presence of AHLs, carbon chains ranging from 6-14 carbons in length [26]. Prior to

14

the assay, the bacteria was grown in sterilized Luria-Bertani (LB) broth medium with 50

15

µg L-1 spectinomycin and 4.5 µg L-1 tetracycline on a rotary shaker (100 rpm) at 30 °C

16

for 48 h. Antibiotic solutions were filtered through a 0.22 µm filter.

17

The sterilized LB medium of 50 ˚C containing 0.6% (g/g) molten agar mixed with the

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culture of A. tumefaciens A136 (5% v/v) mentioned above and 50 µg L-1 X-gal. This

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soft agar suspension was poured onto Petri-dishes. After solidification of the agar, wells

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(5 mm diameter) are made using a sterile glass bar. To wells in the agar plate, 10 µL of

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concentrated supernatant solution and AHL standards was added. The concentration of

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the selected AHL standards (C6-HSL, C8-HSL, C12-HSL) were 250 mg L-1. The Petri 8

1

dishes were incubated overnight at 30 °C, and the syntheses of blue pigments were

2

examined to identify the existence of AHLs in the anammox culture supernatant.

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2.6 HPLC analysis

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HPLC analysis was applied to identify varies kinds of AHLs in anammox culture

5

supernatant and bacteria. Briefly, 20 µL solutions were applied to a HPLC column

6

(Zorbax SB-Aq 4.6×250, 5 um), eluted with a linear gradient of methanol in water (20

7

to 100%) over a 30-min period at a flow rate of 1 mL min-1 and monitored at 210 nm

8

with DAD detector. For the bacteria extraction contained low concentration of AHLs,

9

the input dosage for HPLC analysis was increased to 100 µL to enhance the AHL peaks.

10

2.7 Batch test

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The NH4 +-N and NO2--N removal profile of the anammox culture were determined in

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four 100 mL anaerobic serum bottles. The biomass was washed and re-suspended in the

13

cultivation medium. Incubation temperature was kept at 37 oC and anaerobic condition

14

was maintained. The initial biomass was approximately 0.025 g VSS for each bottle.

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After an experiment cycle, which last for 20-25 h depending on the bacteria activities,

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the supernatant was poured and fresh medium was added. Samples were collected per 5

17

h using a syringe. The four bottles were incubated three experimental cycles until the

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bacterial activity stabilized. After that, C6-HSL, C8-HSL and C12-HSL (30 mg L-1) were

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added to the three bottles respectively. The batch tests with the exogenous addition of

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AHLs were conducted for three experimental cycles to get the average value. Finally,

21

the nitrogen removal rate, the NO2-N removal/NH4 -N removal and the bacterial activity

22

were measured and calculated to determine and compare the various effects. Bacterial 9

1

VSS was measured in every begin (S0) and the end (S1) of experiment cycle, and the

2

growth rate of bacteria was defined as the specific growth rate (u=(S1- S0)/S0.t; t is the

3

time of one experiment cycle). The test was calculated by comparing the changes of

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NH4+-N removal rate, bacterial activity, bacterial growth rate and NO2--N removal/

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NH4+-N removal of the anammox culture after the introduction of C6-HSL, C8-HSL and

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C12-HSL. Every experiment was repeated for three times and the significance level of

7

data was analyzed through Independent-Samples T Test.

8

2.8 Real-time PCR analysis

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DNA was extracted using a Fast DNA spin kit (Applied Biosystems, USA) according

10

to the manufacturer’s instructions. Real-time PCR assays were conducted as follows.

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The primers for anammox and eubacteria were 694F (forward primer)-960R (reverse

12

primer) [27] and 314F (forward primer)-518R (reverse primer) [28], respectively. Each

13

PCR working solution (20 µL) contained 12.5 µL of SYBR Green PCR master mix

14

(Applied Biosystems, USA), 1 µL each of forward and reverse primers, 3 µL of

15

template DNA (the DNA extraction was diluted 100 times) and 2.5 µL of ddH2O. PCR

16

amplification and detection were performed with an ABI Prism 7000 sequence detection

17

system (Applied Biosystems, USA). The PCR temperature program for both anammox

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and eubacterial DNA extracts was initiated with 2 min at 50 °C and 10 min at 95 °C

19

followed by 30 cycles of 10 s at 95 °C and 1 min at 59 °C.

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3. Results and Discussion

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3.1 Bio-fermentor operation profile for anammox bacteria cultivation

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The bio-fermentor was operated for 400 days after inoculating 1.74 g VSS anammox 10

1

bacteria. The reactor nitrogen removal performance was measured and showed in Fig.

2

1(a). During the 400 days’ operation, the nitrogen removal rate (NRR) was increased

3

from 20 mg N L-1day-1 to 650 mg N L-1day-1, indicating the efficient bacteria cultivation

4

and enrichment. The curve was similar to pervious reports [29], namely including phase

5

I with initially steady nitrogen removal performance and phase II with rapidly increased

6

nitrogen removal performance. To further identify the apparent divergence of these two

7

phases, the bacteria VSS and nitrogen removal activity during the operation were

8

measured and recorded in Fig. 1(b). Besides the nitrogen removal performance, it

9

clearly shows the abrupt increase of the bacteria VSS and bacteria activity in phase II of

10

the bio-fermentor operation, from day 350 to day 400. Compared to the bacteria

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behavior in phase I of the operation, it seems that bacteria status was changed and all

12

the bacteria became more active in phase II. It is very strange about what happened in

13

this phase. Actually, this is a typical anammox reactor start-up performance [29].

14

However, the involved mechanism for the abrupt increase in phase II is significantly

15

worthy to be further investigated, which might be useful to accelerate anammox reactor

16

start-up and solve the key restricted problem for anammox process application.

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The phenomenon during the anammox reactor start-up indicates both of the activity

18

and growth rate of anammox bacteria depend on the bacteria amounts. From this, QS

19

system for the bacteria communication comes to our view as the key points to regulate

20

the bacteria behavior. Its identification and verification need further investigation.

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3.2 Identification of AHLs release in anammox culture

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In order to provide the direct evidence about the existence of QS system in anammox 11

1

bacteria, we firstly tried to detect the release of AHLs in anammox culture. Because

2

QS-regulated bacteria release AHLs into the surroundings, it is necessary to detect

3

AHLs in the supernatant. At first, the agar-plate based bioassay was applied by using

4

biosensor A. tumefaciens A136 to identify the QS signals in the supernatant from the

5

fermentation at the end of the cultivation cycle on the day 400 of the operation period.

6

As results, the bacteria could efficiently display blue color in the C6-HSL, C8-HSL and

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C12-HSL test wells in the agar-plate. These AHLs signals have been detected in several

8

bacterial communities in water treatment facilities [12, 30]. The most worthy to be

9

noticed is that, the concentrated supernatant of anammox culture also showed a blue

10

color in the test wells, strongly verifying the presence of AHLs in the supernatant of

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anammox culture.

12

With the aim to further clarify the AHLs species in the supernatant of anmmox

13

culture, especially whether it contains C6-HSL, C8-HSL and C12-HSL, HPLC was

14

explored in this investigation. The C6-HSL, C8-HSL and C12-HSL standard were used

15

and the key retention times were determined as 16.02, 29.39 and 44.51 min in the HPLC

16

fingerprint, respectively (Fig. 2). Here we use “Standard addition method” to identify

17

the existence of signals in the concentrated culture supernatant. Standard addition

18

method is an analysis approach used in analytical chemistry, which was conducted by

19

directly adding standard to the analyzed sample with the aim to reduce the interference

20

effects on the analysis results [31]. The sample “standards” are actually “mixture of

21

standards and concentrated culture supernatant”. By comparing the spectrogram peaks

22

of this sample to that of “concentrated culture supernatant”, we can see the peak at 12

1

44.51 min clearly increased after dosage C12-HSL. Thus, the peak at 44.51 min in Fig.2

2

(c) represented C12-HSL. Other peaks came from the concentrated culture supernatant,

3

which have no relationship with C12-HSL. In this part, we mainly paid attention to the

4

release of C12-HSL by the biomass, so we did not check other peaks. For the anammox

5

culture supernatant, the corresponding peaks also appeared, suggesting the simultaneous

6

existence of C6-HSL, C8-HSL and C12-HSL here. We established a linear relationship

7

between peak area and AHLs concentration to quantify the concentration of AHLs in the

8

extraction. At this extraction time, it was calculated that C6-HSL, C8-HSL and C12-HSL

9

in the supernatant was 0.33, 0.18 and 0.33 mg L-1, respectively.

10 11

3.3 Effects of AHLs on anammox culture Although we obtained the direct evidence about the appear release of QS signals in

12

anammox culture, more crucial question about whether these AHLs affect anammox

13

bacteria profile is still necessary to be revealed. It is very important to clarify the

14

important roles of QS communication system in anammox culture. To confirm the effect

15

of the identified AHLs in anammox culture, exogenous C6-HSL, C8-HSL and C12-HSL

16

with concentration of 30 mg L-1 (30 mg L-1 was chosen as the target concentration due

17

to solubility limitations of C12-HSL) were added to Vials B, C and D, respectively. Vial

18

A was considered as control sample without any addition of exogenous signals. As

19

expected, NH4+-N removal rate, bacterial growth rate, activity and NO2--N

20

removal/NH4+-N removal of the anammox culture were visibly affected upon their

21

addition, which was clearly present in Fig.3.

22

The NH4 +-N removal rate was most worthy to be concerned since it could directly 13

1

impress the performance of anammox reactor. Thereby, effects of the exogenous

2

signaling molecules C6-HSL, C8-HSL and C12-HSL on the NH4 +-N removal rate were

3

firstly investigated. The quantitative analysis results were clearly in different levels and

4

showed in Fig. 3 (a). For the original anammox culture (Vial A), the NH4 +-N removal

5

rate was a little increased from 63 mg NH4+-N L-1 day -1 to 71 mg NH4+-N L-1 day -1

6

after the tested few days. A comparison of the anammox culture treated with different

7

signaling molecules showed significant difference. Every experiment was repeated for

8

three times for the statistical test. The significant (p<0.05) increase of NH4+-N removal

9

rate occurred when cultivating with signaling molecules C6-HSL (Vial B) and C8-HSL

10

(Vial C), with change from 55 mg NH4+-N L-1 day -1 to 74 mg NH4+-N L-1 day -1 and 60

11

mg NH4+-N L-1 day -1 to 72 mg NH4+-N L-1 day -1 on the average, respectively, resulting

12

in the increased NH4+-N removal performance by 35% and 20%, respectively. However,

13

the nitrogen removal performance of anammox culture when incubated with C12-HSL

14

(Vial D) did not have significant difference (p>0.05) with that of the control sample

15

without any exogenous AHLs.

16

The effects of exogenous AHLs on the activities of anammox bacteria were also

17

analyzed (Fig. 3(b)). In the control experiment, the calculated activity was a little

18

increased from the average value of 239 mg NH4+-N gVSS-1 day-1 to 258 mg NH4+-N

19

gVSS-1 day-1 after the incubation. Specifically, a promising increase of bacterial activity

20

occurred when incubated with 30 mg L-1 C6-HSL, in which significant (p<0.05)

21

increase occurred from the average value of 208 mg NH4+-N gVSS-1 day-1 to 247 mg

22

NH4+-N gVSS-1 day-1 after the addition of the signaling molecules. The nearly 20% 14

1

enhancement indicated the positive effects of C6-HSL on anammox activity. Addition of

2

C8-HSL also produced a notable increase of the bacteria activity. However, when

3

incubated with C12-HSL, the bacterial activity significantly decreased (p<0.005)

4

comparing with the control sample without any exogenous AHLs, suggesting that

5

C12-HSL produced the negative effects on anammox activity.

6

The variance of bacteria growth rate also indicated the effects of signaling molecules

7

on anammox culture (Fig.3(c)). The cultivation without exogenous signaling molecules

8

expressed an average growth rate of 0.0027 h-1. Addition of C6-HSL produced a

9

significant increase of the bacteria special growth rate from 0.0025 h-1 to 0.0.032 h-1,

10

meaning 28% increase. The maximum growth-promoting effects were observed when

11

the culture incubated with C12-HSL, which was identified as 0.0038 h-1 with 32%

12

increase contrast to the control sample according to the analysis. The potential effects of

13

signaling molecules on accelerating bacteria growth rate were verified and the signaling

14

molecular, especially C6-HSL and C12-HSL, was thus regarded to act on accelerating the

15

growth.

16

Although both of the signaling molecules C6-HSL and C12-HSL could increase the

17

bacteria growth rate, there is an important question about which bacteria species could

18

be accelerated in the culture community. To identify it, the nitrogen removal profile with

19

the NO2--N removal/ NH4 +-N removal was calculated and the results are shown in Fig.

20

3(d). The ratio of NO2--N removal and NH4+-N removal has been verified to be 1.32 for

21

anammox reaction. After the dosage of C12-HSL, we found remarkable increase of the

22

NO2--N removal/ NH4+-N removal from 1.23 to 1.64, implying the potential occurrence 15

1

of the growth of other bacteria and other nitrogen removal process to remove NO2--N.

2

With the supply of C6-HSL and C8-HSL, the ratio kept almost stable at 1.32 [29],

3

indicating the functional bacteria (anammox bacteria) kept unchanged. The variation of

4

anammox bacteria percentage in the consortia after supplying C6-HSL, C8-HSL and

5

C12-HSL could be verified by RT-PCR analysis.

6

For the purpose to further identify, RT-PCR was performed to count the change of

7

anammox percentage in the culture community after the addition of exogenous signals

8

(Fig.4). Signaling molecules were verified to have profound effects on the anammox

9

percentages in the culture, namely the disturbing of bacteria inter-species

10

communication by signaling molecules also break the steady and balance of the original

11

microbiological community. Notably, when following supply of C12-HSL, we found

12

remarkable reduction of anammox percentage from 81% to 58%, indicating the

13

potential growth of other bacteria. This clear decrease of anammox percentage was

14

identified as important effects of C12-HSL on anammox culture. In contrast, it seems

15

that the C6-HSL brought a little increased anammox percentage from 81% to 88%,

16

compared to the control experiment. It could be easily understood that the quick growth

17

of anammox bacteria obtained by C6-HSL and correspondingly high nitrogen removal

18

performance. The whole process seems as biomass dependent, which has also been

19

reported by other studies [15]. These results are in agreement with the theory that the

20

AHL signals are the quorum sensing systems and they could exert more effects at higher

21

biomass concentration.

22

The different signals for inter-species communication were confirmed to capably 16

1

lead to different behaviors of anammox culture. From the study here, we can attest that

2

C6-HSL has important functions to increase the anammox bacteria activity and growth

3

rate. C6-HSL and C8-HSL have positive effects on anammox culture and significantly

4

increase the nitrogen removal performance.

5

The dosage of C12-HSL resulted in the evident increase of bacteria total amount

6

(VSS). According to the calculation formula u=(S1- S0)/S0.t, the growth rate of the

7

whole bacteria community increased after the introduction of C12-HSL (Fig.3).

8

Meanwhile, the RT-PCR analysis results (Fig.4) showed that the dosage of C12-HSL

9

decreased anammox bacteria percentage in the bacteria community. Combined with the

10

nitrogen removal results, it could be inferred that some heterotrophic bacteria grew,

11

which inevitably decreased anammox activity. Thus, although the dosage of C12-HSL

12

could increase the growth rate of the whole bacteria community, the anammox activity

13

decreased since it accelerated the growth of heterotrophic bacteria.

14

These results are a little discrepant with other study carried out by De Clippeleir et al.

15

[14], who demonstrated that C12-HSL was useful to increase the nitrogen removal

16

performance of OLAND biomass. Actually, the response of different bacteria cultures to

17

AHLs might be different. These effects are also determined by bacteria community

18

composition and biomass concentration. The addition of C12-HSL promoted the growth

19

of heterotrophic bacteria in this study with the appear phenomena about the outstanding

20

increase of NO2 removal/NH4 removal , suggesting some bacteria in the anammox

21

cultures in this study might have the ability to utilize C12-HSL to remove nitrite.

22

C12-HSL has opposite effects on anammox culture comparing to C6-HSL and C8-HSL, 17

1

because it prominently inspired the growth of heterotrophic bacteria in the anammox

2

culture.

3

In all, the advantage of having QS system in anammox bacteria was huge. On one

4

hand, it is well-known that the slow growth rate of anammox bacteria and

5

corresponding long start-up period of anammox process was the main limiting factor for

6

the application of anammox process. According to the results proposed in this study,

7

anammox bacterial growth rate, activity and nitrogen removal rate could be increased

8

and then start-up period could be shortened by the introduction of AHLs. This provided

9

a promising solution for the existing problem of anammox process. On the other hand,

10

the verification of the existence of QS system in anammox bacteria could well explain

11

the intrinsic characteristics of anammox bacteria, such as bacteria aggregation and

12

density-dependent activity (Anammox bacteria do not take on activity until the cell

13

concentration is higher than 10 10–1011 cell mL-1).

14

3.4 Induced release of AHLs by exogenous AHLs

15

With the aim to make clear how the exogenous signals affect anammox culture,

16

C6-HSL was added to the medium of anammox culture and the C6-HSL was tracked by

17

real-time monitoring the concentrations of this signal in the biomass and supernatant.

18

The result is shown in Fig.5. Surprisingly, the exogenous signal C6-HSL could not

19

sustain in the supernatant of the anammox culture for long time. It was rapidly degraded

20

and concentration of C6-HSL in the supernatant decreased in the first 12 h from 35 mg

21

L-1 to 10 mg L-1 with a good linear correlation (R2=0.97). In fact, the degradation of

22

AHLs depends on the pH of the supernatant and is caused by chemical degradation and 18

1

bacteria degradation. It was reported that 3-oxo-C6-HSL became unstable in a pH range

2

of 7 to 8 without other interference factors [32]. The pH of 7.8-8.2 of the medium was

3

regulated for anammox bacteria growth. So, it was also confirmed in this study that the

4

degradation of C6-HSL could be performed in the anammox culture medium even

5

without bacteria (Fig.6 a). In addition, the degradation was accelerated under the effects

6

of bacteria (Fig.6 b).

7

In this case, we all wondered how the exogenous signals worked after their addition.

8

Through the analysis of C6-HSL concentration in the biomass (Fig. 5), we can see

9

although very low amount of C6-HSL was initially detected in the biomass, after the

10

addition of exogenous C6-HSL, the amount of C6-HSL in the biomass increased

11

significantly. This result is similar to that occurs during QS regulation. The increasing

12

concentration of AHLs in the surroundings (the supernatant) reached the threshold value

13

of the QS system and activated the expression of related genes, such as LuxI, the gene

14

encoding AHL synthase [8], which could release more AHLs. Therefore, the amount of

15

C6-HSL in the biomass inevitably increased. For the following12–32 h, a reduced

16

degradation rate of C6-HSL in the supernatant was observed (Fig. 5), corresponding to

17

the increased concentration of C6-HSL in the biomass. The stable amount of C6-HSL in

18

the biomass is attributed to the counterbalance of degradation and production. Taken all

19

together, the addition of exogenous C6-HSL has crucial roles in enhancing the

20

production of endogenous C6-HSL by biomass, and then to accelerate the nitrogen

21

removal process.

22

4. Conclusion 19

1

In this study, the potential existence of QS communication system in anammox

2

culture was verified by the definite release of QS signals C6-HSL, C8-HSL and C12-HSL

3

and the obvious effects of them on anammox bacteria behavior. C6-HSL could increase

4

anammox bacteria activity and growth rate, and C8-HSL only activate on the promotion

5

of anammox bacteria activity. However, addition of C12-HSL decreased anammox

6

activity (Fig. 3). The function pathway that exogenous AHLs activated the bacteria by

7

inducing the production of endogenous signals released by anammox culture was

8

proposed finally, useful to provide a new approach to accelerate the anammox process

9

start-up.

10

Acknowledgements

11

The authors are grateful to the National Natural Science Foundation of China

12

(No.51308007 and No. 21261140336) for financial support.

13 14

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1

Figure legends

2

Fig. 1 (a) The Nitrogen removal rate (NRR) of anammox bio-fermentor operation

3

during 400 days’ period. (b) The activity and the total amount (VSS) of anammox

4

bacteria in the bio-fermentor during the operation period.

5

Fig. 2 HPLC analysis of AHLs extracted from culture supernatant. The detection of (a)

6

C6-HSL, (b) C8-HSL and (c) C12-HSL in concentrated culture supernatant depended on

7

the retention times, which were derived from the retention time of the standards.

8

Fig. 3 Effects of exogenous C6-HSL, C8-HSL and C12-HSL (30 mg/L) on the (a)

9

NH4+-N removal rate, (b) activity, (c) growth rate and (d) NO2-N/NH4-N (ratio of nitrite

10

nitrogen and ammonia nitrogen removal) of anammox bacteria. The experiments were

11

carried out for three times to get the average values at the time points before (red) and

12

after (black) the addition of AHLs. One or two asterisks indicate a significant increase

13

compared to no treatment with either (p<0.05) or (p<0.01), respectively.

14

Fig. 4 Effects of exogenous C6-HSL, C8-HSL and C12-HSL (30 mg/L) on the proportion

15

of anammox bacteria in the community. The proportions of anammox bacteria were

16

measured three times before (black) and after (red) the addition of AHLs by RT-PCR.

17

Fig. 5 The variation of C6-HSL concentrations in supernatant and biomass. The

18

concentration of C6-HSL in biomass over long cultivation times is represented by three

19

phases: (1) initial phase, (2) increasing phase, and (3) stable phase.

20

Fig. 6 HPLC analysis of the degradation of C6-HSL in cultivation medium (a) without

21

anammox culture and (b) with anammox culture.

26

22 23

24 25 26 27

28 29 30 31 32 33 34 35 36

Fig. 1

27

37

38

39 40

Fig. 2

41

28

42

43 44

Fig. 3

29

45 46

Fig. 4

47

30

48 49

Fig. 5

50

31

51 52

Fig. 6

53

32

56

Highlights

57 58




Verified the existence of QS communication system among anammox bacteria.

59




Confirmed the release of C6-HSL, C8-HSL, C12-HSL by anammox culture.

60




C6-HSL and C8-HSL increased activity and growth rate of anammox bacteria.

61




Additional signals activated bacteria by inducing endogenous signals in bacteria.

62




Opened a new window to accelerate the start-up period of anammox reactor.

63

34