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Research ArticleSimultaneous Heterotrophic Nitrification and AerobicDenitrification by Chryseobacterium sp R31 Isolated fromAbattoir Wastewater

Pradyut Kundu1 Arnab Pramanik2 Arpita Dasgupta12

Somnath Mukherjee1 and Joydeep Mukherjee2

1 Department of Civil Engineering Jadavpur University Kolkata 700 032 India2 School of Environmental Studies Jadavpur University Kolkata 700 032 India

Correspondence should be addressed to Joydeep Mukherjee joydeep envstuschooljdvuacin

Received 24 February 2014 Accepted 29 April 2014 Published 2 June 2014

Academic Editor Eugenio Ferreira

Copyright copy 2014 Pradyut Kundu et alThis 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

A heterotrophic carbon utilizing microbe (R31) capable of simultaneous nitrification and denitrification (SND) was isolatedfrom wastewater of an Indian slaughterhouse From an initial COD value of 5830mgL 9554 was removed whilst from astarting NH

4

+-N concentration of 557mgL 9587 was removed after 48 h contact The concentrations of the intermediateshydroxylamine nitrite and nitrate were low thus ensuring nitrogen removal Aerobic denitrification occurring during ammoniumremoval by R31 was confirmed by utilization of both nitrate and nitrite as nitrogen substrates Glucose and succinate weresuperior while acetate and citrate were poor substrates for nitrogen removal Molecular phylogenetic identification supportedby chemotaxonomic and physiological properties assigned R31 as a close relative of Chryseobacterium haifense The NH

4

+-Nutilization rate and growth of strain R31 were found to be higher at CN = 10 in comparison to those achieved with CN ratios of 5and 20 Monod kinetic coefficients half saturation concentration (119870

119904) maximum rate of substrate utilization (119896) yield coefficient

(119884) and endogenous decay coefficient (119870119889) indicated potential application of R31 in large-scale SND process This is the first

report on concomitant carbon oxidation nitrification and denitrification in the genus Chryseobacterium and the associated kineticcoefficients

1 Introduction

Conventionally ammonium from abattoir wastewater isremoved in sequential operations through alternate aerobicand anoxic periods over time [1ndash3] Majority of the carbonis degraded during the aerobic nitrification phase and theresidual carbon is used up during the anoxic denitrificationphase [4] However such systems are prone to operationalhindrances due to reduced rate of nitrification and thedifficulty to separate nitrification and denitrification reactionprocesses Meyer et al [5] noted that nitrification anddenitrification would be incompletely coupled to each otherdue to nonformation of aerobicanoxic zones within themicrobial aggregates resulting in buildup of NO

119909

minus in thereactor In comparison with the conventional nitrificationand denitrification of abattoir wastewater several advantages

of a shortcut nitrogen removal method via nitrite throughpartial nitrification followed by denitrification (PND) havebeen reported recently [6]

Zheng et al [7] observed that both nitrification anddenitrification could take place concomitantly in a singlereactor under identical reaction conditions through a pro-cess known as simultaneous nitrification and denitrification(SND) This procedure neither requires large reactor volumenor incurs high energy costs necessary for circulating liquidbetween aerobic and anoxic systems Furthermore SNDprocesses are characterized by increased nitrogen removalrates and decreased reaction time The key point in thePND process is maintaining the production rate of nitriteby ammonium oxidizing bacteria (AOB) higher than theproduction rate of nitrate by nitrite oxidizing bacteria(NOB) so that nitrite accumulation can be achieved [6]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 436056 12 pageshttpdxdoiorg1011552014436056

2 BioMed Research International

In contrast maintenance of a population balance is notnecessary in SND as the two reactions (nitrification anddenitrification) proceed under identical reaction conditionsThus SND is advantageous over PND Furthermore theSND process prevents accumulation of NO

119909

minus in the sys-tem [8] Discovery of several bacteria capable of carryingout SND has generated considerable scientific interest indeveloping efficient biological nitrogen removal process ina single reaction vessel [7] Examples of such organismsisolated from different sources include Agrobacterium sp [9]Rhodococcus sp [10] Alcaligenes faecalis [11] Diaphorobactersp [12] Bacillus subtilis [13] Bacillus methylotrophicus [14]Acinetobacter calcoaceticus [15] Marinobacter sp [7] andKlebsiella pneumoniae [16] Careful review of the literatureshows that any bacterium capable of performing SND hasnot been isolated from slaughterhouse wastewater It isfurther observed that there are gaps in understanding therole of microorganisms in the nitrogen removal processesoccurring in abattoir wastewater For example Yilmaz et al[8] reported that phosphate accumulating organisms (mainlyAccumulibacter spp) occurring in abattoir wastewater wereresponsible for the denitrification However microorganismsthat reduced nitrate to nitrite before Accumulibacter sppcould carry out denitrification from nitrite to nitrogen gascould not be identified by the authors

Thus investigations on isolation of nitrifying and denitri-fying bacteria from slaughterhouse wastewater establishingtheir taxonomical identity characterizing their metabolicpatterns and identifying their potential application in large-scale nitrogen removal processes are necessary In our pre-vious study [17] we isolated a microbe from slaughterhousewastewater and identified it as Achromobacter xylosoxidansThis bacterium previously known as a clinical isolate wasfor the first time demonstrated to have nitrification activityInterestingly anaerobic denitrification a property which isregarded as an intrinsic characteristic of the Achromobactergenus was conserved In continual pursuance of our searchfor novel nitrifiersdenitrifiers the present paper describesthe isolation of a new nitrifying-denitrifying bacterium capa-ble of performing SND from wastewater emanating from asmall-size rural slaughterhouse in India This communica-tion further reports its taxonomical identification nitrogenremoval pathway characterization and kinetics of substrateutilization and growth

2 Materials and Methods

21 Seed Acclimatization Sludge collected from the ponddischarging wastes of Ms Mokami Slaughterhouse situatedin Nazira South 24 Parganas West Bengal India wasacclimatized to the laboratory conditions by cultivation in anaerated vessel for three months as described [17]

22 Isolation of the Bacterium and Development of PureCulture Ten predominant nitrifying bacteria were isolatedfrom the seed acclimatization vessel as described [17]Nitrification activities and preliminary taxonomic char-acterization of the isolates are presented as Supplemen-tary File 1 in Supplementary Material available online at

httpdxdoiorg1011552014436056 Amongst them thebacterium showing maximum nitrification activity (strainR31) was selected for further studies

23 Molecular Phylogenetic Analysis of Strain R31 Extractionof DNA amplification by polymerase chain reaction (PCR)and sequencing of the 16S rRNA gene were outsourced toScigenome Labs Kochi India DNA was extracted fromthe liquid culture of R31 grown in Luria broth followingSambrook et al [19] Amplification of the 16S rRNA geneof strain R31 was done with forward primer 27F (51015840-AGAGTTTGATCMTGGCTCAG-31015840) and reverse primer 1492R(51015840-GGT TAC CTT GTT ACG ACT T-31015840) [20] PCR wasperformed in a 50 120583L reaction mixture containing 5 120583L of10x PCR buffer with 15mM MgCl

2 4 120583L of 10mM dNTP

50 pmol of each oligonucleotide primers and 1120583L of 3U TaqDNA polymerase The conditions of PCR were initial denat-uration of 5min at 94∘C subsequently 30 incubation cycleseach comprising 30 seconds denaturation at 94∘C 45 secannealing at 57∘C 15min elongation at 72∘C and a final7min elongation at 72∘C The amplicon was electrophoresedin a 1 agarose gel and visualized under UV light Next theamplicon was purified using Nucleospin purification column(Macherey-Nagel Germany) The amplicon was sequencedusing an Applied Biosystems (USA) ABi 3730XL GeneticAnalyzer The PCR primers used for amplification wereapplied for sequencing the amplicon Phylogenetic analysis ofthe 16S rRNA gene sequence was done according to Kundu etal [17]

24 Fatty Acid Methyl Ester (FAME) Analysis of Strain R31FAME analysis of the isolate was outsourced to the MicrobialType Culture Collection and Gene Bank (MTCC) Instituteof Microbial Technology Chandigarh India Analysis wasdone using standard gas chromatography and applying theSherlock MIS system

25 Determination of Physiological Characteristics of StrainR31 Physiological characteristics of the strain were deter-mined following the Bergeyrsquos manual [21] Presence of flag-ella anaerobic growth sodium chloride tolerance aesculinhydrolysis indole production catalase activity gelatin liq-uefaction Tween 80 hydrolysis Simmons citrate utilizationdeoxyribonuclease activity Voges-Proskauer test methyl redtest starch hydrolysis 120573-galactosidase activity urease testand tyrosine hydrolysis were carried out following the meth-ods described in Kundu et al [17] Nitrate reduction test wasperformed to determine whether the isolate (R31) elaboratedthe enzymes nitrate reductase and nitrite reductase Thesetwo enzymes catalyze two reactions required for convertingnitrate to the end product nitrogen gas Aliquot from a pureculture of the isolate R31 was aseptically inoculated to a steriletube containing nitrate brothwith an invertedDurhamrsquos tubeThe inoculated tube was incubated at 37∘C for 24 hoursFinally the reductions of nitrate and nitrite were ascertainedby addition of naphthylamine and sulphanilic acid solutionsto the nitrate broth followed by adding a small amount of zincdust [22] Acid production from various carbohydrates wasdone following Hinz et al [23] and utilization of sole carbon

BioMed Research International 3

nitrogen and amino acids was done according to Gutnicket al [24] while growth in various media different temper-atures and pH as well as testing of antibiotic resistance andsusceptibility were done in accordance with standard pro-cedures as follows To determine the optimum temperatureand pH for growth the isolate was cultured in Luria-Bertanimedium and incubated at 37∘C with shaking (100 rpm) for48 hours Bacterial growth was confirmed by measuring theoptical density of the culture at 600 nm Susceptibility toantibiotics was determined by the routine agar-diffusion platetechnique using disks impregnatedwith following antibioticsvancomycin (30 120583g) lincomycin (15120583g) kanamycin (30 120583g)neomycin (30 120583g) oleandomycin (15120583g) ampicillin (10 120583g)benzylpenicillin (10 units) streptomycin (10 120583g) polymyxinB (300 units) gentamicin (50120583g) tetracycline (30 120583g) car-benicillin (100 120583g) and chloramphenicol (30 120583g) Lysine andarginine decarboxylase activities were measured followingMoslashller [25] whilst production of flexirubin pigment wastested according to Fautz and Reichenbach [26] Hydrogensulphide production was detected by visualizing the black-ening of SIM medium [27] All tests were done thrice intriplicate sets

26 Carbon Oxidation and Ammonium Stabilization by StrainR31 Carbon oxidation and ammonium stabilization wereassessed in batch cultures by cultivating strain R31 in abasal medium (all units gL) K

2HPO414 KH

2PO46

MgSO4sdot7H2O 02 trace mineral solution 2mL pH (at 25∘C)

= 7 plusmn 02 The trace mineral solution consisted of (allunits gL) EDTA 001 ZnSO

4sdot7H2O 00001 CaCl

2sdot2H2O

01 MnCl2sdot2H2O 0008 FeCl

3sdot6H2O 071 (NH

4)6Mo7O24

000011 CuSO4sdot5H2O 00001 CoCl

2sdot6H2O 02 The car-

bon (glucose) concentration was 1251 gL (carbon sub-strate 500mgL) while nitrogen (ammonium chloride) was0191 gL (nitrogen substrate 50mgL) forming CN ratio(ww) of 10 The sterilized media were inoculated with 1(vv) of the isolate and incubated at 37∘C with shaking(100 rpm) for 48 h Aliquots were removed from each Erlen-meyer flask at four hour intervals and growth was recordedby measuring OD

600 Next the liquid was centrifuged at

10000 rpm for 10 minutes and the supernatant was analyzedfor COD (by closed refluxmethod using dichromate) ammo-nia nitrite and nitrate nitrogen by using an expandable ionelectrode analyzer (Orion EA 940 Thermo Fisher ScientificUSA) Medium pH was measured using Cyberscan 510 pHmeter (Eutech Instruments Singapore) Hydroxylamine wasmeasured spectrophotometrically according to Frear andBurrell [28] Experiments were done thrice in triplicate sets

27 Influence of Carbon and Nitrogen Substrates on NitrogenRemoval by Strain R31 Effect of different types of carbonand nitrogen substrates and CN ratios on nitrogen removalis an important consideration in wastewater treatment Sothe influence of the nature of substrates and their relativeconcentrations on nitrogen removal ability of isolate R31 wasinvestigated through batch experiments

271 Carbon Substrates From a survey of literature [7 9 11ndash15 29ndash33] on the use of various carbon sources for bacteria

carrying out SND the most common substrates applied byother investigators appeared to be glucose sodium succinatesodium acetate and sodium citrate The influence of vari-ous carbon substrates was considered at constant ammonianitrogen concentration of 50mgL andCN ratio of 100 Glu-cose (1251 gL) sodium succinate (1688 gL) sodium acetate(1709 gL) and trisodium citrate (1792 gL) were used asdifferent carbon substrates in the basal medium described inSection 26 taking into account the fixed ammonia nitrogenconcentration and the corresponding constant CN ratio

272 Nitrogen Substrates Sodium nitrite (0303 gL) andsodium nitrate (0303 gL) were used in place of ammoniumchloride in the basal medium described in Section 26 toascertain the denitrification pathway of R31 The carbonsubstrate concentration and CN ratio were fixed at 500mgLand 100 respectively

273 CN Ratios The influence of various CN ratiosnamely 5 10 and 20 on heterotrophic nitrification-aerobicdenitrification by strain R31 was considered at constantammonium nitrogen concentration of 50mgL The CNratio was fixed by adjusting the amount of the carbon sub-strateThus the amounts of glucose usedwere 0625 gL (CN= 5) 1251 gL (CN= 10) and 2502 gL (CN= 20) Each flaskcontaining sterilized media was inoculated with the isolate(1 vv) and incubated at 37∘C with shaking (100 rpm) for48 h Aliquots were removed from each Erlenmeyer flask atfour hour intervals and growth was recorded by measuringOD600

The liquid was centrifuged at 10000 rpm for 10minutes and analyzed for ammonia nitrogen All experimentswere done thrice in triplicate sets

28 Evaluation of Kinetic Parameters To study simultaneouscarbon oxidation nitrification and denitrification kineticsexperiments were carried out in four different sets whereineach set consisted of twelve Erlenmeyer flasks The CNratio in each flask was maintained at 100 with appropri-ate amounts of glucose (carbon substrate) and ammoniumchloride (nitrogen substrate) All flasks containing sterilizedbasal media were inoculated with the isolate (1 vv) andincubated at 37∘C with shaking (100 rpm) for 48 h One flaskfrom each set was removed from the shaker every four hoursThe culture was centrifuged at 10000 rpm for 10 minutes andthe supernatant was analyzed for COD ammonia nitrogennitrate nitrogen pH and MLVSS (mixed liquor volatilesuspended solids) which was measured gravimetrically in amuffle furnace at 550 plusmn 50∘C) Experiments were done thrice

281 Kinetics of Substrate Removal and Growth Estimationof the kinetic constants for carbon and ammonia utilizationsuch as half saturation concentration (119870

119904) and maximum

substrate utilization rate (119896) as well as determination of thegrowth kinetic constants such as yield coefficient (119884) and theendogenous decay coefficient (119870

119889) were done by applying

Lawrence and McCartyrsquos modified Monod equation [34]The results so obtained were fitted in to a straight line inaccordance with the Lineweaver-Burk plot for determinationof the kinetic constants


10098

95

92

55

86

57

99

100

100

89

69

86

100

66

55

78 100lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

Candidatus Amoebinatus massiliae 7Ebis (AF531765)

Chryseobacterium sp R31 (KF751764)

Chryseobacterium anthropi NF 1366T (AM982786)

Chryseobacterium haifense H38T (EF204450)

Chryseobacterium carnis NCTC 13525T (JX100817)

Chryseobacterium arothri P2K6T (EF554408)

Chryseobacterium hominis NF802T (AM261868)

Chryseobacterium molle DW3T (AJ534853)

Chryseobacterium hispanicum VP48T (AM159183)

Chryseobacterium tructae 1084-08T (FR871429)

Chryseobacterium nakagawai NCTC 13529T (JX100822)

Chryseobacterium taihuense THMBM1T (JQ283114)

Chryseobacterium hispalenseEpilithonimonas lactis H1T (EF204460)

Riemerella columbipharyngis 8151T (HQ286278)

Elizabethkingia anophelis 25T (EF426425)

Chryseobacterium pallidum 26-3St2bT (AM232809)

Elizabethkingia meningoseptica ATCC 13253T (AJ704540)

Soonwooa buanensis HM0024T (FJ713810)Cruoricaptor ignavus IMMIB L-12475T (HE614680)

Salinirepens amamiensis

Flavobacterium yonginense HMD1001T (GQ144413)

Spongiimonas flava J36A-7T (AB742039)

Bacillus subtilis DSM10T (AJ276351)

Luteibaculum oceani CC-AMWY-103BT (KC169812)

Nonlabens ulvanivorans PLRT (GU902979)

01

AM11-6T (AB517714)

AG13T (EU336941)

Figure 1Unrooted phylogenetic tree obtained by the neighbor-joining (NJ)methodbased on 16S rRNAgene sequences depicting the positionof strain Chryseobacterium sp R31 amongst its phylogenetic neighbors Numbers at nodes designate levels of bootstrap support () basedon a NJ analysis of 1000 resampled datasets only values higher than 50 are displayed Asterisks denote branches that were obtained usingthe maximum parsimony and maximum likelihood algorithms NCBI accession numbers are provided in parentheses Bar = 01 nucleotidesubstitutions per site The sequence of Bacillus subtilis DSM 10 (AJ276351) was applied as an outgroup

3 Results and Discussion

31 Basic Taxonomical Identification of Strain R31 The phys-iological characteristics of isolate R31 are listed in Supple-mentary File 2 Small circular colonies with entire edgeswere observed when R31 was grown on nutrient agar platesThe colonies were glossy raised and yellowish-golden incolor On the basis of nucleotide homology and phylogeneticanalysis the isolate was shown to be significantly similarto Chryseobacterium haifense Identity analysis on the EZtaxon server [35] revealed that the 16S rRNA gene sequencehad the closest similarity (9825) to the gene sequence of

the type strain of Chryseobacterium haifense (SupplementaryFile 3) The phylogenetic position of the isolate (R31) havingNCBI Genbank accession number KF751764 is shown in thedendogram (Figure 1) Strain R31 emerged as a distinctivephylogenetic line from the cluster containing the type ofstrains of Chryseobacterium species shown in SupplementaryFile 3 This differentiation was corroborated by considerablebranch length and a high bootstrap value (95) The phy-logenetic position of strain R31 was further supported bythe maximum parsimony (MP) and maximum likelihood(ML) analyses An analogous delineation of the strain wasfound applying the MP and ML algorithms which was again


confirmed by high bootstrap values (70 for MP and 98for ML) and significant branch lengthsThe result of the fattyacidmethyl ester (FAME) analysis is shown in SupplementaryFile 4The predominant cellular fatty acids of isolate R31 were150 iso (4040) 150 anteiso (1944) and 170 iso 3-OH(1113) which closely matched those of ChryseobacteriumhaifenseT 150 iso (416) 150 anteiso (166) and 170 iso 3-OH (103) [36] In the nitrate reduction test gas formationwas observed in Durhamrsquos tube indicating that the isolateR31 had the ability to convert nitrate to nitrite and then tonitrogen gas The isolate (R31) was found positive for bothnitrate reductase and nitrite reductase Similar conclusionswere drawn by Fernandez et al [37]

A number of physiological characteristics correspondedwith the standard species description of C haifense such ashavingGram-negative reaction being nonmotile without anyflagella being obligately aerobic having temperature rangesof growth having growth at 4∘C having NaCl requirementsfor growth being oxidase positive being urease negativebeing lysine decarboxylase negative being aesculin andgelatin hydrolysis positive and being flexirubin productionnegative On the other hand the physiological traits of strainR31 that did not agree the standard species description ofC haifense were having acid production from carbohydratesbeing catalase negative being nitrate reduction positivebeing 120573-galactosidase negative being indole production neg-ative and being H

2S production positive Strain R31 may be

a new member of the Chryseobacterium genus and furtherconfirmation can bemade throughDNA-DNA hybridizationtests

32 Simultaneous COD and Nitrogen Removal by Strain R31

321 Carbon Oxidation and Ammonium Stabilization byStrain R31 Figure 2 shows the profile of COD removal withrespect to the time of contact After 48 h 9554 of CODwas removed from the initial concentration of 5830mgLindicating heterotrophic nutritional characteristic of isolateR31 Stabilization of the utilizable component of the basalmedium within the reaction period was indicated by theasymptotic nature of the curve after 48 h Growth kineticscorresponding to carbon oxidation is also shown in Figure 2The lag phase lasted for approximately 4 h following whichisolate R31 grew exponentially for 30 h Highest biomassproduction was observed around 48 h following which themicroorganism attained the stationary phase of growth Yanget al [13] demonstrated growth of heterotrophic nitrifying-denitrifying Bacillus subtilis A1 and the nitrogen removalprocess consumed most of the organic substrates in themedia yielding COD removal efficiencies of 710 plusmn 05671 plusmn 0 645 plusmn 15 and 639 plusmn 18 for acetate glucosecitrate and succinate respectively Khardenavis et al [12]attained COD removal of 85ndash93 using Diaphorobacter spThese values were lower than that obtained in this study

The profile of ammoniacal nitrogen utilization by isolateR31 with relation to time is also shown in Figure 3 After acontact period of 48 h 9587 of NH

4

+-N was consumeddemonstrating the heterotrophic nitrification ability of

002040608

112141618

0 8 16 24 32 40 48Time (h)

0102030405060708090100

Opt

ical

den

sity

at600

nm

COD

rem

oval

()

Figure 2 Time profile of carbon oxidation and growth of Chry-seobacterium sp R31 Growth (filled diamonds) and COD removal() (filled squares) Error bars represent one SD (119899 = 9)

0

10

20

30

40

50

60

0 8 16 24 32 40 48Time (h)

02468101214

NH

4+-N

and

NO

2minus-N

and

NO

3minus-N

(mg

L)

hydr

oxyl

amin

e (m

gL)

Figure 3 Time profile of ammonium oxidation by Chryseobac-terium sp R31 Ammonium nitrogen (filled diamonds) nitritenitrogen (filled squares) nitrate nitrogen (filled triangles) andhydroxylamine (filled circle) Error bars represent one SD (119899 = 9)

the isolate Figure 3 further depicts that the initial ammo-nia utilization rate was high and maximum stabilizationof ammonium in solution was attained within 32 h afterwhich the utilization declined and ammonium was removedmarginally Exhaustion of ammonium led to reduced avail-ability of the nitrogen substrate to the microorganismFigure 3 also represents the corresponding nitrite nitrogen(NO2

minus-N) and nitrate nitrogen (NO3

minus-N) concentrationswith respect to time This figure shows that ammoniumwas substantially converted to nitrite and after a reactionperiod of 24 h the nitrite level in the reactor was maximumcorresponding to 7199 ammonium utilization Nitrateconcentration initially increased with time confirming het-erotrophic nitrification by strain R31 Nitrate concentrationreached its peak value at 32 h when 8779 of ammoniumwas consumed The decrease in nitrate concentration afterreaching a peak value ascertained the denitrification pro-cess Nitrate was utilized by the isolate after ammoniumwas exhausted Hydroxylamine was detected in reducedamounts possibly due to the unstable nature of hydroxy-lamine and rapid transformation to the next intermediatenitrite [14] Simultaneous utilization of organic matter andammonia degradation indicated R31 to be competent ofheterotrophic nitrification as well as aerobic denitrificationHydroxylamine nitrite and nitrate accumulation occurred


0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50Time (h)

NH

4+-N

(mg

L)

(b)

Figure 4 (a) Influence of various carbon substrates on growth of Chryseobacterium sp R31 Sodium succinate (filled diamonds) glucose(filled squares) trisodium citrate (filled triangles) and sodium acetate (filled circles) Error bars represent one SD (119899 = 9) (b) Influence ofvarious carbon substrates on ammonium removal by Chryseobacterium sp R31 Sodium succinate (filled diamonds) glucose (filled squares)trisodium citrate (filled triangles) and sodium acetate (filled circles) Error bars represent one SD (119899 = 9)

with the decrease of ammonia nitrogen The concentrationsof the three intermediates were low thus ensuring nitrogenremoval The profile of hydroxylamine nitrite and nitrateformationwas similar to that observed during SNDprocessesoccurring in Agrobacterium sp [9] as well as Klebsiellapneumonia [16]The amount of ammonium removed (9587in 48 h) was higher than that striped off by Bacillus subtilis(584 plusmn 43 in 60 h) [13] Bacillus methylotrophicus (5103in 54 h) [14] and Marinobacter sp F6 (4862) [7] Theammonium utilization rate (115mgLh) was similar to thatobtained for Pseudomonas alcaligenes sp AS-1 (115mgLh)[10] and higher than the corresponding value of Bacillus spLY (043mgLh) [10]

322 Influence of Carbon Substrates on Nitrogen Removal byStrain R31 The influence of various carbon substrates ongrowth of strain R31 and ammonium stabilization by theisolate is represented in Figures 4(a) and 4(b) respectivelyThe plot depicts abundant growth when glucose or succinatewas used whereas lesser biomass was formedwith citrate andacetate as substrates indicating that these carbon sourceswerenot utilizable by the isolate Within 48 h 9587 and 9107of NH

4

+-N were removed in the presence of glucose andsodium succinate respectively whilst reduced nitrificationoccurred in the presence of citrate and acetate It was alsoinferred that simultaneous COD removal nitrification anddenitrification would not be totally achieved in wastewatercontaining these substrates Glucose and sodium succinatewere superior carbon substrates for nitrogen removal byMarinobacter sp [7] and Bacillus methylotrophicus [14] whilstcitrate and acetate were inferior substrates analogous to ourresults On the other hand citrate and acetate proved tobe better substrates for nitrogen removal by A faecalis [11]The four substrates glucose acetate citrate and succinateremoved nitrogen with equal efficiency during simultaneousnitrification-denitrification by Bacillus subtilis [13]

A specific membrane transporter citrate lyase activ-ity and oxaloacetate decarboxylase activity are required

for bacterial citrate utilization [38] There are two routesfor the metabolic interconversion of acetate and acetyl-CoA acetyl-CoA synthetase pathway and acetate kinase-phosphotransacetylase pathway Growth on acetate furtherentails the glyoxylate shunt thus bypassing the decarboxy-lation steps of the tricarboxylic acid cycle This in turnneeds two enzymes isocitrate lyase andmalate synthase [39]It appeared that the metabolic pathways andor enzymesrequired for citrate and acetate utilization were either par-tially inactive or repressed when isolate R31 was fed withcitrate and acetate Succinate was used as efficiently as glucosefor growth and ammonium stabilization by isolate R31 Suc-cinate is a bifunctional substrate of succinate dehydrogenasean enzyme that is required in both the TCA cycle andthe electron transport chain coupling carbon flow to ATPsynthesis [40]

323 Influence of Nitrogen Substrates on Nitrogen Removalby Strain R31 The influence of various nitrogen substrateson growth of isolate R31 and nitrogen removal is shown inFigures 5(a) and 5(b) respectively It is evident from thefigures that similar growth was attained with ammoniumchloride nitrate and nitrite as substrates Nitrogen removalwas nearly alike for all three substrates Thus the isolatewas capable of utilizing ammonium nitrate and nitrite asnitrogen substrates and heterotrophic nitrification-aerobicdenitrification was independent of the nature of the nitrogensubstrate Growth with nitrite was sufficient and 75 nitritewas utilized It appeared that the enzymes required forammonium utilization such as ammonium monooxygenaseand hydroxylamine oxidoreductase as well as nitritenitratereductase required for nitrate utilization were all active whenthe organism was fed with these substrates This experimentfurther confirmed the nitrification and denitrification abil-ity of the isolate Aerobic denitrification occurring duringammonium removal by R31 demonstrated by utilization ofboth nitrate and nitrite was similar to the process occurringin Agrobacterium strain LAD9 as reported by Chen and Ni


0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)

Figure 5 (a) Influence of various nitrogen substrates on growth of Chryseobacterium sp R31 Ammonium chloride (filled squares) sodiumnitrate (filled triangles) and sodium nitrite (filled circles) Error bars represent one SD (119899 = 9) (b) Influence of various nitrogen substrateson nitrogen removal by Chryseobacterium sp R31 Ammonium chloride (filled squares) sodium nitrate (filled triangles) and sodium nitrite(filled circles) Error bars represent one SD (119899 = 9)

[9] The nitrate removal rate was 10mgLh which is higherthan that recorded forRhodococcus (093mgLh) as reportedby Chen et al [10] Aerobic denitrifiers utilize nitrite byintracellular assimilation or extracellular reduction pathwaysSimilar to the observations for Klebsiella pneumoniae [16]concomitant cell density increase was noted in our exper-iments along with consumption of nitrite indicating thatextracellular reduction had occurred Aerobic denitrificationability of strain R31 was confirmed by the positive resultsobtained in the nitrate and nitrite reductase assays (theseenzyme activities were absent in the type of strain of Chaifense)

Two pathways are implicated in the simultaneous nitri-fication and denitrification processes the first throughnitrite and nitrate and the second via hydroxylamine [41]Thiosphaera pantotropha [32] Bacillus subtilis [13] Agrobac-terium sp [9] Rhodococcus sp [10] K pneumoniae [16] andMarinobacter sp [7] possess a complete nitrification and den-itrification pathway (NH

4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash

N2) Contrastingly neither nitrite nor nitrate was detected

as intermediates in Alcaligenes faecalis [11] and Acinetobactercalcoaceticus [15] Furthermore nitrite or nitrate reductaseactivities were not detected in these two bacteria Denitri-fication in A faecalis and A calcoaceticus occurs throughhydroxylamine not requiring nitrite or nitrate (NH

4

+ndashNH2OHndashN

2OndashN2) Results as described in this section

in relation to the reports of previous workers indicatethat Chryseobacterium sp R31 has a complete nitrificationand denitrification pathway Conventionally denitrificationoccurs under anoxic conditions but strain R31 is obligatelyaerobic Microorganisms capable of SND possess respiratoryand fermentative types of metabolism Bacteria belonging tothe genera Agrobacterium [42] Rhodococcus [43] Alcaligenes[44]Acinetobacter [45] andMarinobacter [46] are obligatelyaerobic Bacteria of the Bacillus genus [47] are aerobic whichmay be strict or facultative while the genus Klebsiella [48]comprises facultative anaerobic bacteria

324 Influence of Different CN ratios on Growth and Ammo-nium Removal by Strain R31 Figures 6(a) and 6(b) showthat at CN ratio of 10 the growth rate of isolate R31 wasthe highest and maximum ammonia nitrogen was removed(9587)within 48 h Figure 6(b) also demonstrates that after48 h of contact NH

4

+-N utilization nearly ceased indicatingexhaustion of the nutrient or inhibition of key enzyme(s)The NH

4

+-N utilization rate (Figure 6(b)) and growth (Fig-ure 6(a)) of strain R31 were found to be the higher at CN= 10 in comparison to those achieved with CN ratios of 5and 20 The rate of nitrogen removal at CN = 10 acceleratedbeyond 8 h and reached an equilibrium level after 40 h AtCN ratio of 5 the consumption of ammoniacal nitrogenpractically stopped after 36 h of incubation resulting in6311 removal At CN = 5 heterotrophic nitrification couldnot be performed efficiently for residual NH

4

+-N beyond2025mgL after 36 h perhaps due to early exhaustion of thecarbon source On the other hand at CN = 10 the growthcurve shows that higher biomass was reached probably dueto enhanced amounts of utilizable C and N nutrients At CN= 20 the overall removal of NH

4

+-N was close to the valueattained at CN = 10 but initial consumption was delayed by atime lag Appreciable removal at this ratio ensued after 24 hJoo et al [11] reported similar trend of ammoniacal nitrogenutilization by the heterotrophic nitrifier (Alcaligenes faecalisNo 4) Authors observed that at lower CN ratio such as5 40 unconsumed NH

4

+-N remained in the reactor dueto dearth of the carbonaceous substrate At CN ratio of 10authors noted that both carbon andNH

4

+-N levels decreasedin a well-balanced fashion with respect to time along withappreciable amount of growth of the microorganism ForBacillus subtilisA1 optimalCN ratiowas 60 and ammoniumand acetate were consumed at a proportionate rate [13] AtCN = 2 the consumption of NH

4

+-N stopped because thecarbon source was depleted The NH

4

+-N removal efficiencywas higher at CN = 6 and CN = 12 than at CN = 2 and CN= 26 at 60 h


0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)

Figure 6 (a) Influence of different CN ratios on growth of Chryseobacterium sp R31 CN = 5 (filled diamonds) CN = 10 (filled squares)and CN = 20 (filled triangles) Error bars represent one SD (119899 = 9) (b) Influence of different CN ratios on ammonium removal byChryseobacterium sp R31 CN = 5 (filled diamonds) CN = 10 (filled squares) and CN = 20 (filled triangles) Error bars represent oneSD (119899 = 9)

The optimal values of CN ratioswere 80-90 forAgrobac-terium [9] and 150 for Bacillus sp LY [10] andMarinobactersp [7] Increasing the CN ratio enhanced nitrate removalrates of Pseudomonas putida whereas nitrogen assimilationinto the cellmass was not changedThe optimal CN ratiowas8 [33] CN ratio did not influence heterotrophic nitrificationby Bacillus methylotrophicus [14] In general for a givenCN range an enhancement in organic carbon concentrationresulted in increased denitrification efficiency of aerobicdenitrifiers [7] Our observations support this statementLower value of CN ratio is advantageous in wastewatertreatment processes as operating costs can be reduced [9]Considering this nitrogen removal using Chryseobacteriumsp R31 may be more economical than Bacillus sp LY andMarinobacter sp and as cost-effective asA faecalis In light ofthe recommendations of Joo et al [11] the CN ratio can beused as a criterion of control when considering ammoniumremoval in continuous culture by R31 and the addition ofexternal carbon would be necessary in the treatment of high-strength ammonium wastewater at CN less than 10

325 Substrate Removal and Growth Kinetics of Simultane-ous Carbon Oxidation Nitrification and Denitrification byStrain R31 Substrate removal kinetics for carbon oxidation

nitrification and denitrification as well as growth kineticsfor carbon oxidation nitrification and denitrification werestudied applying Lawrence and McCartyrsquos modified Monodequation [34] The data fitted reasonably well following theLineweaver-Burk equation (Figures 7(a)ndash7(f)) From theseplots 119870

119904 119896 119884 and 119896

119889were estimated as enumerated in

Table 1 As shown in Table 1 the values of the coefficientswere similar to those obtained for the other slaughter-house wastewater isolate A xylosoxidans [17] and thosedetermined for aerobic-anoxic process for slaughterhousewastewater [1] Although belonging to different genera thekinetics of substrate utilization displayed by the two isolates(A xylosoxidans and Chryseobacterium sp) was essentiallysimilar It may be noted that the 119870

119904values for carbon oxi-

dation (22011mgL) and nitrification (219mgL) obtained inthis study were higher than those conventionally accepted fordomestic wastewater (for comparison see Table 1) Enhancedvalues were attained due to the higher initial CODandNH

4

+-N levels found in slaughterhouse wastewater compared tothat present in domestic wastewater [18] Values indicatethat R31 had low affinity for carbonaceous substrate as wellas for ammonia The high 119870

119904value may be an effect of

high substrate concentration [49] The other kinetic coeffi-cients (119896 119884 and 119896

119889) for carbon oxidation and nitrification

were however comparable to that reported for domestic


08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01

1S (1mgL of NO3minus-N)

1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12

U (mg of CODdaymg of MLVSS)

R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25

U (mg of NH4+-Ndaymg of MLVSS)

R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)

U (mg of NO3minus-Ndaymg of MLVSS)

R2 = 09998

y = 07856x minus 00361

(f)

Figure 7 Lineweaver-Burk plot for determination of substrate utilization kinetic constants for (a) carbon oxidation (b) nitrification and (c)denitrification and growth kinetic constants for (d) carbon oxidation (e) nitrification and (f) denitrification by Chryseobacterium sp R31Each data point represents the mean value of nine determinations Error is within one SD of the mean

Table 1 Summarized values of kinetic coefficients obtained in the present study our previous study [17] and domestic wastewater [18]

Kinetic coefficientsCarbon oxidation Nitrification Denitrification

Presentstudy

Kundu etal [17]

Metcalf andEddy [18]

Presentstudy

Kundu etal [17]


Presentstudy


119896 (dayminus1) 298 224 2ndash10 1466 1366 1ndash30 070 023ndash288119870119904(mgL) 22011 2320 25ndash100 219 213 02ndash05 013 006ndash020119884 (mgmg) 054 044 04ndash08 028 024 01ndash03 078 04ndash09119896119889(dayminus1) 003 006 0025ndash0075 003 005 003ndash006 003 004ndash008


wastewater [18] For municipal and industrial wastewaterthe coefficient values represent the net effect of microbialkinetics on the simultaneous degradation of a variety ofdifferent wastewater constituents The kinetic coefficientsdetermined for denitrification corroborated with the typicalvalues obtained for domestic wastewater (for comparison seeTable 1) The constants determined are suitable for CODand nitrogen removal similar to the conclusions of Palaand Bolukbas [50] Authors reported that the Monod kineticconstants (119884 119896

119889 and 119870

119904) were indicative of efficient COD

andnitrogen removal frommunicipal wastewaterThekineticcoefficients obtained in this study are supportive of thosecalculated by Kundu et al [1]This proves that R31 has similarcapacity to perform in terms of COD and nitrogen removalfrom slaughterhouse wastewater if used in a large-scaleSND treatment plant Kinetic coefficients for simultaneouscarbon oxidation nitrification and denitrification by anyChryseobacterium species are being determined for the firsttime Analysis of the kinetic coefficients is the bestmethod forprediction of potential large-scale application of pure culturesand this approach has been described for the first time forbacteria capable of SND

The Chryseobacterium genus was first described by Van-damme et al [51] with six species and following the reviseddescription of the family Flavobacteriaceae by Bernardet et al[52] more than sixty novel species of the genus Chryseobac-terium have been identified from a large number of sourcessuch as wastewater [53] contaminated soils [54] plantrhizosphere [55] human clinical source [56] chicken [57]fish [58] mosquito [59] glacier [60] and beermanufacturing[61] However most of the novel species have been reportedfrom bovine milk [36 62 63] Our isolate was obtained fromslaughterhouse wastewater for the first time and to the bestof our knowledge nitrification and denitrification propertywithin this genus is also being recorded for the first timeTheNitrosomonas genus having ammonia-oxidizing bacteria andthe Nitrobacter genus comprising nitrite oxidizing bacteriaare regarded as the principal genera of chemolithotrophicnitrifying bacteria Accordingly the probes required forfluorescence in situ hybridization (FISH) NSO1225 andNIT3 were selected to stain 120573-proteobacteria sp AOB andNitrobacter sp NOB respectively by Pan et al [6] duringtheir study on nitrogen removal from abattoir wastewaterthrough partial nitrification and subsequent denitrificationin intermittently aerated sequencing batch reactors Authorsjustified the selection stating that both 120573-proteobacteriasp AOB and Nitrobacter sp NOB were widely found inwastewater systems However Yilmaz et al [8] noted thatcommon NOBs (Nitrobacter and Nitrospira) were not foundin the sequential batch reactor during simultaneous nitri-fication denitrification and phosphate removal of abattoirwastewater In consonance with the finding of Yilmaz et al[8] and similar to our previous study [17] NitrosomonasNitrobacter and Nitrospira were not recovered as the mostactive nitrifiersdenitrifiers in the current investigation Ourtwo studies assert that novel microorganisms should beconsidered for simultaneous COD reduction nitrificationand denitrification in slaughterhouse wastewater

4 Conclusions

Through the present investigation a bacterium isolated fromslaughterhouse wastewater was identified as Chryseobac-terium sp which successfully stabilized COD as well asammonium nitrogen The bacterium was capable of utiliz-ing NO

3

minus-N aerobically in presence of NH4

+-N Nitrogenremoval was dependent on the nature of the carbon substratebut independent of the type of the nitrogen substrate Thisbacterium is a newmember in the group of microbes capableof simultaneous removal of organic carbon andnitrogen fromwastewater Further taxonomic investigations are warrantedto establish the strain as a new speciesThe kinetic coefficientsof substrate removal and growth for concomitant carbonoxidation nitrification and denitrification are favorable andcorroborative with the results of earlier researchers Thekinetic coefficients may be considered for the design ofbioreactors thus opening up the possibility of SND processin treatment of slaughterhouse wastewater

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Contributions of Pradyut Kundu Arnab Pramanik andArpita Dasgupta are equal in all respects

Acknowledgments

Financial support fromDST-PURSE and TEQIP programs ofJadavpur University is thankfully acknowledged

References

[1] P Kundu A Debsarkar and S Mukherjee ldquoTreatment ofslaughter house wastewater in a sequencing batch reactorperformance evaluation and biodegradation kineticsrdquo BioMedResearch International vol 2013 Article ID 134872 11 pages2013

[2] C F Bustillo-LecompteMMehrvar andEQuinones-BolanosldquoCombined anaerobic-aerobic and UVH

2O2processes for the

treatment of synthetic slaughterhouse wastewaterrdquo Journal ofEnvironmental Science andHealth Part A vol 48 no 9 pp 1122ndash1135 2013

[3] J B R Mees S D Gomes S D M Hasan B M Gomes andM A V Boas ldquoNitrogen removal in a SBR operated with andwithout pre-denitrification effect of the carbon nitrogen ratioand the cycle timerdquo Environmental Technology vol 35 no 1 pp115ndash123 2014

[4] Y Filali-Meknassi M Auriol R D Tyagi Y Comeau andR Y Surampalli ldquoDesign strategy for a simultaneous nitri-ficationdenitrification of a slaughterhouse wastewater in asequencing batch reactor ASM2d modeling and verificationrdquoEnvironmental Technology vol 26 no 10 pp 1081ndash1100 2005

[5] R L Meyer R J Zeng V Giugliano and L L BlackallldquoChallenges for simultaneous nitrification denitrification andphosphorus removal in microbial aggregates mass transfer


limitation and nitrous oxide productionrdquo FEMS MicrobiologyEcology vol 52 no 3 pp 329ndash338 2005

[6] M Pan L G Henry R Liu X Huang and X Zhan ldquoNitrogenremoval from slaughterhouse wastewater through partial nitri-fication followed by denitrification in intermittently aeratedsequencing batch reactors at 11∘Crdquo Environmental Technologyvol 35 pp 470ndash477 2014

[7] H-Y Zheng Y Liu X-Y Gao G-M Ai L-L Miao andZ-P Liu ldquoCharacterization of a marine origin aerobicnitrifying-denitrifying bacteriumrdquo Journal of Bioscience andBioengineering vol 114 no 1 pp 33ndash37 2012

[8] G Yilmaz R Lemaire J Keller and Z Yuan ldquoSimultaneousnitrification denitrification and phosphorus removal fromnutrient-rich industrial wastewater using granular sludgerdquoBiotechnology and Bioengineering vol 100 no 3 pp 529ndash5412008

[9] Q Chen and J Ni ldquoAmmonium removal by Agrobacterium spLAD9 capable of heterotrophic nitrification-aerobic denitrifica-tionrdquo Journal of Bioscience and Bioengineering vol 113 no 5 pp619ndash623 2012

[10] P Chen J Li Q X Li et al ldquoSimultaneous heterotrophic nitri-fication and aerobic denitrification by bacterium Rhodococcussp CPZ24rdquo Bioresource Technology vol 116 pp 266ndash270 2012

[11] H-S Joo M Hirai and M Shoda ldquoCharacteristics of ammo-nium removal by heterotrophic nitrification-aerobic denitrifi-cation by Alcaligenes faecalis no 4rdquo Journal of Bioscience andBioengineering vol 100 no 2 pp 184ndash191 2005

[12] A A Khardenavis A Kapley and H J Purohit ldquoSimultaneousnitrification and denitrification by diverse Diaphorobacter sprdquoApplied Microbiology and Biotechnology vol 77 no 2 pp 403ndash409 2007

[13] X-P Yang S-M Wang D-W Zhang and L-X Zhou ldquoIso-lation and nitrogen removal characteristics of an aerobic het-erotrophic nitrifying-denitrifying bacterium Bacillus subtilisA1rdquo Bioresource Technology vol 102 no 2 pp 854ndash862 2011

[14] Q-L Zhang Y Liu G-MAi L-LMiaoH-Y Zheng andZ-PLiu ldquoThe characteristics of a novel heterotrophic nitrification-aerobic denitrification bacterium Bacillus methylotrophicusstrain L7rdquo Bioresource Technology vol 108 pp 35ndash44 2012

[15] B Zhao Y L He J Hughes and X F Zhang ldquoHeterotrophicnitrogen removal by a newly isolatedAcinetobacter calcoaceticusHNRrdquo Bioresource Technology vol 101 no 14 pp 5194ndash52002010

[16] S K Padhi S Tripathy R Sen A S Mahapatra S Mohantyand N K Maiti ldquoCharacterisation of heterotrophic nitrifyingand aerobic denitrifyingKlebsiella pneumoniaeCF-S9 strain forbioremediation of wastewaterrdquo International Biodeteriorationand Biodegradation vol 78 pp 67ndash73 2013

[17] P Kundu A Pramanik SMitra J D Choudhury J Mukherjeeand S Mukherjee ldquoHeterotrophic nitrification by Achromobac-ter xylosoxidans S18 isolated from a small-scale slaughterhousewastewaterrdquo Bioprocess and Biosystems Engineering vol 35 no5 pp 721ndash728 2012

[18] Metcalf and Eddy Inc Wastewater Engineering TreatmentDisposal Reuse Tata McGraw-Hill 3rd edition 1995

[19] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory Press ColdSpring Harbor NY USA 2nd edition 1989

[20] A V Piterina J Bartlett and J Tony Pembroke ldquoMolecularanalysis of bacterial community DNA in sludge undergoingautothermal thermophilic aerobic digestion (ATAD) pitfallsand improved methodology to enhance diversity recoveryrdquoDiversity vol 2 no 4 pp 505ndash526 2010

[21] J F Bernardet C J Hugo and B Bruun ldquoGenus XChryseobac-teriumVandamme Bernardet Segers Kersters and Holmesrdquo inBergeyrsquos Manual of Systematic Bacteriology W B Whitman NR Krieg J T Staley et al Eds vol 4 pp 180ndash192 2nd edition2010

[22] G Barrow and R K A Feltham Cowan and Steelrsquos Manualfor the Identification of Medical Bacteria Cambridge UniversityPress Cambridge Mass USA 3rd edition 1993

[23] K-H Hinz M Ryll and B Kohler ldquoDetection of acid produc-tion from carbohydrates by Riemerella anatipestifer and relatedorganisms using the buffered single substrate testrdquo VeterinaryMicrobiology vol 60 no 2-4 pp 277ndash284 1998

[24] D Gutnick JM Calvo T Klopotowski and B N Ames ldquoCom-pounds which serve as the sole source of carbon or nitrogen forSalmonella typhimurium LT-2rdquo Journal of Bacteriology vol 100no 1 pp 215ndash219 1969

[25] V Moslashller ldquoSimplified tests for some amino acid decarboxylasesand for the arginine dihydrolase systemrdquo Acta Pathologica etMicrobiologica Scandinavica vol 36 no 2 pp 158ndash172 1955

[26] E Fautz and H Reichenbach ldquoA simple test for flexirubin-typepigmentsrdquo FEMS Microbiology Letters vol 8 no 2 pp 87ndash911980

[27] P R Murray E J Baron M A Pfaller F C Tenover and R HYolken Manual of Clinical Microbiology American Society forMicrobiology Washington DC USA 6th edition 1995

[28] D S Frear and R C Burrell ldquoSpectrophotometric method fordetermining hydroxylamine reductase activity in higher plantsrdquoAnalytical Chemistry vol 27 no 10 pp 1664ndash1665 1955

[29] M Shoda and Y Ishikawa ldquoHeterotrophic nitrification andaerobic denitrification of high-strength ammonium in anaer-obically digested sludge by Alcaligenes faecalis strain No 4rdquoJournal of Bioscience and Bioengineering vol 117 no 6 pp 737ndash741 2014

[30] J-J Su B-Y Liu and C-Y Liu ldquoComparison of aerobicdenitrification under high oxygen atmosphere by Thiosphaerapantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newlyisolated from the activated sludge of a piggery wastewatertreatment systemrdquo Journal of Applied Microbiology vol 90 no3 pp 457ndash462 2001

[31] B Zhao Q An Y L He and J S Guo ldquoN2O andN

2production

during heterotrophic nitrification by Alcaligenes faecalis strainNRrdquo Bioresource Technology vol 116 pp 379ndash385 2012

[32] L A Robertson and J G Kuenen ldquoHeterotrophic nitrificationin Thiosphaera pantotropha oxygen uptake and enzyme stud-iesrdquo Journal of General Microbiology vol 134 pp 857ndash863 1988

[33] M Kim S-Y Jeong S J Yoon et al ldquoAerobic denitrification ofPseudomonas putida AD-21 at Different CN ratiosrdquo Journal ofBioscience and Bioengineering vol 106 no 5 pp 498ndash502 2008

[34] A W Lawrence and P L McCarty ldquoUnified basis for biologicaltreatment design and operationrdquo Journal of the Sanitary Engi-neering Division-ASCE vol 96 pp 757ndash778 1970

[35] O-S Kim Y-J Cho K Lee et al ldquoIntroducing EzTaxon-e aprokaryotic 16s rRNA gene sequence database with phylotypesthat represent uncultured speciesrdquo International Journal ofSystematic and EvolutionaryMicrobiology vol 62 no 3 pp 716ndash721 2012

[36] E Hantsis-Zacharov and M Halpern ldquoChryseobacteriumhaifense sp nov a psychrotolerant bacterium isolated fromraw milkrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 57 no 10 pp 2344ndash2348 2007

[37] L A Fernandez E B Perotti M A Sagardoy and M AGomez ldquoDenitrification activity of Bradyrhizobium sp isolatedfrom Argentine soybean cultivated soilsrdquo World Journal of


Microbiology and Biotechnology vol 24 no 11 pp 2577ndash25852008

[38] N G Quintans V Blancato G Repizo C Magni and P LopezldquoCitrate metabolism and aroma compound production in lacticacid bacteriardquo in Molecular Aspects of Lactic Acid Bacteria forTraditional and NewApplications Research Signpost B Mayo PLopez and G P Martinez Eds pp 65ndash88 Kerala India 2008

[39] B Wei S Shin D Laporte A J Wolfe and T Romeo ldquoGlobalregulatory mutations in csrA and rpoS cause severe centralcarbon stress in Escherichia coli in the presence of acetaterdquoJournal of Bacteriology vol 182 no 6 pp 1632ndash1640 2000

[40] H Eoh andKY Rhee ldquoMultifunctional essentiality of succinatemetabolism in adaptation to hypoxia in Mycobacterium tuber-culosisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 110 no 16 pp 6554ndash6559 2013

[41] D J Richardson J-M Wehrfritz A Keech et al ldquoThediversity of redox proteins involved in bacterial heterotrophicnitrification and aerobic denitrificationrdquo Biochemical SocietyTransactions vol 26 no 3 pp 401ndash408 1998

[42] L D Kuykendall ldquoOrder VI Rhizobiales ord novrdquo in BergeyrsquosManual of Systematic Bacteriology (the Proteobacteria) PartC (the Alpha- Beta- Delta- and Epsilonproteobacteria) D JBrenner N R Krieg J T Staley and G M Garrity Eds vol2 pp 324ndash340 2nd edition 2005

[43] K S Bell J C Philp D W J Aw and N Christofi ldquoA reviewthe genusRhodococcusrdquo Journal of AppliedMicrobiology vol 85no 2 pp 195ndash210 1998

[44] H J Busse and G Auling ldquoGenus I Alcaligenesrdquo in BergeyrsquosManual of Systematic Bacteriology (the Proteobacteria) PartC (the Alpha- Beta- Delta- and Epsilonproteobacteria) D JBrenner N R Krieg J T Staley and G M Garrity Eds vol2 pp 653ndash658 2nd edition 2005

[45] K Towner ldquoThe genus Acinetobacterrdquo in The Prokaryotes AHandbook on the Biology of Bacteria Proteobacteria GammaSubclass M Dworkin S Falkow E Rosenberg K H Schleiferand E Stackebrandt Eds vol 6 pp 746ndash758 3rd edition 2006

[46] J P Bowman and T A Mcmeekin ldquoGenus VIIMarinobacterrdquoin Bergeyrsquos Manual of Systematic Bacteriology (the Proteobac-teria) Part B (the Gammaproteobacteria) D J Brenner N RKrieg J T Staley and G M Garrity Eds vol 2 pp 459ndash4642nd edition 2005

[47] R A Slepecky and H E Hemphill ldquoThe genus Bacillus-Nonmedicalrdquo in The Prokaryotes A Handbook on the Biologyof Bacteria Bacteria Firmicutes Cyanobacteria M DworkinS Falkow E Rosenberg K H Schleifer and E StackebrandtEds vol 4 pp 530ndash562 3rd edition 2006

[48] P A D Grimont and F Grimont ldquoGenus XVI Klebsiellardquo inBergeyrsquos Manual of Systematic Bacteriology (the Proteobacteria)Part B (the Gammaproteobacteria) D J Brenner N R KriegJ T Staley and G M Garrity Eds vol 2 pp 685ndash693 2ndedition 2005

[49] K Cho D X Nguyen S Lee and S Hwang ldquoUse of real-time QPCR in biokinetics and modeling of two differentammonia-oxidizing bacteria growing simultaneouslyrdquo Journalof Industrial Microbiology and Biotechnology vol 40 pp 1015ndash1022 2013

[50] A Pala and O Bolukbas ldquoEvaluation of kinetic parameters forbiological CNP removal from a municipal wastewater throughbatch testsrdquo Process Biochemistry vol 40 no 2 pp 629ndash6352005

[51] P Vandamme J-F Bernardet P Segers K Kersters and BHolmes ldquoNew perspectives in the classification of the flavobac-teria description of Chryseobacterium gen nov Bergeyella gen

nov and Empedobacter nom revrdquo International Journal ofSystematic Bacteriology vol 44 no 4 pp 827ndash831 1994

[52] J-F Bernardet Y Nakagawa B Holmes et al ldquoProposed min-imal standards for describing new taxa of the family Flavobac-teriaceae and emended description of the familyrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 52 no3 pp 1049ndash1070 2002

[53] J-H Yoon S-J Kang and T-K Oh ldquoChryseobacteriumdaeguense sp nov isolated from wastewater of a textile dyeworksrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 57 no 6 pp 1355ndash1359 2007

[54] S Szoboszlay B Atzel J Kukolya et al ldquoChryseobacteriumhungaricum sp nov isolated from hydrocarbon-contaminatedsoilrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 58 no 12 pp 2748ndash2754 2008

[55] C-C Young P Kampfer F-T Shen W-A Lai and A BArun ldquoChryseobacterium formosense sp nov isolated from therhizosphere of Lactuca sativa L (garden lettuce)rdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 55 no1 pp 423ndash426 2005

[56] A F Yassin H Hupfer C Siering and H-J Busse ldquoChry-seobacterium treverense sp nov isolated from a human clinicalsourcerdquo International Journal of Systematic and EvolutionaryMicrobiology vol 60 no 9 pp 1993ndash1998 2010

[57] H de Beer C J Hugo P J Jooste et al ldquoChryseobacteriumvrystaatense sp nov isolated from raw chicken in a chicken-processing plantrdquo International Journal of Systematic and Evo-lutionary Microbiology vol 55 no 5 pp 2149ndash2153 2005

[58] H De Beer C J Hugo P J Jooste M Vancanneyt T Coenyeand P Vandamme ldquoChryseobacterium piscium sp nov isolatedfrom fish of the South Atlantic Ocean off South Africardquo Interna-tional Journal of Systematic and Evolutionary Microbiology vol56 no 6 pp 1317ndash1322 2006

[59] P Kampfer K Chandel G B K S Prasad Y S Shouche andV Veer ldquoChryseobacterium culicis sp nov isolated from themidgut of the mosquito Culex quinquefasciatusrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no10 pp 2387ndash2391 2010

[60] F Bajerski L Ganzert K Mangelsdorf L Padur A Lipski andD Wagner ldquoChryseobacterium frigidisoli sp nov a psychro-tolerant species of the family Flavobacteriaceae isolated fromsandy permafrost from a glacier forefieldrdquo International Journalof Systematic and Evolutionary Microbiology vol 63 no 7 pp2666ndash2671 2013

[61] P Herzog I Winkler D Wolking P Kampfer and A Lip-ski ldquoChryseobacterium ureilyticum sp nov Chryseobacteriumgambrini sp nov Chryseobacterium pallidum sp nov andChryseobacterium molle sp nov isolated from beer-bottlingplantsrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 58 no 1 pp 26ndash33 2008

[62] E Hantsis-Zacharov T Shaked Y Senderovich and MHalpern ldquoChryseobacterium oranimense sp nov a psychro-tolerant proteolytic and lipolytic bacterium isolated from rawcowrsquosmilkrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 58 no 11 pp 2635ndash2639 2008

[63] E Hantsis-Zacharov Y Senderovich and M Halpern ldquoChry-seobacterium bovis sp nov isolated from raw cowrsquos milkrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 58 no 4 pp 1024ndash1028 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


In contrast maintenance of a population balance is notnecessary in SND as the two reactions (nitrification anddenitrification) proceed under identical reaction conditionsThus SND is advantageous over PND Furthermore theSND process prevents accumulation of NO

119909

minus in the sys-tem [8] Discovery of several bacteria capable of carryingout SND has generated considerable scientific interest indeveloping efficient biological nitrogen removal process ina single reaction vessel [7] Examples of such organismsisolated from different sources include Agrobacterium sp [9]Rhodococcus sp [10] Alcaligenes faecalis [11] Diaphorobactersp [12] Bacillus subtilis [13] Bacillus methylotrophicus [14]Acinetobacter calcoaceticus [15] Marinobacter sp [7] andKlebsiella pneumoniae [16] Careful review of the literatureshows that any bacterium capable of performing SND hasnot been isolated from slaughterhouse wastewater It isfurther observed that there are gaps in understanding therole of microorganisms in the nitrogen removal processesoccurring in abattoir wastewater For example Yilmaz et al[8] reported that phosphate accumulating organisms (mainlyAccumulibacter spp) occurring in abattoir wastewater wereresponsible for the denitrification However microorganismsthat reduced nitrate to nitrite before Accumulibacter sppcould carry out denitrification from nitrite to nitrogen gascould not be identified by the authors

Thus investigations on isolation of nitrifying and denitri-fying bacteria from slaughterhouse wastewater establishingtheir taxonomical identity characterizing their metabolicpatterns and identifying their potential application in large-scale nitrogen removal processes are necessary In our pre-vious study [17] we isolated a microbe from slaughterhousewastewater and identified it as Achromobacter xylosoxidansThis bacterium previously known as a clinical isolate wasfor the first time demonstrated to have nitrification activityInterestingly anaerobic denitrification a property which isregarded as an intrinsic characteristic of the Achromobactergenus was conserved In continual pursuance of our searchfor novel nitrifiersdenitrifiers the present paper describesthe isolation of a new nitrifying-denitrifying bacterium capa-ble of performing SND from wastewater emanating from asmall-size rural slaughterhouse in India This communica-tion further reports its taxonomical identification nitrogenremoval pathway characterization and kinetics of substrateutilization and growth

2 Materials and Methods

21 Seed Acclimatization Sludge collected from the ponddischarging wastes of Ms Mokami Slaughterhouse situatedin Nazira South 24 Parganas West Bengal India wasacclimatized to the laboratory conditions by cultivation in anaerated vessel for three months as described [17]

22 Isolation of the Bacterium and Development of PureCulture Ten predominant nitrifying bacteria were isolatedfrom the seed acclimatization vessel as described [17]Nitrification activities and preliminary taxonomic char-acterization of the isolates are presented as Supplemen-tary File 1 in Supplementary Material available online at

httpdxdoiorg1011552014436056 Amongst them thebacterium showing maximum nitrification activity (strainR31) was selected for further studies

23 Molecular Phylogenetic Analysis of Strain R31 Extractionof DNA amplification by polymerase chain reaction (PCR)and sequencing of the 16S rRNA gene were outsourced toScigenome Labs Kochi India DNA was extracted fromthe liquid culture of R31 grown in Luria broth followingSambrook et al [19] Amplification of the 16S rRNA geneof strain R31 was done with forward primer 27F (51015840-AGAGTTTGATCMTGGCTCAG-31015840) and reverse primer 1492R(51015840-GGT TAC CTT GTT ACG ACT T-31015840) [20] PCR wasperformed in a 50 120583L reaction mixture containing 5 120583L of10x PCR buffer with 15mM MgCl

2 4 120583L of 10mM dNTP

50 pmol of each oligonucleotide primers and 1120583L of 3U TaqDNA polymerase The conditions of PCR were initial denat-uration of 5min at 94∘C subsequently 30 incubation cycleseach comprising 30 seconds denaturation at 94∘C 45 secannealing at 57∘C 15min elongation at 72∘C and a final7min elongation at 72∘C The amplicon was electrophoresedin a 1 agarose gel and visualized under UV light Next theamplicon was purified using Nucleospin purification column(Macherey-Nagel Germany) The amplicon was sequencedusing an Applied Biosystems (USA) ABi 3730XL GeneticAnalyzer The PCR primers used for amplification wereapplied for sequencing the amplicon Phylogenetic analysis ofthe 16S rRNA gene sequence was done according to Kundu etal [17]

24 Fatty Acid Methyl Ester (FAME) Analysis of Strain R31FAME analysis of the isolate was outsourced to the MicrobialType Culture Collection and Gene Bank (MTCC) Instituteof Microbial Technology Chandigarh India Analysis wasdone using standard gas chromatography and applying theSherlock MIS system

25 Determination of Physiological Characteristics of StrainR31 Physiological characteristics of the strain were deter-mined following the Bergeyrsquos manual [21] Presence of flag-ella anaerobic growth sodium chloride tolerance aesculinhydrolysis indole production catalase activity gelatin liq-uefaction Tween 80 hydrolysis Simmons citrate utilizationdeoxyribonuclease activity Voges-Proskauer test methyl redtest starch hydrolysis 120573-galactosidase activity urease testand tyrosine hydrolysis were carried out following the meth-ods described in Kundu et al [17] Nitrate reduction test wasperformed to determine whether the isolate (R31) elaboratedthe enzymes nitrate reductase and nitrite reductase Thesetwo enzymes catalyze two reactions required for convertingnitrate to the end product nitrogen gas Aliquot from a pureculture of the isolate R31 was aseptically inoculated to a steriletube containing nitrate brothwith an invertedDurhamrsquos tubeThe inoculated tube was incubated at 37∘C for 24 hoursFinally the reductions of nitrate and nitrite were ascertainedby addition of naphthylamine and sulphanilic acid solutionsto the nitrate broth followed by adding a small amount of zincdust [22] Acid production from various carbohydrates wasdone following Hinz et al [23] and utilization of sole carbon




2HPO414 KH

2PO46




2sdot2H2O


3sdot6H2O 071 (NH

4)6Mo7O24














119904) and maximum





10098

95

92

55

86

57

99

100

100

89

69

86

100

66

55

78 100lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

























01

AM11-6T (AB517714)

AG13T (EU336941)













4


002040608

112141618

0 8 16 24 32 40 48Time (h)

0102030405060708090100

Opt

ical

den

sity

at600

nm

COD

rem

oval

()


0

10

20

30

40

50

60

0 8 16 24 32 40 48Time (h)

02468101214

NH

4+-N

and

NO

2minus-N

and

NO

3minus-N

(mg

L)

hydr

oxyl

amin

e (m

gL)






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50Time (h)

NH

4+-N

(mg

L)

(b)




4






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)




4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash



4

+ndashNH2OHndashN




4


4


4


4


4


4


4


4



0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2HPO414 KH

2PO46




2sdot2H2O


3sdot6H2O 071 (NH

4)6Mo7O24














119904) and maximum





10098

95

92

55

86

57

99

100

100

89

69

86

100

66

55

78 100lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

























01

AM11-6T (AB517714)

AG13T (EU336941)













4


002040608

112141618

0 8 16 24 32 40 48Time (h)

0102030405060708090100

Opt

ical

den

sity

at600

nm

COD

rem

oval

()


0

10

20

30

40

50

60

0 8 16 24 32 40 48Time (h)

02468101214

NH

4+-N

and

NO

2minus-N

and

NO

3minus-N

(mg

L)

hydr

oxyl

amin

e (m

gL)






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50Time (h)

NH

4+-N

(mg

L)

(b)




4






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)




4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash



4

+ndashNH2OHndashN




4


4


4


4


4


4


4


4



0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10098

95

92

55

86

57

99

100

100

89

69

86

100

66

55

78 100lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

























01

AM11-6T (AB517714)

AG13T (EU336941)













4


002040608

112141618

0 8 16 24 32 40 48Time (h)

0102030405060708090100

Opt

ical

den

sity

at600

nm

COD

rem

oval

()


0

10

20

30

40

50

60

0 8 16 24 32 40 48Time (h)

02468101214

NH

4+-N

and

NO

2minus-N

and

NO

3minus-N

(mg

L)

hydr

oxyl

amin

e (m

gL)






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50Time (h)

NH

4+-N

(mg

L)

(b)




4






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)




4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash



4

+ndashNH2OHndashN




4


4


4


4


4


4


4


4



0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4


002040608

112141618

0 8 16 24 32 40 48Time (h)

0102030405060708090100

Opt

ical

den

sity

at600

nm

COD

rem

oval

()


0

10

20

30

40

50

60

0 8 16 24 32 40 48Time (h)

02468101214

NH

4+-N

and

NO

2minus-N

and

NO

3minus-N

(mg

L)

hydr

oxyl

amin

e (m

gL)






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50Time (h)

NH

4+-N

(mg

L)

(b)




4






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)




4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash



4

+ndashNH2OHndashN




4


4


4


4


4


4


4


4



0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50Time (h)

NH

4+-N

(mg

L)

(b)




4






0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)




4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash



4

+ndashNH2OHndashN




4


4


4


4


4


4


4


4



0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

orN

O2minus

-N o

rNO

3minus-N

(mg

L)

(b)




4

+ndashNH2OHndashNO

2

minusndashNO3

minusndashN2Ondash



4

+ndashNH2OHndashN




4


4


4


4


4


4


4


4



0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12

14

16

0 10 20 30 40 50Time (h)

Opt

ical

den

sity

at600

nm

(a)

0

10

20

30

40

50

60

0 10 20 30 40 50Time (h)

NH

4+-N

(mg

L)

(b)





119904 119896 119884 and 119896





4







08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


08

07

06

05

04

03

02

01

00 0001 0002 0003 0004 0005 0006

1U

(mg

of M

LVSS

day

mg

COD

)

y = 73804x + 03353

R2 = 09597

1S (1mgL of COD)

(a)

0077

0076

0075

0074

0073

0072

0071

007

00690 001 002 003 004 005

1S (1mgL of NH4+-N)

1U

(mg

of M

LVSS

day

mg

ofN

H4+

-N)

y = 0174x + 0067

R2 = 0998

(b)

1446

1444

1442

144

1438

1436

1434004 005 006 007 008 009 01


1U

(mg

of M

LVSS

day

mg

ofN

O3minus

-N)

y = 01898x + 14253

R2 = 09776

(c)

7

6

5

4

3

2

1

00 2 4 6 8 10 12


R2 = 09985

1120579

C(p

er d

ay)

y = 05421x minus 00338

(d)

7

6

5

4

3

2

1

00 5 10 15 20 25


R2 = 09952

1120579

C(p

er d

ay)

y = 02898x minus 00312

(e)

12

1

08

06

04

02

004 06 08 1 12 14

1120579

C(p

er d

ay)


R2 = 09998

y = 07856x minus 00361

(f)




Presentstudy

Kundu etal [17]


Presentstudy

Kundu etal [17]


Presentstudy





119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119889 and 119870




4 Conclusions


3







Acknowledgments


References




































2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























2production












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Simultaneous Heterotrophic …downloads.hindawi.com/journals/bmri/2014/436056.pdfSimultaneous Heterotrophic Nitrification and Aerobic Denitrification by ... tary File