removal of nitrate in simulated water at low temperature

Research ArticleRemoval of Nitrate in Simulated Water at LowTemperature by a Novel Psychrotrophic and Aerobic BacteriumPseudomonas taiwanensis Strain J

Tengxia He Qing Ye Quan Sun Xi Cai Jiupai Ni Zhenlun Li and Deti Xie

Chongqing Key Laboratory of Soil Multiscale Interfacial Process Southwest University Chongqing 400716 China

Correspondence should be addressed to Zhenlun Li lizhlun4740sinacom and Deti Xie xdtswueducn

Received 23 September 2017 Revised 9 December 2017 Accepted 10 January 2018 Published 28 March 2018

Academic Editor Raf Dewil

Copyright copy 2018 Tengxia He et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Low temperatures and high pH generally inhibit the biodenitrification Thus it is important to explore the psychrotrophicand alkali-resisting microorganism for degradation of nitrogen This research was mainly focused on the identification ofa psychrotrophic strain and preliminary explored its denitrification characteristics The new strain J was isolated using thebromothymol blue solidmediumand identified as Pseudomonas taiwanensis on the basis ofmorphology and phospholipid fatty acidas well as 16S rRNA gene sequence analyses which is further testified to work efficiently for removing nitrate fromwastewater at lowtemperature circumstances This is the first report that Pseudomonas taiwanensis possessed excellent tolerance to low temperaturewith 15∘C as its optimum and 5∘C as viable The Pseudomonas taiwanensis showed unusual ability of aerobic denitrification withthe nitrate removal efficiencies of 100 at 15∘C and 5161 at 5∘C Single factor experiments showed that the optimal conditionsfor denitrification were glucose as carbon source 15∘C shaking speed 150 rmin CN 15 pH ge 7 and incubation quantity 20 times106 CFUmL The nitrate and total nitrogen removal efficiencies were up to 100 and 9379 at 15∘C when glucose is served ascarbon source These results suggested that strain J had aerobic denitrification ability as well as the notable ability to tolerate thelow temperature and high pH

1 Introduction

Nitrate due to its highwater-soluble characteristic is possiblythe major nitrogen contaminant in the water [1 2] Highconcentration nitrate could contribute to water eutroph-ication [3] and even impose a serious threat to humanhealth such asmalformation carcinoma andmutationwhenthe nitrate transformed into nitrosamines [4] The WorldHealth Organization (WHO) has recommended that thenitrate concentration in drinking water should be lower than10mgL [5] and the same value was also proposed by China[6] Unfortunately the nitrate concentration was higher than10mgL in numerous aquifers and even exceeded 30mgL insome groundwater in China [7ndash9] The nitrate remediationthereby is a big challenge and has become a matter of greatconcern in the recent years

Previous researches have shown that the most commonlymethods to remove nitrate fromwastewater included biologi-cal denitrification with microorganism and physicochemical

reduction using ion exchange electrodialysis reverseosmosis zero-valent iron and zero-valent magnesium[3 10] Several reports demonstrated that bioremediationhas much higher efficiency has lower cost and is easier toimplement compared with physicochemical methods fornitrate removal from wastewater [11 12] Likewise numerousresearchers discovered that the biological denitrification isthe most promising approach because the bacteria couldreduce the nitrate to the harmless nitrogen gas [13 14]Therefore there are a host of papers dedicated to isolationand identification of nitrate reduction bacteria such asStenotrophomonas sp ZZ15 Oceanimonas sp YC13 [15]Bacillus sp YX-6 [16] Psychrobacter sp S1-1 [17] Zoogloeasp N299 [18] and Alcaligenes sp TB [19] while the reporteddenitrifying bacteria including these mentioned above arealmost mesophilic bacteria and the optimum denitrificationtemperatures are between 20 and 35∘C However thesemesophilic denitrifying bacteria might face great challengesin winter months because the low temperatures generally

HindawiBioMed Research InternationalVolume 2018 Article ID 4984087 9 pageshttpsdoiorg10115520184984087

2 BioMed Research International

drastically inhibit their denitrification ability cell growth andproliferation especially when the temperature was at 10∘C orless [20 21]Thus it is important to explore the bacteriawhichcould effectively remove nitrate nitrogen at low temperatures

Additionally nitrate biodegradation may generate OHminuswhich could inhibit the denitrification process and enzymeactivity [22] Zhang et al [23] discovered that neither celldensity increase nor nitrate reduction was found if the pH isgreater than 85 Li [22] reported that the nitrate reductionwas completely inhibited when the pH reached about 95Therefore the optimal pH of most new isolated aerobicdenitrifiers ranged from 65 to 70 such as Ochrobactrum sp(65ndash70) [24] Alcaligenes sp S84S3 (70) [25] Psychrobactersp (70) [17] and Pseudomonas mendocina 3ndash7 (70) [26]It may be difficult for these microorganisms to meet therequirements of alkaline sewage treatment

In this study a novel bacterial strain for candidate ofaerobic denitrifier capable of aerobic denitrification withnitrate was identified as Pseudomonas taiwanensis namedJ To the best of our knowledge few nitrate denitrifica-tion studies have been performed to focus on the speciesof Pseudomonas taiwanensis In this research the impactresistance of strain J to low temperature extreme alkalinityand high CN ratio were investigated using nitrate as solenitrogen source The experimental results showed that thestrain J possessed excellent tolerance to low temperatureswith 15∘C as its optimum and 5∘C as viable Furthermoreit has been found that the nitrogen removal efficiency didnot decrease obviously with temperature between 15∘C and40∘C Significantly high CN ratio and strong alkalinity werenot the limiting factors for cell density increase and nitratedenitrification performance Accordingly the strain J couldbe used to treat the high CN ratio wastewater and thealkaline wastewater in four seasons

2 Materials and Methods

21 Bacterium and Media The psychrotrophic and aerobicdenitrifying bacterium Pseudomonas taiwanensis strain J wasstocked in 30 glycerol solution at minus20∘C

We followed the methods of He and Li [27] Thebromothymol blue (BTB) solid medium per liter (pH =72) comprised NaNO

31 g KH

2PO41 g FeCl

2-6H2O 05 g

MgSO4-7H2O 1 g CaCl

2-7H2O 02 g sodium succinate 85 g

BTB reagent 1mL [15 in ethanol] and agar 20 g [28] Themodified denitrificationmedium (MDM) per liter comprisedthe following (ultrapure water per liter) NaNO

3031 g

CH3COONa 256 g KH

2PO415 g Na

2HPO4042 gMgSO

4-

7H2O 1 g Fe SO

4-7H2O 005 g and pH 72 plusmn 01 [16] LB

medium (pH 70 per liter) included tryptone 100 g yeastextract 50 g andNaCl 10 g For preparation of LB solid plates2 (wv) agar powder was added

22 Identification of the Psychrotrophic and Aerobic Denitrify-ing Bacterium Strain J Colony morphologies of strain J weremonitored on the BTBmediumplates after incubating at 15∘Cfor 3 d Cell morphologies of strain J were observed underthe HITACHI S-3000N scanning electron microscope andatomic force microscopy Phospholipid fatty acids (PLFAs)

were extracted by using about 40mg pure culture of strain Jafter incubating 48 h at 15∘C Each type of PLFAswas analyzedby Agilent 6850

The nearly full-length of 16S rRNA gene sequence wasamplified using DNA as template which was extracted bygenomic DNA purification kit (Thermo scientific) Theuniversal primers 27F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and 1492R (51015840-GGTTACCTTGTTACGACTT-31015840) wereused for polymerase chain reaction (PCR) amplificationThe PCR amplification was conducted in the 50mL volumecontaining 2 120583L DNA 2 120583L primer 25120583L 2x Taq PCRMasterMix and 19 120583L sterile water The PCR conditions weredenaturation for 5min at 94∘C 30 cycles of 1min at 94∘C 30 sat 555∘C and 1min at 72∘C and extension for 10min at 72∘CThe 15 kb product was separated on a 15 agarose gel andpurified by BioSpin gel extraction kit (BioFlux) The purifiedproduct was cloned into pMD20-T vector (Takara) andthen sequenced by Invitrogen company Then the 16S rRNAsequence of strain J was submitted to NCBI for accessionnumber Sequence alignment and multiple alignment wereperformedusingNCBI SearchTool program (BLAST httpsblastncbinlmnihgovBlastcgiPROGRAM=blastnampPAGETYPE=BlastSearchampLINK LOC=blasthome) and CLUSTALW And the phylogenetic tree was constructed using MEGA60 software by neighbor-joining distance method andbootstrap analyses of 1000 replicates

23 Effects of Culturing Conditions on the DenitrificationAbility of Strain J Effects of six cultivation conditionsincluding temperature (5∘C 10∘C 15∘C 20∘C 25∘C 30∘C35∘C and 40∘C) shaking speed (0 rmin 50 rmin 100 rmin150 rmin and 200 rmin) pH (65 70 80 90 and 100)inoculation quantity (05 times 108 CFU 10 times 108 CFU 15 times108 CFU 20 times 108 CFU and 25 times 108 CFU within 100mLDM) and carbon source (sodium citrate sodium succinatesodium acetate sucrose and glucose) on denitrificationperformance of strain J were determined by the single factortests The amount of carbon was changed to adjust the CNratio to 0 5 10 15 20 and 25 by fixing the amount of NaNO

3

as the nitrogen source 10 times 108 CFU precultured bacterialsuspension was inoculated into 250mL sterilized conicalflask which contained 100mL DM broth medium for testingexcept for inoculation quantity which will be specified laterAfter incubating for 48 hours nitrate and total nitrogen werethen determined for these samples in order to analyze theinfluences of the various factors indicated above

The nitrate and total nitrogen removal efficiencies werecalculated by the equation Rv = (119879

1minus 1198792)1198791times 100 to

assess the denitrification ability of strain J Note that Rv 1198791

and 1198792represent nitrate or total nitrogen removal efficiency

the initial concentration of nitrate or total nitrogen in MDMbroth medium and the final concentration of nitrate ortotal nitrogen in MDM broth medium after incubation for48 hours respectively All experiments were conducted intriplicate

24 Analytical Methods The cell optical density (OD600

) wasmonitored by measuring the absorbance at the wavelengthof 600 nm using a spectrophotometer (DU800 BECKMAN

BioMed Research International 3

(a) (b)

(c)

D

001 height sensor

49 Ｇ

minus1350 ＨＧ

1350 ＨＧ

(d)

Figure 1 The morphologies of the strain J Colony on BTB plates (a) unicell morphology of strain J (10 times 100) (b) cells under the scanningelectron microscope (10000x) (c) and atomic force microscopy (AFM) profile of strain J (d)

COULTER USA) Total nitrogen was calculated by theabsorbance value at 220 nm subtracting the two times back-ground absorbance value at 275 nm after alkaline potas-sium persulfate digestion Nitrate was detected using thesupernatant after samples centrifuged at 8000 rpm for 8minNO3

minus-N was calculated by the absorbance value at 220 nmsubtracting the two times background absorbance value at275 nm [29] The value of pH was measured by pH-Meter(PHS-3D Shanghai Precision and Scientific Instrument Cor-poration)

The final results were obtained by the average at leastthree independent experiments and were presented as meansplusmn SD (standard deviation of means) All statistical analyseswere carried out by one-way ANOVA with Tukeyrsquos HSD test(119875 lt 005) using Excel and SPSS Statistics 22 and graphicalworks were carried out by Origin 86 software

3 Results and Discussions

31 Identification of Strain J The colony morphologies ofpure strain J are bluewith a small white sport convex smoothwith wet surfaces regular edge and opaque on BTB medium(Figure 1(a)) The strain J was gram-negative rod-shapednonspore and without flagellum (Figures 1(b) 1(c) and 1(d))

Phospholipid fatty acids (PLFAs) as a key componentof cell membrane are an important indicator for identifying

bacteria and fungiThePLFAs is difference in differentmicro-bial groups but is relatively constant in the same microbialpopulation which have been proven to be relatively simplefast and inexpensive to be analyzed by gas chromatography[30 31] The PLFAs of strain J showed 0627 similarity indexwith Pseudomonas-syringae-syringae in the version 60 ofSherlock Microbial identification system As depicted inTable 1 the value of 0627 is greater than 05 which indicatedthat the strain J belongs to being a typical Pseudomonas-syringae-syringae according to the principle of PLFAs iden-tification However the temperature growth phase carbonsubstrate and so on maybe affect the PLFA compositionTherefore we further identified the strain J by using 16SrRNA gene sequence which could enhance the credible of theidentification result

The 16S rRNA gene sequence of strain J (1399 bp) wasdetermined and exhibited 99 similarity with PseudomonastaiwanensisThe phylogenetic treewas constructed byMEGA60 software which showed that the evolutionary divergenceof strain J was also closely related to Pseudomonas taiwanensisinstead of Pseudomonas-syringae-syringae (Figure 2) Thisdifference identification results between PLFAs and 16S rRNAgene sequence might be that the version 60 of SherlockMicrobial identification system did not contain the speciesof Pseudomonas taiwanensis Although the species of Pseu-domonas taiwanensis has been reported by Volmer et al [32]


Table 1 The result of the specific PLFAs identification for strain J

Library Sim Index Entry name

RTSBA6621

0627 Pseudomonas-syringae-syringae0600 Pseudomonas-fluorescens-biotype B0595 Pseudomonas-fluorescens-biotype A0558 Pseudomonas-putida-biotype Bvancouverensis0512 Pseudomonas-syringae-glycinea0508 Pseudomonas-putida-biotype A0485 Pseudomonas-syringae-phaseolicola0480 Pseudomonas-fluorescens-biotype Gtaetrolens0452 Herbaspirillum autotrophicum0425 Pseudomonas-syringae-tabaci

RCLIN6620 0581 Chromobacterium-violaceum0489 Pseudomonas-putida-biotype A

MI7H10380 (No match)

Pseudomonas taiwanensis strain ECAe22 (JF9113841)Pseudomonas taiwanensis strain EPAe52 (JF9113821)

Pseudomonas syringae strain MHGNU B101 (KU8860021) Pseudomonas taiwanensis strain BF-S2 (EU8574171)

Strain J (KY927411)Pseudomonas taiwanensis strain BCRC 17751 (NR 1161721)

Pseudomonas putida strain SP2 (GQ2008221)Pseudomonas syringae strain NBRC 14076

100

9961

64

05

Figure 2 Phylogenetic tree of the strain J Numbers in parentheses represent the sequencesrsquo in GenBank The number at each branch pointis percentage supported by bootstrap (1000 resamplings) Bar 01 sequence divergence

and Schmutzler et al [33] whether the strain of Pseudomonastaiwanensis could conduct aerobic denitrification has notbeen researched The accession number of strain J in Gen-Bank nucleotide sequence databases is KY927411 Taking thephysiological phospholipid fatty acid and 16SrRNA genesequence analyses into account the strain J was identified asPseudomonas taiwanensis andnamed JUp to date the speciesof Pseudomonas taiwanensis was hardly ever reported as thepsychrotrophic aerobic denitrifier

32 Effect of Temperature on Nitrogen Removal of the Strain JTemperature is usually regarded as an environmental stressfactor for the survival of bacteria because too high tem-perature could induce nucleic acid or protein denaturationwhereas too low temperature could result in inhibition of cellgrowth and proliferation alter protein expression patternsand weaken metabolic activity [34] Generally different bac-teria have diverse temperatures for cell growth and functionalactivity The effects of different temperatures on cell growthand nitrate removal of strain J in MDMmedium were shownin Figure 3(a) Pseudomonas taiwanensis strain J was ableto grow and conduct aerobic denitrification with nitrate asnitrogen source at a broad temperature range from 5 to40∘C which may expand its application scope in differentseasons The strain J possessed tolerance to low temperature

with 15∘C as its optimum and 5∘C as viable which suggestedthat strain J was a psychrotrophic aerobic bacterium Datain Figure 3(a) showed that the nitrate nitrogen removalefficiency was enhanced with the increased temperaturein a certain range The nitrate and total nitrogen removalpercentage increased from 5161 and 876 at 5∘C to 100and 8448 at 15∘C under the condition of 150 rmin withinitial nitrate nitrogen concentration of about 50mgL after48 h cultivation These results demonstrated that highertemperature could promote the nitrate removal efficiency ofstrain J (119875 lt 005) The nitrite nitrogen accumulation wasdistinctly observed at 5∘C and 10∘C with the concentrationsof 150 and 2012mgL respectively (data were not shownin Figure 3) It was interesting that the nitrite-nitrogen wasnot detected when the temperature is higher than 15∘C oreven below the nitrite detection limit when nitrate was usedas sole nitrogen source These results indicated that too lowtemperature might result in high nitrite accumulation Thesimilar results were reported that the nitrite could be accu-mulated as the temperature below 15∘C [8 35] By contrastthe nitrate and total nitrogen removal efficiencies decreasedcontinuously when the temperature further increased from15∘C to 40∘C and the nitrate and total nitrogen removalefficiencies decreased to 70 and 4061 at 40∘C whichdemonstrated that the denitrification ability of strain J could


0 5 10 15 20 25 30 35 40 450

20

40

60

80

100

Removal percentage of total nitrogen

A

A

BCD

B

EE D DE D

DECD

CDBCD

BB

BC

000102030405060708

Temperature (∘C)

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

Removal percentage of 3minus-N

＄600The value of

(a)

0 50 100 150 200Shaking speed (rmin)

BA

AB

CC

D

DC C

0001020304050607

0

20

40

60

80

100

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

Removal percentage of total nitrogenRemoval percentage of 3

minus-N

＄600The value of

(b)

Carbon source

B BC CBC

C C

A A

CB

00020406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

N

rem

oval

effici

enci

es (

)

sodi

um ci

trat

e

sodi

um su

ccin

ate

gluc

ose

sucr

ose

sodi

um ac

etat

e


minus-N

＄600The value of

(c)

0 5 10 15 20 25CN ratio

A A

B

B

C

C

DD

DE

DE

0200

0406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(d)

60 65 70 75 80 85 90 95 100 105pH

A A

CB

BB

CB

BB

000102030405060708

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

＄

600


minus-N

＄600The value of

(e)

05 10 15 20 25

AA

BB

C

C

CD

C

C

0001020304050607

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

Inoculated quantity (times106 ５Ｇ)


minus-N

＄600The value of

(f)

Figure 3 Growth of strain J and nitrate removal at different culturing conditions Temperature (a) shaking speed (b) carbon source (c) CNratio (d) pH (e) and inoculated quantity (f) Values are means plusmn SD (errors bars) for three replicates

be slightly inhibited by high temperatures Accompaniedwiththe reduction of nitrate and total nitrogen the OD

600value of

strain J increased firstly from 004 to 069 corresponding toan average growth rate of 0345 dminus1 and then decreased to035 at 40∘C Above all results suggested that the temperaturehad a pronounced effect on the nitrogen removal efficienciesand cell growth of strain J (119875 lt 005)

It is notable that nearly all the reported aerobic denitrify-ing bacteria were mesophiles and the optimum temperatureswere ranged from 20 to 37∘C in which the cell growth andnitrogen denitrification were severely or totally inhibited atthe temperature as low as 10∘C [16 36 37] As a result thestrain J could conduct aerobic denitrification efficiently at 5∘Cdemonstrating that the strain J has much more tolerance to


the cold condition which may offer a new cold-adaptationaerobic denitrifier for nitrogen removal in winter

33 Effect of Dissolved Oxygen on Nitrogen Removal of theStrain J It has been widely accepted that the dissolvedoxygen (DO) concentration in solution could be adjustedby the rotation speed of the shaker The faster the shakingspeed is the higher the DO level can be obtained In theaerobic denitrification process both of the dissolved oxygenand nitrate nitrogen could act as electron accepters A largeamount of papers showed that a certain DO concentrationwas beneficial to bacterial growth and nitrogen removal[8 38 39] The influence of different shaking speed oncell growth and nitrogen removal by strain J is shown inFigure 3(b) Significant differences were observed amongshaking speed of 0ndash200 rmin (119875 lt 005) Poor nitrogenremoval efficiencies were obtained in static cultivation withonly 1836 nitrate and 284 total nitrogen removal effi-ciencies after 48 h incubation The higher rotation speedsignificantly promoted the cell reproduction and nitrogenremoval efficiencies The optimal shaking speed for strain Jgrowth and denitrification was 150 rmin which is equivalentto 625mgL DO concentration [37] The peak of nitrate andtotal nitrogen removal efficiencies were 100 and 8475under the conditions of 150 rmin and 15∘C Subsequentlythe cell growth and nitrogen removal ability were inhibitedat the shaking speed of 200 rmin which may be that thenitrate reductase is sensitive to high concentration of dissolveoxygen The value of OD

600showed similar change with

the nitrogen removal efficiencies and no obvious nitriteaccumulation in the experiment Apparently the excessivelow or high concentration of DOwas unfavourable for bacte-rial growth and denitrification which indicated that properaeration could improve the nitrogen removal efficiencies ofstrain J in the practice application

34 Effect of Carbon Source on Nitrogen Removal of the StrainJ Carbon sources with different chemical structures andmolecular weights usually served as the electron donor andenergy for heterotrophic aerobic denitrification process Gen-erally carbon sources with simpler structures and lowmolec-ular weight were more beneficial for aerobic bacterial den-itrification [19] Figure 3(c) showed that the different carbonsources hadmarkedly affected nitrogen reduction efficiencies(119875 lt 005) at 15∘C and 150 rmin The experimental resultsshowed that sodium citrate sodium succinate glucose andsodium acetate could well support the growth of the strain Jand promote nitrate reduction Strain J exhibited the highestnitrogen removal abilitywhen the glucosewas used as the solenitrogen source with the nitrate and total nitrogen removalpercentage of 100 and 9379 respectively Glucose dueto its simple and small molecular structure being the bestcarbon source for aerobic denitrification was also discoveredin the previous paper such as the strain of AnoxybacilluscontaminansHA [34] Nevertheless the sucrose was not goodfor both cell growth and nitrogen removal ability of strain JThe accumulation of nitrite nitrogen was not observed in thiscarbon source experiments Therefore it could be concludedhere that a variety of carbon sources were beneficial for strain

J conducting aerobic denitrification implying that the carbonsources were not the limiting factors for strain J in the nitratereduction process at low temperature

35 Effect of CN Ratio on Nitrogen Removal of the StrainJ Previous reports indicated that the high CN ratio couldpromote the nitrogen reduction [40 41] Sodium succinatelocated in central position of tricarboxylic acid cycle (TCA)was used as carbon source by fixing the nitrogen of NaNO

3

which may provide electron donor and energy rapidly forcell multiplication and aerobic denitrification The effectsof different CN ratio on cell growth and nitrogen removalby strain J were shown in Figure 3(d) The significant dif-ferences in nitrogen removal percentage and cell growthwere obtained among CN ratio of 0ndash15 (119875 lt 005)Little nitrogen reduction and bacterial reproduction wereobserved when the CN ratio was 0 suggesting that strainJ was not an autotrophic denitrifier With the CN ratioincreasing from 0 to 15 the nitrate and total nitrogen removalpercentage reached the peak value of 100 and 8824However although CN ratio further increased from 15 to25 the removal efficiencies of nitrate and total nitrogenwere comparatively constant whichmanifested that althoughhigh CN ratio could not accelerate nitrogen reduction italso could not inhibit the nitrogen removal ability of strainJ The results dramatically differ from the previous studiesthat much higher CN could lower the nitrogen removalefficiency For instance Zhang et al [8] reported that theammonium removal rate decreased at the CN of 20 byMicrobacterium sp SFA13 and Huang et al [18] found thatthe nitrogen removal efficiency yielded a slight decrease asthe CN ratio higher than 2 with the strain of Zoogloea spN299 It might be concluded that the strain J could tolerate amuch higher concentration of carbon source and the optimalCN ratio was 15 which was consistent with the strain ofMarinobacter sp possessing the optimal CN ratio of 15 [42]Moreover the bacterial proliferation increased continuouslywith increasing of CN ratio corresponding to a peak OD

600

value of 111 at CN ratio of 25 and no nitrite was detected inall CN ratio solutions

36 Effect of pH on Nitrogen Removal of the Strain J Thenitrate and total nitrogen removal using the strain J atdifferent pH in theMDMwas investigatedwith 150 rmin and15∘C as shown in Figure 3(e) This figure depicted that thedifferent pH also had a great effect on nitrogen reduction andbacterial growth (119875 lt 005) The growth and denitrificationactivities of the strain J were almost unchanged at the initialpH value of 65 implying that slightly acidic was harmful tothe cell reproduction and metabolic activities But above pH7 the nitrogen removal efficiencies and the value of OD

600

were increased obviously The maximum nitrate removalefficiency of 100 was obtained at pH 70 When the pHincreased from 70 to 100 the nitrate removal percentagewas lower than 100 but higher than 9051 This resultwas consistence with the strain of Acinetobacter sp CN86in which the nitrate removal rate was lower slightly whenthe pH increased from 70 to 90 [43] but conflicted withanother papers which reported that the nitrate reduction


occurred under circumneutral pH or weak acidic conditions[44 45] Compared with the degradation of nitrate the valueof pH arranging from 70 to 100 had no distinctively effecton total nitrogen removal ability of strain J with the removalpercentage about 840 Meanwhile all values of OD

600

were almost equivalent to 067 except that the value of pHwas 65 Apparently the neutral and alkaline environmentswere conducive to heterotrophic aerobic denitrification andthe strong alkaline was not the limiting factor for aerobicdenitrification

37 Effect of Inoculation Quantity on Nitrogen Removal of theStrain J The nitrate and total nitrogen removal efficiencieswere directly affected by the amount of strain J (119875 lt005) as described in Figure 3(f) The aerobic denitrificationpercentage increased with the increase of the inoculationamount from 05 times 106 CFUmL to 20 times 106 CFUmL at15∘C But further increase of incubation quantity to 25 times106 CFUmL could result in a distinct decrease of the nitrogenremoval efficienciesThemaximumnitrate and total nitrogenremoval efficiencies were 100 and 8315 and themaximumvalue of OD

600was 065 with inoculation amount of 20

times 106 CFUmL It thus could be suggested that the cellgrowth space oxygen supply and nitrogen reduction mightbe influenced when inoculation quantity was higher than 20times 106 CFUmL Furthermore there was no nitrite nitrogenaccumulation in this inoculation quantity experimentsThustaking the nitrogen removal efficiencies and cell growth intoconsideration the optimal inoculation quantity was 20 times106 CFUmL This optimized inoculation quantity of strain Jcould provide a reference for the actual wastewater treatment

In this research the nitrate nitrogen was used as the solenitrogen source to preliminary explore whether strain J hada denitrification ability The removal efficiencies of nitratenitrogen and total nitrogen denoted the denitrification abilityof the strain J and the denitrification products (NON

2O and

N2) should be further studied with nitrate as sole nitrogen

source

4 Conclusion

The novel psychrotrophic aerobic bacterium named strainJ was identified as Pseudomonas taiwanensis based on mor-phology and phospholipid fatty acid as well as 16S rRNAgene sequence Approximately 1000 of nitrate nitrogen wasremoved at 15∘C in different single factor experiment Thetotal nitrogen removal efficiency was higher than 8315Thestrain J possessed excellent cold resistance and was able toremove nitrate at 5∘C with the removal percentage of 5161The low temperature (below 15∘C)was the only factor to resultin nitrite accumulation Most carbon sources could supportaerobic denitrification except sucrose Moreover high CNratio and alkalinity condition were conducive to the celldensity increase and nitrate denitrification

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (41671291) and the Scientific ResearchInnovation Project of Chongqing China (no CYB17063)

References

[1] L Guo Q Chen F Fang et al ldquoApplication potential of a newlyisolated indigenous aerobic denitrifier for nitrate and ammo-nium removal of eutrophic lake waterrdquo Bioresource Technologyvol 142 pp 45ndash51 2013

[2] S Tang Q Yang H Shang and T Sun ldquoRemoval of nitrate byautosulfurotrophic denitrifying bacteria Optimization kinet-ics and thermodynamics studyrdquo Fresenius Environmental Bul-letin vol 19 no 12 B pp 3193ndash3198 2010

[3] A Bhatnagar and M Sillanpaa ldquoA review of emerging adsor-bents for nitrate removal from waterrdquo Chemical EngineeringJournal vol 168 no 2 pp 493ndash504 2011

[4] Y Zhao C Feng Q Wang Y Yang Z Zhang and NSugiura ldquoNitrate removal from groundwater by cooperatingheterotrophic with autotrophic denitrification in a biofilm-electrode reactorrdquo Journal of Hazardous Materials vol 192 no3 pp 1033ndash1039 2011

[5] W Xing D Li J Li Q Hu and S Deng ldquoNitrate removaland microbial analysis by combined micro-electrolysis andautotrophic denitrificationrdquo Bioresource Technology vol 211 pp240ndash247 2016

[6] J F SuMMa T L Huang et al ldquoCharacteristics of autotrophicand heterotrophic denitrification by the strain pseudomonas spH117rdquo Geomicrobiology Journal vol 34 no 1 pp 45ndash52 2016a

[7] J-F Su J-X Shi T-L Huang F Ma J-S Lu and S-F YangldquoEffect of nitrate concentration pH and hydraulic retentiontime on autotrophic denitrification efficiency with Fe(II) andMn(II) as electron donorsrdquo Water Science and Technology AJournal of the International Association on Water PollutionResearch vol 74 no 5 pp 1ndash8 2016b

[8] D Zhang W Li X Huang Q Wen and L Miao ldquoRemovalof ammonium in surface water at low temperature by anewly isolated Microbacterium sp strain SFA13rdquo BioresourceTechnology vol 137 no 6 pp 147ndash152 2013a

[9] X Zhang Z Xu X Sun W Dong and D Ballantine ldquoNitratein shallow groundwater in typical agricultural and forest ecosys-tems in Chinardquo Journal of Environmental Sciences vol 25 no 5pp 1007ndash1014 2004

[10] R Sierra-Alvarez R Beristain-Cardoso M Salazar J GomezE Razo-Flores and J A Field ldquoChemolithotrophic denitrifica-tion with elemental sulfur for groundwater treatmentrdquo WaterResearch vol 41 no 6 pp 1253ndash1262 2007

[11] J C Leyva-Dıaz A Gonzalez-Martınez J Gonzalez-LopezM M Munıo and J M Poyatos ldquoKinetic modeling andmicrobiological study of two-step nitrification in a membranebioreactor and hybrid moving bed biofilm reactor-membranebioreactor for wastewater treatmentrdquo Chemical EngineeringJournal vol 259 pp 692ndash702 2015

[12] M Shrimali and K P Singh ldquoNew methods of nitrate removalfromwaterrdquo Environmental Pollution vol 112 no 3 pp 351ndash3592001

[13] VMateju S Cizinska J Krejcı and T Janoch ldquoBiological waterdenitrification-A reviewrdquo Enzyme and Microbial Technologyvol 14 no 3 pp 170ndash183 1992


[14] R D Hauck and D R Bouldin ldquoDistribution of isotopicnitrogen in nitrogen gas during denitrificationrdquoNature vol 191no 4791 pp 871-872 1961

[15] L Yu Y Liu and G Wang ldquoIdentification of novel denitrifyingbacteria Stenotrophomonas sp ZZ15 and Oceanimonas spYC13 and application for removal of nitrate from industrialwastewaterrdquo Biodegradation vol 20 no 3 pp 391ndash400 2009

[16] Z-F Song J An G-H Fu and X-L Yang ldquoIsolation andcharacterization of an aerobic denitrifying Bacillus sp YX-6from shrimp culture pondsrdquo Aquaculture vol 319 no 1-2 pp188ndash193 2011

[17] H Zheng Y Liu G Sun XGaoQ Zhang andZ Liu ldquoDenitri-fication characteristics of a marine origin psychrophilic aerobicdenitrifying bacteriumrdquo Journal of Environmental Sciences vol23 no 11 pp 1888ndash1893 2011

[18] T-L Huang S-L Zhou H-H Zhang S-Y Bai X-X He andX Yang ldquoNitrogen removal characteristics of a newly isolatedindigenous aerobic denitrifier from oligotrophic drinking waterreservoir Zoogloea sp N299rdquo International Journal of Molecu-lar Sciences vol 16 no 5 pp 10038ndash10060 2015

[19] J Chen S Gu H Hao and J Chen ldquoCharacteristics andmetabolic pathway of Alcaligenes sp TB for simultaneous het-erotrophic nitrification-aerobic denitrificationrdquo Applied Micro-biology and Biotechnology vol 100 no 22 pp 9787ndash9794 2016

[20] A Rodriguez-Caballero S Hallin C Pahlson M Odlareand E Dahlquist ldquoAmmonia oxidizing bacterial communitycomposition and process performance in wastewater treatmentplants under low temperature conditionsrdquo Water Science andTechnology vol 65 no 2 pp 197ndash204 2012

[21] N Sundaresan and L Philip ldquoPerformance evaluation of var-ious aerobic biological systems for the treatment of domesticwastewater at low temperaturesrdquoWater Science and TechnologyA Journal of the International Association on Water PollutionResearch vol 58 no 4 p 819 2008

[22] W Li ldquoStudy on characteristics in the removal process ofammonia nitrogen and nitrate nitrogen by an isolated het-erotrophic nitrification-aerobic denitrification strainRhodococ-cus sprdquo Journal of Environmental Protection vol 4 no 1 pp 74ndash79 2013

[23] H Zhang H Wang K Yang Y Sun J Tian and B Lv ldquoNitrateremoval by a novel autotrophic denitrifier (Microbacterium sp)using Fe(II) as electron donorrdquo Annals of Microbiology vol 65no 2 pp 1ndash10 2015

[24] Q Mahmood B Hu J Cai et al ldquoIsolation of OchrobactrumspQZ2 from sulfide and nitrite treatment systemrdquo Journal ofHazardous Materials vol 165 no 1-3 pp 558ndash565 2009

[25] Y Angar S Kebbouche-Gana N-E Djelali and S Khemili-Talbi ldquoNovel approach for the ammonium removal by simul-taneous heterotrophic nitrification and denitrification using anovel bacterial species co-culturerdquo World Journal of Microbiol-ogy and Biotechnology vol 32 no 3 pp 1ndash14 2016

[26] L Zhu W Ding L-J Feng X Dai and X-Y Xu ldquoCharacteris-tics of an aerobic denitrifier that utilizes ammonium and nitratesimultaneously under the oligotrophic nicherdquo EnvironmentalScience and Pollution Research vol 19 no 8 pp 3185ndash3191 2012

[27] T-X He and Z-L Li ldquoIdentification and denitrificationcharacterization of a novel hypothermia and aerobic nitrite-denitrifying bacterium Arthrobacter arilaitensis strain Y-10rdquoDesalination and Water Treatment vol 57 no 41 pp 1ndash9 2015

[28] Q Shao and Y-U Xiao-Bin ldquoIsolation and Characterization ofA Strain Denitrobacteriardquo Biotechnology vol 18 no 3 pp 63ndash65 2008

[29] APHA Standard Methods for the Examination of Water andWastewater American Public Health Association WashingtonDC USA 2012

[30] B Pratt R Riesen and C G Johnston ldquoPLFA Analyses ofMicrobial Communities Associated with PAH-ContaminatedRiverbank SedimentrdquoMicrobial Ecology vol 64 no 3 pp 680ndash691 2012

[31] J Trogl I Jirkova P Zemankova et al ldquoEstimation of thequantity of bacteria encapsulated in Lentikats Biocatalyst viaphospholipid fatty acids content A preliminary studyrdquo FoliaMicrobiologica vol 58 no 2 pp 135ndash140 2013

[32] J Volmer C Neumann B Buhler andA Schmid ldquoEngineeringof Pseudomonas taiwanensis VLB120 for constitutive solventtolerance and increased specific styrene epoxidation activityrdquoApplied and Environmental Microbiology vol 80 no 20 pp6539ndash6548 2014

[33] K Schmutzler O N Kracht A Schmid and K BuehlerldquoTrophic regulation of autoaggregation in Pseudomonas taiwa-nensis VLB120rdquo Applied Microbiology and Biotechnology vol100 no 1 pp 347ndash360 2016

[34] J Chen J Zheng Y Li H-H Hao and J-M Chen ldquoChar-acteristics of a novel thermophilic heterotrophic bacteriumAnoxybacillus contaminans HA for nitrificationndashaerobic deni-trificationrdquo Applied Microbiology and Biotechnology vol 99 no24 pp 10695ndash10702 2015

[35] A Andersson P Laurent A Kihn M Prevost and P ServaisldquoImpact of temperature on nitrification in biological activatedcarbon (BAC) filters used for drinking water treatmentrdquoWaterResearch vol 35 no 12 pp 2923ndash2934 2001

[36] J Zhang P Wu B Hao and Z Yu ldquoHeterotrophic nitrificationand aerobic denitrification by the bacterium Pseudomonasstutzeri YZN-001rdquo Bioresource Technology vol 102 no 21 pp9866ndash9869 2011

[37] S Yao J Ni T Ma and C Li ldquoHeterotrophic nitrification andaerobic denitrification at low temperature by a newly isolatedbacterium Acinetobacter sp HA2rdquo Bioresource Technology vol139 pp 80ndash86 2013

[38] J Ye B Zhao Q An and Y-S Huang ldquoNitrogen removal byProvidencia rettgeri strain YL with heterotrophic nitrificationand aerobic denitrificationrdquo Environmental Technology (UnitedKingdom) vol 37 no 17 pp 2206ndash2213 2016

[39] L Yu Y Wang H Liu C Xi and L Song ldquoA novelheterotrophic nitrifying and aerobic denitrifying bacteriumZobellella taiwanensis DN-7 can remove high-strength ammo-niumrdquo Applied Microbiology and Biotechnology vol 100 no 9pp 4219ndash4229 2016

[40] A P Miqueleto C C Dolosic E Pozzi E Foresti andM ZaiatldquoInfluence of carbon sources and CN ratio on EPS productionin anaerobic sequencing batch biofilm reactors for wastewatertreatmentrdquoBioresource Technology vol 101 no 4 pp 1324ndash13302010

[41] M Kim S-Y Jeong and J-Y Su ldquoAerobic Denitrification ofPseudomonas putida AD-21 at Different CN Ratiosrdquo Journal ofBioscience and Bioengineering vol 106 no 5 pp 498ndash502 2008

[42] H-Y Zheng Y Liu X-Y Gao G-M Ai L-L Miao and Z-P Liu ldquoCharacterization of a marine origin aerobic nitrifying-denitrifying bacteriumrdquo Journal of Bioscience and Bioengineer-ing vol 114 no 1 pp 33ndash37 2012

[43] J-F Su J-X Shi T-L Huang and F Ma ldquoKinetic analysisof simultaneous denitrification and biomineralization of novelAcinetobacter sp CN86rdquoMarine Pollution Bulletin vol 109 no1 pp 87ndash94 2016


[44] J-F Su K Zhang T-L Huang G Wen L Guo and S-FYang ldquoHeterotrophic nitrification and aerobic denitrificationat low nutrient conditions by a newly isolated bacteriumAcinetobacter sp SYF26rdquo Microbiology (United Kingdom) vol161 no 4 pp 829ndash837 2015

[45] Y Wen and C-H Wei ldquoHeterotrophic nitrification and aerobicdenitrification bacterium isolated from anaerobicanoxicoxictreatment systemrdquo African Journal of Biotechnology vol 10 no36 pp 6985ndash6990 2011

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



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BioMed Research International

Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


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MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


drastically inhibit their denitrification ability cell growth andproliferation especially when the temperature was at 10∘C orless [20 21]Thus it is important to explore the bacteriawhichcould effectively remove nitrate nitrogen at low temperatures

Additionally nitrate biodegradation may generate OHminuswhich could inhibit the denitrification process and enzymeactivity [22] Zhang et al [23] discovered that neither celldensity increase nor nitrate reduction was found if the pH isgreater than 85 Li [22] reported that the nitrate reductionwas completely inhibited when the pH reached about 95Therefore the optimal pH of most new isolated aerobicdenitrifiers ranged from 65 to 70 such as Ochrobactrum sp(65ndash70) [24] Alcaligenes sp S84S3 (70) [25] Psychrobactersp (70) [17] and Pseudomonas mendocina 3ndash7 (70) [26]It may be difficult for these microorganisms to meet therequirements of alkaline sewage treatment

In this study a novel bacterial strain for candidate ofaerobic denitrifier capable of aerobic denitrification withnitrate was identified as Pseudomonas taiwanensis namedJ To the best of our knowledge few nitrate denitrifica-tion studies have been performed to focus on the speciesof Pseudomonas taiwanensis In this research the impactresistance of strain J to low temperature extreme alkalinityand high CN ratio were investigated using nitrate as solenitrogen source The experimental results showed that thestrain J possessed excellent tolerance to low temperatureswith 15∘C as its optimum and 5∘C as viable Furthermoreit has been found that the nitrogen removal efficiency didnot decrease obviously with temperature between 15∘C and40∘C Significantly high CN ratio and strong alkalinity werenot the limiting factors for cell density increase and nitratedenitrification performance Accordingly the strain J couldbe used to treat the high CN ratio wastewater and thealkaline wastewater in four seasons

2 Materials and Methods

21 Bacterium and Media The psychrotrophic and aerobicdenitrifying bacterium Pseudomonas taiwanensis strain J wasstocked in 30 glycerol solution at minus20∘C

We followed the methods of He and Li [27] Thebromothymol blue (BTB) solid medium per liter (pH =72) comprised NaNO

31 g KH

2PO41 g FeCl

2-6H2O 05 g

MgSO4-7H2O 1 g CaCl

2-7H2O 02 g sodium succinate 85 g

BTB reagent 1mL [15 in ethanol] and agar 20 g [28] Themodified denitrificationmedium (MDM) per liter comprisedthe following (ultrapure water per liter) NaNO

3031 g

CH3COONa 256 g KH

2PO415 g Na

2HPO4042 gMgSO

4-

7H2O 1 g Fe SO

4-7H2O 005 g and pH 72 plusmn 01 [16] LB

medium (pH 70 per liter) included tryptone 100 g yeastextract 50 g andNaCl 10 g For preparation of LB solid plates2 (wv) agar powder was added

22 Identification of the Psychrotrophic and Aerobic Denitrify-ing Bacterium Strain J Colony morphologies of strain J weremonitored on the BTBmediumplates after incubating at 15∘Cfor 3 d Cell morphologies of strain J were observed underthe HITACHI S-3000N scanning electron microscope andatomic force microscopy Phospholipid fatty acids (PLFAs)

were extracted by using about 40mg pure culture of strain Jafter incubating 48 h at 15∘C Each type of PLFAswas analyzedby Agilent 6850

The nearly full-length of 16S rRNA gene sequence wasamplified using DNA as template which was extracted bygenomic DNA purification kit (Thermo scientific) Theuniversal primers 27F (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and 1492R (51015840-GGTTACCTTGTTACGACTT-31015840) wereused for polymerase chain reaction (PCR) amplificationThe PCR amplification was conducted in the 50mL volumecontaining 2 120583L DNA 2 120583L primer 25120583L 2x Taq PCRMasterMix and 19 120583L sterile water The PCR conditions weredenaturation for 5min at 94∘C 30 cycles of 1min at 94∘C 30 sat 555∘C and 1min at 72∘C and extension for 10min at 72∘CThe 15 kb product was separated on a 15 agarose gel andpurified by BioSpin gel extraction kit (BioFlux) The purifiedproduct was cloned into pMD20-T vector (Takara) andthen sequenced by Invitrogen company Then the 16S rRNAsequence of strain J was submitted to NCBI for accessionnumber Sequence alignment and multiple alignment wereperformedusingNCBI SearchTool program (BLAST httpsblastncbinlmnihgovBlastcgiPROGRAM=blastnampPAGETYPE=BlastSearchampLINK LOC=blasthome) and CLUSTALW And the phylogenetic tree was constructed using MEGA60 software by neighbor-joining distance method andbootstrap analyses of 1000 replicates

23 Effects of Culturing Conditions on the DenitrificationAbility of Strain J Effects of six cultivation conditionsincluding temperature (5∘C 10∘C 15∘C 20∘C 25∘C 30∘C35∘C and 40∘C) shaking speed (0 rmin 50 rmin 100 rmin150 rmin and 200 rmin) pH (65 70 80 90 and 100)inoculation quantity (05 times 108 CFU 10 times 108 CFU 15 times108 CFU 20 times 108 CFU and 25 times 108 CFU within 100mLDM) and carbon source (sodium citrate sodium succinatesodium acetate sucrose and glucose) on denitrificationperformance of strain J were determined by the single factortests The amount of carbon was changed to adjust the CNratio to 0 5 10 15 20 and 25 by fixing the amount of NaNO

3

as the nitrogen source 10 times 108 CFU precultured bacterialsuspension was inoculated into 250mL sterilized conicalflask which contained 100mL DM broth medium for testingexcept for inoculation quantity which will be specified laterAfter incubating for 48 hours nitrate and total nitrogen werethen determined for these samples in order to analyze theinfluences of the various factors indicated above

The nitrate and total nitrogen removal efficiencies werecalculated by the equation Rv = (119879

1minus 1198792)1198791times 100 to

assess the denitrification ability of strain J Note that Rv 1198791

and 1198792represent nitrate or total nitrogen removal efficiency

the initial concentration of nitrate or total nitrogen in MDMbroth medium and the final concentration of nitrate ortotal nitrogen in MDM broth medium after incubation for48 hours respectively All experiments were conducted intriplicate

24 Analytical Methods The cell optical density (OD600

) wasmonitored by measuring the absorbance at the wavelengthof 600 nm using a spectrophotometer (DU800 BECKMAN


(a) (b)

(c)

D

001 height sensor

49 Ｇ

minus1350 ＨＧ

1350 ＨＧ

(d)













RTSBA6621








100

9961

64

05






0 5 10 15 20 25 30 35 40 450

20

40

60

80

100


A

A

BCD

B

EE D DE D

DECD

CDBCD

BB

BC

000102030405060708

Temperature (∘C)

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


＄600The value of

(a)


BA

AB

CC

D

DC C

0001020304050607

0

20

40

60

80

100

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(b)

Carbon source

B BC CBC

C C

A A

CB

00020406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

N

rem

oval

effici

enci

es (

)

sodi

um ci

trat

e

sodi

um su

ccin

ate

gluc

ose

sucr

ose

sodi

um ac

etat

e


minus-N

＄600The value of

(c)

0 5 10 15 20 25CN ratio

A A

B

B

C

C

DD

DE

DE

0200

0406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(d)

60 65 70 75 80 85 90 95 100 105pH

A A

CB

BB

CB

BB

000102030405060708

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

＄

600


minus-N

＄600The value of

(e)

05 10 15 20 25

AA

BB

C

C

CD

C

C

0001020304050607

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()



minus-N

＄600The value of

(f)



600value of











3


600



600




600






2O and


source

4 Conclusion




Acknowledgments


References


















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(a) (b)

(c)

D

001 height sensor

49 Ｇ

minus1350 ＨＧ

1350 ＨＧ

(d)













RTSBA6621








100

9961

64

05






0 5 10 15 20 25 30 35 40 450

20

40

60

80

100


A

A

BCD

B

EE D DE D

DECD

CDBCD

BB

BC

000102030405060708

Temperature (∘C)

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


＄600The value of

(a)


BA

AB

CC

D

DC C

0001020304050607

0

20

40

60

80

100

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(b)

Carbon source

B BC CBC

C C

A A

CB

00020406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

N

rem

oval

effici

enci

es (

)

sodi

um ci

trat

e

sodi

um su

ccin

ate

gluc

ose

sucr

ose

sodi

um ac

etat

e


minus-N

＄600The value of

(c)

0 5 10 15 20 25CN ratio

A A

B

B

C

C

DD

DE

DE

0200

0406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(d)

60 65 70 75 80 85 90 95 100 105pH

A A

CB

BB

CB

BB

000102030405060708

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

＄

600


minus-N

＄600The value of

(e)

05 10 15 20 25

AA

BB

C

C

CD

C

C

0001020304050607

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()



minus-N

＄600The value of

(f)



600value of











3


600



600




600






2O and


source

4 Conclusion




Acknowledgments


References


















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





RTSBA6621








100

9961

64

05






0 5 10 15 20 25 30 35 40 450

20

40

60

80

100


A

A

BCD

B

EE D DE D

DECD

CDBCD

BB

BC

000102030405060708

Temperature (∘C)

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


＄600The value of

(a)


BA

AB

CC

D

DC C

0001020304050607

0

20

40

60

80

100

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(b)

Carbon source

B BC CBC

C C

A A

CB

00020406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

N

rem

oval

effici

enci

es (

)

sodi

um ci

trat

e

sodi

um su

ccin

ate

gluc

ose

sucr

ose

sodi

um ac

etat

e


minus-N

＄600The value of

(c)

0 5 10 15 20 25CN ratio

A A

B

B

C

C

DD

DE

DE

0200

0406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(d)

60 65 70 75 80 85 90 95 100 105pH

A A

CB

BB

CB

BB

000102030405060708

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

＄

600


minus-N

＄600The value of

(e)

05 10 15 20 25

AA

BB

C

C

CD

C

C

0001020304050607

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()



minus-N

＄600The value of

(f)



600value of











3


600



600




600






2O and


source

4 Conclusion




Acknowledgments


References


















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0 5 10 15 20 25 30 35 40 450

20

40

60

80

100


A

A

BCD

B

EE D DE D

DECD

CDBCD

BB

BC

000102030405060708

Temperature (∘C)

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


＄600The value of

(a)


BA

AB

CC

D

DC C

0001020304050607

0

20

40

60

80

100

＄

600

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(b)

Carbon source

B BC CBC

C C

A A

CB

00020406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

N

rem

oval

effici

enci

es (

)

sodi

um ci

trat

e

sodi

um su

ccin

ate

gluc

ose

sucr

ose

sodi

um ac

etat

e


minus-N

＄600The value of

(c)

0 5 10 15 20 25CN ratio

A A

B

B

C

C

DD

DE

DE

0200

0406081012

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()


minus-N

＄600The value of

(d)

60 65 70 75 80 85 90 95 100 105pH

A A

CB

BB

CB

BB

000102030405060708

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()

＄

600


minus-N

＄600The value of

(e)

05 10 15 20 25

AA

BB

C

C

CD

C

C

0001020304050607

＄

600

0

20

40

60

80

100

Nitr

ate a

nd T

Nre

mov

al effi

cien

cies

()



minus-N

＄600The value of

(f)



600value of











3


600



600




600






2O and


source

4 Conclusion




Acknowledgments


References


















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










3


600



600




600






2O and


source

4 Conclusion




Acknowledgments


References


















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




600






2O and


source

4 Conclusion




Acknowledgments


References


















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


removal of nitrate in simulated water at low temperature

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