Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.

11' 2#13

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Investigation of the Effect of Initial Biomass on Nitrate and Phosphate Removal from Synthetic Wastewater by Selected Bacteria Isolates
%laolu ()1' *+ or %,1- an. *.eri/e ,I1'2 1.)e art0ent of ,iological Sciences' 1an.0ar+ 2ni3ersit/' %0u *ran' 4wara State' Nigeria 2. )e art0ent of 5icro6iolog/' 7+iti State 2ni3ersit/' *.o 7+iti' 7+iti State' Nigeria - 8orres on.ing author9 a+ or.oghenero6or:l0u.e.u.ng The research is financed by the College of Science and Engineering, Landmark University, Omu Aran, K ara State, !igeria Abstract *lthough nitrogen an. hos horus are i0 ortant nutrients to the e;istence of li3ing organis0s' their resence in e;cessi3e a0ounts in wastewater coul. ha3e .etri0ental effects to hu0ans an. other li3ing organis0s. (he resent in3estigation was ai0e. at ascertaining the effect of initial 6io0ass concentration on the nutrient re0o3al efficienc/ of four 6acterial s ecies in s/nthetic wastewater un.er sha+ing flas+s con.itions. <our .ifferent initial 6io0asses =2.$1 ; 1#8 cfu>01' ".82 ; 1#8 cfu>01' 8.?3 ; 1#8 cfu>01 an. 1.16 ; 1#$ cfu>01 (Klebsiella s .!@ 6.31 ; 1#8 cfu>01' 1.26 ; 1#$ cfu>01' 1.8$ ; 1#$ cfu>01 an. 2."2 ; 1#$ cfu>01 ("seudomonas s .!@ 1.?" ; 1#8 cfu>01' 3.4$ ; 1#8 cfu>01' ".24 ; 1#8 cfu>01 an. 6.$8 ; 1#8 cfu>01 (Lysinibacillus s .!' an. ?.1 ; 1#8 cfu>01' 1.42 ; 1#$ cfu>01' 2.13 ; 1#$ cfu>01 an. 2.84 ; 1#$ cfu>01 (Sta#hylococcus s .!A were use. for the stu./. <or nutrient re0o3al stu.ies' the res ecti3e initial 6io0asses of the test isolates were inoculate. into flas+s containing the wastewater 0e.ia. *liBuot sa0 les were ta+en at the 6eginning of the stu./ an. e3er/ 24 h for $6 h for the esti0ation of growth rate' C' hos hate an. nitrate concentrations in the wastewater' using stan.ar. 0etho.s. (he results re3eale. onl/ slight hos hate .ecreases in the wastewater after the e; iration of incu6ation. *ll the test isolates showe. significant nitrate re0o3al a6ilit/ e;ce t the Lysinibacillus s . *fter $6 h incu6ation 68.36 to $#.6? D' $1.8# to $".2$ D' 3.2# to 11.48 D an. 86.?? to $4.33 D of nitrate was re0o3e. in the resence of the Klebsiella s ., "seudomonas s ., Lysinibacillus s . an. Sta#hylococcus s ., res ecti3el/. (he stu./ was a6le to re3eal the hos hate an. nitrate re0o3al a6ilit/ of the isolates at the .ifferent initial 6io0asses use. for the in3estigation. Keywords9 ,acteria' wastewater' nutrient re0o3al' hos hate' nitrate ! Introd"ction Nitrogen an. hos horus are +nown to 6e two the 0ost i0 ortant nutrients to li3ing organis0s@ howe3er an e;cess of these nutrients causes se3eral water-Bualit/ ro6le0s. So0e of these ro6le0s inclu.e the .egra.ation of aBuatic ha6itat' ollution of .rin+ing water an. the .e letion in the econo0ic an. recreational 3alue of water (2SES' 1$$6!. (he resence of these nutrients in high a0ounts in recei3ing water 6o.ies is also +nown to lea. to eutro hication. 7utro hication results 6ecause of the e; losi3e thri3e of hotos/nthetic algae' there6/ re.ucing the a0ount of .issol3e. o;/gen a3aila6le to other aBuatic organis0s' thus lea.ing to their .eath. (he resence of e;cessi3e a0ount of algae .ue to eutro hication coul. also lea. to the 0assi3e conta0ination of water .ue to the ro.uction of h/coto;ins. In wastewater treat0ent' nitrate an. hos hate are consi.ere. riorit/ targets for re0o3al (7P*' 2##2@ Far. et al.' 2##"@ Jalal et al.' 2#11!. (he ossi6le entr/ of nitrate an. hos hate ions into aBuatic en3iron0ent is through househol. sewage water an. in.ustrial effluents- articularl/ fertiliGer an. soa in.ustries. It is ostulate. that onl/ ortho hos hate can 6e che0icall/ reci itate.. )uring 6iological treat0ent' 0ost of the organic hos horus an. ol/ hos hates are con3erte. to the ortho hos hate for0 (4rishnaswa0/ et al.' 2#11!. (here is the in.ication that nitrate conta0ination is a glo6al ro6le0' which is re orte. to stan. as the secon. 0ost .angerous ollutant after estici.es. *t high concentrations' nitrate is a threat to infants an. other susce ti6le in.i3i.uals. ,esi.es' its carcinogenic effects ha3e also 6een re orte.. Si0ilarl/' hos hate' which in water is generall/ resent as ol/ hos hate an. ortho hos hate' is in.icate. to cause .igesti3e ro6le0s in hu0ans when resent in e;tre0el/ high concentrations. *t concentrations a6o3e 1.# 0g>1' it is in.icate. to interfere with coagulation in water treat0ent lants ()e6Ro/ et al.' 2#12!. 8on3entionall/' che0ical an. 6iological rocesses ha3e 6een e0 lo/e. for the re0o3al of nutrients fro0 wastewaters. ,ecause' 6iological treat0ent rocesses are +nown to 6e cost-effecti3e 0etho.s for wastewater' 0icro6ial strategies for the re0o3al of en3iron0ental ollutants fro0 waste strea0s or conta0inate. sites can ro3i.e attracti3e alternati3es to other tra.itional 0etho.s (4rishnaswa0/ et al.' 2#11!. In recent /ears' 6ecause

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.11' 2#13

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of the 0an/ negati3e i0 acts' such as increase. 3olu0e of slu.ge' ro.uction of slu.ge with oor settling an. .ewatering characteristics an. the .e ression of the C' 6iological treat0ent rocesses is 6eing a.3ocate. (Era/' 2##2@ *+ or' 2#11!. ,ecause 6iological nutrient re0o3al techniBues in3ol3ing the use of 0icroorganis0s ha3e shown to offer great otential in the re0o3al of nutrients fro0 wastewater@ it is a.3ocate. for use in recent ti0es. So0e of the a.3antages of 6iological treat0ent rocesses inclu.e' low o erating costs' eco-frien.l/ an. cost-effecti3e alternati3e of con3entional techniBues an. efficienc/ at lower le3els of conta0ination (Sri3asta3ia an. 5aHu0.er 2##8!. )uring treat0ent' the nutrients are use. 6/ the 0icroorganis0s for the construction of cell 0e06ranes' cellular 0aintenance' s/nthesis of nucleic aci.s an. che0ical energ/ transfer reactions within cells. In so0e instances' so0e of the nutrients are also store. for future use 6/ the cells (4rishnaswa0/ et al.' 2#11!. * nu06er of 0icroorganis0s (6acteria' fungi' algae an. rotoGoa! ha3e 6een i0 licate. in the re0e.iation of nitrogen an. hos horus ollute. wastewaters. It is howe3er argue. that the e;tent of re0o3al is .e en.ent on the net ro.uction of li3ing 6io0ass ((,itton' 1$$4@ Ra0otho+ang et al.' 2##6@ 56wele' 2##6' 4rishnaswa0/ et al.' 2#11!. (his stu./ was therefore ai0e. at in3estigating the nutrient re0o3al efficienc/ of four 6acterial s ecies ("seudomonas s ., Klebsiella s ., Lysinibacillus s $ an. Sta#hylococcus s .! using initial 6io0ass concentrations for inoculation. #! $aterials and $ethods 2.1 (est 6acterial s ecies <our 6acterial ("seudomonas s .$, Klebsiella s $, Lysinibacillus s $ an. Sta#hylococcus s $! were use. for this in3estigation. (he/ were o6taine. fro0 the 5icro6iolog/ )e art0ent of the 7+iti State 2ni3ersit/' *.o 7+iti' 7+iti State' Nigeria. 7ach of the test isolates were store. in nutrient agar slants an. incu6ate. at 4 o8 until nee.e.. Prior use' each isolate was first strea+e. in nutrient agar slants an. incu6ate. at 3? o8 for 24 h to ascertain their urit/' after which the isolates were su6-culture. into nutrient 6roth. <or each e; eri0ent' onl/ 18-24 h ol. 6roth cultures of the isolates were use. for nutrient re0o3al stu./. 2.2 S/nthetic wastewater (he co0 osition of the s/nthetic wastewater use. for the stu./ was as follows9 so.iu0 acetate (" g>1!' 0agnesiu0 sulfate (#." g>1!' otassiu0 nitrate (#.2 g>1!' otassiu0 .ih/.rogen hos hate (#." g>1!' 0eat e;tract (1 g>1!' e tone (1 g>1! so.iu0 chlori.e(#." g>1!. 7ach of the co0 onents of the s/nthetic wastewater was first .issol3e. se aratel/ in "# 01 of .eioniGe. water' 6efore 0i;ing with the other co0 onents an. 0a+ing u to the 0ar+. ,efore usage' the s/nthetic wastewater was first .is ense. in 2## 01 Buantit/ in 2"# 01 7rlen0e/erIs flas+s. *ll flas+s containing the s/nthetic wastewater were cotton- lugge. an. steriliGe. at 121o8 for 1"0in at 1" si in an autocla3e. *fter steriliGation' flas+s were first incu6ate. for 24 h to ascertain that there was no growth fro0 an/ conta0inant. 2.3 Nutrient re0o3al stu./ (o the sterile s/nthetic wastewater in 7rlen0e/erIs flas+s' a +nown o ulation of the res ecti3e isolates was inoculate.. In this stu./' four .ifferent initial 6io0asses =2.$1 ; 1#8 cfu>01' ".82 ; 1#8 cfu>01' 8.?3 ; 1#8 cfu>01 an. 1.16 ; 1#$ cfu>01 (Klebsiella s .!@ 6.31 ; 1#8 cfu>01' 1.26 ; 1#$ cfu>01' 1.8$ ; 1#$ cfu>01 an. 2."2 ; 1#$ cfu>01 ("seudomonas s .!@ 1.?" ; 1#8 cfu>01' 3.4$ ; 1#8 cfu>01' ".24 ; 1#8 cfu>01 an. 6.$8 ; 1#8 cfu>01 (Lysinibacillus s .!' an. ?.1 ; 1#8 cfu>01' 1.42 ; 1#$ cfu>01' 2.13 ; 1#$ cfu>01 an. 2.84 ; 1#$ cfu>01 (Sta#hylococcus s .!A were use. for in3estigation. (he esti0ation of the initial 6io0asses was carrie. out using stan.ar. lating techniBues (*PC*' 2#12!. *fter inoculation' the inoculate. flas+s were incu6ate. in a rotar/ sha+e at 12# re3olutions er 0inute (r 0! at 2" o8. I00e.iatel/ after inoculation (referre. to as # h in this stu./! an. e3er/ 24 h for $6 h' aliBuot sa0 les were ase ticall/ ta+en fro0 each flas+ for the esti0ation of the C' growth rate' hos hate an. nitrate concentrations in the wastewater' using stan.ar. roce.ures. In all cases' hos hate an. nitrate concentrations in the wastewater were anal/se. using the ascor6ic aci. 0etho. an. the salic/late 0etho.s' res ecti3el/ (*PC*' 2#12!. *ll e; eri0ent anal/ses were carrie. out in tri licate. *lso' all reagents use. were of anal/tical gra.es. In all cases' an uninoculate. control was also set u for each 6atch of e; eri0ent. 2.4 Statistical anal/sis <or statistical anal/sis' the P*S( ( aleontological statistics software ac+age for e.ucation an. .ata anal/sis! was use. (Ca00er et al.' 2##1!. (he test for the co0 arison of 0eans was .one using the one-wa/ anal/sis of 3ariance (*N%&*! test while the tests for relationshi s were carrie. out using the Pearson correlation in.e;.

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.11' 2#13

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%! Res"lts 3.1 Phos hate re0o3al in resence of the test 6acterial s ecies <igure 1 shows the tren. in the hos hate concentration of the s/nthetic wastewater inoculate. with the Klebsiella s . *s re3eale. in the figure' there were slight .ecreases in the concentration of hos hate in the s/nthetic wastewater 6etween 24 h an. 48 h incu6ation erio.' after which increases were o6ser3e. with ti0e. (his tren. was irres ecti3e of the initial 6io0ass use. for in3estigation. *t the en. of the $6h incu6ation erio.' hos hate concentration in resence of the Klebsiella s . was o6ser3e. to change fro0 1"4.4#0g>1 at # h to 141.?#0g>1' 132.?#0g>1' 1"8.11 0g>1 132.?10g>1' at for 6io0ass concentrations at 2.$1 ; 1#8 cfu>01' ".82 ; 1#8 cfu>01' 8.?3 ; 1#8 cfu>01 an. 1.16 ; 1#$ cfu>01' res ecti3el/ (<ig. 1!. )es ite the o6ser3e. .ifferences in the hos hate concentrations at the .ifferent initial 6io0asses .uring the incu6ation erio.' these .ifferences were not o6ser3e. to 6e significant ( J #.#"!.

<ig. 19 &ariation in hos hate concentration in resence of the .ifferent initial 6io0asses of the Klebsiella s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at 2.$1 ; 1#8 cfu>01' ".82 ; 1#8 cfu>01' 8.?3 ; 1#8 cfu>01 an. 1.16 ; 1#$ cfu>01' res ecti3el/! Phos hate le3els in the wastewater in resence of the "seudomonas s . showe. stea./ .ecreases in concentration 6etween #h an. 48h incu6ation' after which there were stea./ increases with ti0e. (his tren. was irres ecti3e of the initial 6io0ass use. for inoculation. *t the en. of the $6 h incu6ation ti0e' the concentration of hos hate at initial 6io0ass of 6.31 ; 1#8 cfu>01 was o6ser3e. to .ecrease fro0 1"4.40g>1 to 144.?0g>1.*t initial 6io0asses of 1.26 ; 1#$ cfu>01' 1.8$ ; 1#$ cfu>01 an. 2."2 ; 1#$ cfu>01' hos hate concentrations were o6ser3e. e; eriences increases fro0 1"4.4# 0g>1 at # h to 168.?3 0g>1' 1"8.4" 0g>1 an. 1?1.?3 0g>1 (<ig. 2!' res ecti3el/. (hroughout the erio. of incu6ation there were no significant .ecreases in hos hate concentrations in the wastewater at the .ifferent initial 6io0asses. *lso' .es ite the .ifferences in hos hate concentrations at the .ifferent initial 6io0asses' these .ifferences were not o6ser3e. to 6e significant ( J #.#"!.

<ig. 29 &ariation in hos hate concentration in resence of the .ifferent initial 6io0asses of the "seudomonas s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at.6.31 ; 1#8 cfu>01' 1.26 ; 1#$ cfu>01' 1.8$ ; 1#$ cfu>01 an. 2."2 ; 1#$ cfu>01' res ecti3el/!

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In the resence of the Lysinibacillus s .' onl/ slight .ecreases in hos hate concentration were o6ser3e. with the first 48 h of incu6ation' after which there were stea./ increases in concentration with ti0e. (his tren. was irres ecti3e of the .ifferent initial 6io0asses. *t the e; iration of the $6 h incu6ation erio.' hos hate concentration increase. fro0 initial le3el of 1"4.4# 0g>1 to 164."? 0g>1' 1"4."# 0g>1' 182.#2 0g>1 an. 1$6.?# 0g>1' at initial 6io0asses of 1.?" ; 1#8 cfu>01' 3.4$ ; 1#8 cfu>01' ".24 ; 1#8 cfu>01 an. 6.$8 ; 1#8 cfu>01' res ecti3el/ (<ig. 3!. (hroughout the erio. of incu6ation' no significant .ecrease in hos hate concentration was o6ser3e. at the .ifferent initial 6io0asses. *lso' the hos hate concentration in the wastewater was not o6ser3e. to .iffer significantl/ 6etween the .ifferent initial 6io0asses ( J #.#"!. (he tren. in hos hate concentration in the wastewater in the resence of the Sta#hylococcus s showe. onl/ slight .ecreases within the first 48 h incu6ation' after which there were stea./ increases with incu6ation ti0e. (his tren. was irres ecti3e of the initial 6io0ass use. for inoculation. *fter the e; iration of the $6 h incu6ation erio.' hos hate concentration was foun. to 6e 1"$.?4 0g>1' 1"#.2# 0g>1' 141.11 0g>1 an. 141.?# 0g>1' at initial 6io0asses of ?.1 ; 1#8 cfu>01' 1.42 ; 1#$ cfu>01' 2.13 ; 1#$ cfu>01 an. 2.84 ; 1#$ cfu>01' res ecti3el/ (<ig. 4!. (hroughout the erio. of incu6ation' hos hate le3els at the .ifferent initial 6io0asses .i. not .iffer significantl/. *lso' no significant .ecrease in hos hate concentration was o6ser3e. at the .ifferent initial 6io0asses ( J #.#"!.

<ig. 39 &ariation in hos hate concentration in resence of the .ifferent initial 6io0asses of the Lysinibacillus s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at1.?" ; 1#8 cfu>01' 3.4$ ; 1#8 cfu>01' ".24 ; 1#8 cfu>01 an. 6.$8 ; 1#8 cfu>01' res ecti3el/!

<ig. 49 &ariation in hos hate concentration in resence of the .ifferent initial 6io0asses of the Sta#hylococcus s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at?.1 ; 1#8 cfu>01' 1.42 ; 1#$ cfu>01' 2.13 ; 1#$ cfu>01 an. 2.84 ; 1#$ cfu>01' res ecti3el/! Eenerall/' the results re3eale. that after the $6 h incu6ation erio.' hos hate re0o3al rates that range. fro0 8.23 D to 14.#" D an. fro0 2.?2 D to 8.61 D were o6ser3e. in the resence of the Klebsiella s . an.

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Sta#hylococcus s .' res ecti3el/. In the resence of the "seudomonas s . an. Lysinibacillus s .' hos hate increases that range. fro0 2.62 D to 1?.?# D an. fro0 #.#6 D to 2?.3$ D' res ecti3el/ were o6ser3e. after the $6 h incu6ation erio. ((a6le 1!. &able ' (hange in phosphate concentration in the wastewater after )* h inc"bation in the presence of the different isolates at the different initial biomass concentrations Initial P+,%- .mg/01 2inal P+,%-/0 .mg/01 3 change Klebsiella sp! * 1"4.4# 141.?# 8.22 , 1"4.4# 13?.?# 1#.82 8 1"4.4# 1"8.11 -2.4# ) 1"4.4# 132.?1 14.#" Pseudomonas sp! * 1"4.4# 144.6? 6.3# , 1"4.4# 168.?3 -$.28 8 1"4.4# 1"8.4" -2.62 ) 1"4.4# 181.?3 -1?.?# Lysinibacillus sp! * 1"4.4# 16"."? -?.23 , 1"4.4# 1"4."# -#.#6 8 1"4.4# 182.#2 -1?.8$ ) 1"4.4# 1$6.?# -2?.3$ Staphylococcus sp! * 1"4.4# 1"$.?4 -3.46 , 1"4.4# 1"#.2# 2.?2 8 1"4.4# 141.11 8.61 ) 1"4.4# 141.?# 8.22 *ll 3alues are in ercentages. Positi3e an. Negati3e 3alues re resent ercentage .ecreases an. increases' res ecti3el/. *' ,' 8 an. ) re resent the .ifferent initial 6io0asses of the test 6acterial s ecies use. for inoculation 3.2 Nitrate re0o3al in resence of the test 6acterial s ecies *s re3eale. in figure "' nitrate concentration in the resence of the Klebsiella s ' significant .ecreases in concentrations were o6ser3e. with ti0e. (his tren. was irres ecti3e of the initial 6io0ass use.. *t the e; iration of the $6 h incu6ation erio.' the concentrations of nitrate at initial 6io0asses of 2.$1 ; 1#8 cfu>01' ".82 ; 1#8 cfu>01' 8.?3 ; 1#8 cfu>01 an. 1.16 ; 1#$ cfu>01 were foun. 223.110g>1 "2.1#0g>1 at 2#.830g>1 1?.320g>1 an. ?#."$ 0g>1' res ecti3el/ (<ig. "!. *lthough significant nitrate re0o3al was o6ser3e. at the .ifferent initial 6io0asses' no .ifferences were o6ser3e. 6etween nitrate concentrations at the .ifferent 6io0asses ( J #.#"!.

<ig. "9 &ariation in nitrate concentration in resence of the .ifferent initial 6io0asses of the Klebsiella s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at 2.$1 ; 1#8 cfu>01' ".82 ; 1#8 cfu>01' 8.?3 ; 1#8 cfu>01 an. 1.16 ; 1#$ cfu>01' res ecti3el/!

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(he tren. in nitrate concentration in the resence of "seudomonas s . at the 3arious 6io0ass concentrations re3eale. a gra.ual .ecrease in concentration within the first 24 h of incu6ation after which there were shar .ecreases with ti0e after 48 h incu6ation' which was 0aintaine. throughout the $6 h incu6ation erio.. *fter the $6 h incu6ation erio.' nitrate concentration was o6ser3e. to .ecrease fro0 223.11 0g>1 at # h to 8.3#0g' 1#."10g>1' 11.16 0g>1' 11.68 0g>1' at initial 6io0asses 6.31 ; 1#8 cfu>01' 1.26 ; 1#$ cfu>01' 1.8$ ; 1#$ cfu>01 an. 2."2 ; 1#$ cfu>01' res ecti3el/' (<ig. 6!. *t the en. of incu6ation' the .ecreases in nitrate concentration in the resence of the "seudomonas s . were o6ser3e. to 6e significant. Cowe3er there were no significant .ifferences 6etween .ecreases at the .ifferent initial 6io0asses ( J #.#"!.

<ig. 69 &ariation in nitrate concentration in resence of the .ifferent initial 6io0asses of the "seudomonas s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at6.31 ; 1#8 cfu>01' 1.26 ; 1#$ cfu>01' 1.8$ ; 1#$ cfu>01 an. 2."2 ; 1#$ cfu>01' res ecti3el/! *s shown in figure ?' throughout the erio. of incu6ation' nitrate concentration in resence of the Lysinibacillus s . was not o6ser3e. to show an/ significant .ecrease in concentration. *fter the $6 h incu6ation erio.' the concentration of nitrate was o6ser3e. to 6e 21".$8 0g>1' 2#".?$ 0g>1' 1$?.4? 0g>1 an. 1$8.14 0g>1 at initial 6io0asses 1.?" ; 1#8 cfu>01' 3.4$ ; 1#8 cfu>01' ".24 ; 1#8 cfu>01 an. 6.$8 ; 1#8 cfu>01' res ecti3el/ (<ig. ?!. *s was o6ser3e. in the case of hos hate' throughout the erio. of incu6ation' no significant .ifferences were o6ser3e. 6etween the nitrate le3els at the .ifferent initial 6io0asses ( J #.#"!. In the resence of the Sta#hylococcus s .' there was onl/ a slight re.uction in nitrate concentration within the first 24 h of incu6ation' after which there was a .rastic re.uction with ti0e' a tren. that continue. till the en. of the incu6ation erio.. (his o6ser3ation was irres ecti3e of the initial 6io0ass use. for inoculation. Nitrate concentration in the wastewater after the $6 h incu6ation erio. was foun. to .ecrease fro0 223.11 0g>1 at # h to 21."4 0g>1' 2$."2 0g>1' 1".$6 0g>1 an. 12.6" 0g>1' at initial 6io0asses of ?.1 ; 1#8 cfu>01' 1.42 ; 1#$ cfu>01' 2.13 ; 1#$ cfu>01 an. 2.84 ; 1#$ cfu>01' res ecti3el/ (<ig. 8!. *lthough significant .ecrease in nitrate concentration was o6ser3e. at the .ifferent initial 6io0asses' these .ifferences were not o6ser3e. to .iffer 6etween the .ifferent initial 6io0asses use. for inoculation ( J #.#"!.

<ig. ?9 &ariation in nitrate concentration in resence of the .ifferent initial 6io0asses of the Lysinibacillus s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at1.?" ; 1#8 cfu>01' 3.4$ ; 1#8 cfu>01' ".24 ; 1#8 cfu>01 an. 6.$8 ; 1#8 cfu>01' res ecti3el/!

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.11' 2#13

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<ig. 89 &ariation in hos hate concentration in resence of the .ifferent initial 6io0asses of the Sta#hylococcus s . (*' ,' 8 an. ) re resent initial 6io0ass concentrations at?.1 ; 1#8 cfu>01' 1.42 ; 1#$ cfu>01' 2.13 ; 1#$ cfu>01 an. 2.84 ; 1#$ cfu>01' res ecti3el/! *fter the $6 incu6ation erio.' nitrate concentration in the wastewater in the resence of the test isolates showe. re0o3al rates that range. fro0 68.36D to $2.24 D' fro0 $1.8# D to $".2$ D' fro0 3.2# D to 11.4$ D an. fro0 86.?? D to $4.33 D' at the .ifferent initial 6io0asses of the Klebsiella s .' "seudomonas s .' Lysinibacillus s . an. Sta#hylococcus s .' res ecti3el/ ((a6le 2!. &able #' (hange in nitrate concentration in the wastewater after )* h inc"bation in the presence of the different isolates at the different initial biomass concentrations Initial N+%- .mg/01 2inal N+%-/0 .mg/01 3 change Klebsiella s . * 223.11 "2.1# ?6.6" , 223.11 2#.83 $#.6? 8 223.11 1?.32 $2.24 ) 223.11 ?#."$ 68.36 "seudomonas s . * 223.11 18.3# $1.8# , 223.11 1#."1 $".2$ 8 223.11 11.16 $".## ) 223.11 11.68 $4.?? Lysinibacillus s . * 223.11 21".$8 3.2# , 223.11 2#".?$ ?.?6 8 223.11 1$?.4$ 11.4$ ) 223.11 1$8.14 11.2# Sta#hylococcus s . * 223.11 21."4 $#.3" , 223.11 2$."2 86.?? 8 223.11 1".$6 $2.8" ) 223.11 12.6" $4.33 *ll 3alues re resent ercentage .ecreases. *' ,' 8 an. ) re resent the .ifferent initial 6io0asses of the test 6acterial s ecies use. for inoculation 3.3 C an. growth rates in resence of the test 6acterial s ecies *s shown in (a6le 3' in the resence of the test isolates' C of the wastewater was o6ser3e. to increase gra.uall/ with incu6ation ti0e. )es ite the o6ser3e. increases with ti0e' the 3alues were still the near neutral ranges. (his tren. was irres ecti3e of the test isolates or the initial 6io0asses use. for inoculation. *t the en. of the $6 h incu6ation erio.' fro0 the original 3alue of ".$ at # h' C 3alues at the .ifferent initial 6io0asses were o6ser3e. to range fro0 ?.1 to ?.3' ?.# to ?.8' ?.3 to ?6." an. 6.8 to ?."' in the resence of the Klebsiella s .' "seudomonas s .' Lysinibacillus s . an. Sta#hylococcus s .' res ecti3el/ ((a6le 3!.

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.11' 2#13

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&able %' p4 variations d"ring the n"trient removal st"dies in presence of the test isolates at the different initial biomasses "sed for inoc"lation &ime A B ( 5 Klebsiella sp! #h ".$ ".$ ".$ ".$ 24h 6.3 6.4 6.4 6.4 48h 6." 6.$ 6.$ 6.$ ?2h 6.$ ?.4 ?.4 ?.3 $6h ?.1 ?.3 ?.3 ?.3 Pseudomonas sp! A B ( 5 #h ".$ ".$ ".$ ".$ 24h 6.4 6.3 6.3 6.4! 48h ?.# ?.# ?.# ?.# ?2h ?.? ?." ?." ?.6 $6h ?.# ?.6 ?.6 ?.8 Lysinibacillus sp! A B ( 5 #h ".$ ".$ ".$ ".$ 24h 6.4 6.3 6.3 6.4 48h 6.8 6.? 6.? ?.# ?2h ?.2 ?.1 ?.1 ?.4 $6h ?.3 ?.2 ?.2 ?." Staphylococcus sp! A B ( 5 #h ".$ ".$ ".$ ".$ 24h 6.2 6.2 6.2 6.4 48h 6.4 6.4 6.4 ?.# ?2h 6.6 6." 6." ?.4 $6h 6.8 6.6 6.6 ?." *ll 3alues are a3erage of tri licate anal/sis. *' ,' 8 an. ) re resent the .ifferent initial 6io0asses of the test 6acterial s ecies use. for inoculation Fith res ect to growth rates of the isolates .uring the nutrient re0o3al stu.ies' there were stea./ increases with ti0e. (his tren. was irres ecti3e of the test isolates or the initial 6io0ass use. for inoculation. *t the en. of the $6 h incu6ation' growth rates of the isolates at the res ecti3e initial 6io0asses were o6ser3e. to range fro0 1.$? .-1 to 1.$8-1' 1.$6 to 2.## .-1' 1.$? to 2.#8 .-1 an. fro0 1.$8 .-1 to 2.1" .-1' for the Klebsiella s .' "seudomonas s .' Lysinibacillus s . an. Sta#hylococcus s .' res ecti3el/ ((a6le 4!.

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.11' 2#13

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&able %' 6rowth rate .d- 1 of the different isolates d"ring the n"trient removal st"dies at the different initial biomass concentrations 7h #,h ,8h 9#h )*h Klebsiella sp! * # 1."4 1."" 1.$6 1.$? , # 1."4 1.$" 1.$? 1.$$ 8 # 1.$4 1.$" 1.$? 1.$8 ) # 1.$4 1.$6 1.$? 1.$8 Pseudomonas sp! * # 1.$4 1.$6 1.$$ 2.## , # 1."4 1.$6 1.$8 1.$$ 8 # 1.$4 1.$" 1.$6 1.$6 ) # 1.$" 1.$6 1.$$ 2.## Lysinibacillus sp! * # 1.68 2.#4 2.#8 2.#8 , # 1.$4 1.$" 1.$6 1.$? 8 # 1.$? 1.$6 1.$? 1.$? ) # 1.$4 1.$6 1.$? 1.$8 Staphylococcus sp! * # 1.$4 1.$" 1.$? 1.$8 , # 1.?" 1.84 1.$4 1.$$ 8 # 1.84 1.$3 2.#1 2.1" ) # 1.8" 1.$4 2.## 2.#8 *ll 3alues are a3erage of tri licate anal/sis. *' ,' 8 an. ) re resent the .ifferent initial 6io0asses of the test 6acterial s ecies use. for inoculation ,! 5isc"ssion (he resent stu./ re3eale. slight hos hate re0o3al in the resence of Klebsiella s .' "seudomonas s . an. Sta#hylococcus s . In the resence of the Lysinibacillus s . hos hate releases were o6ser3e. at the 3arious initial 6io0asses use. for inoculation. So0e s ecies of Klebsiella s . an. "seudomonas s . ha3e 6een i0 licate. in hos hate re0o3al 6/ re3ious in3estigators (5o06a an. 8loete' 1$$6@ 4rishnaswa0/ et al.' 2#11@ )ie an. 8uc' 2#13!. (he tren. in C in this in3estigation showe. slight increases with ti0e' although all 3alues were within the near neutral ranges. Increases in C .uring nutrient stu.ies ha3e 6een re orte. 6/ so0e in3estigators (8he3alier et al.' 2###@ *+ or et al.' 2##?!. *ccor.ing to *+ or an. co-wor+ers (2##?!' .uring nutrient re0o3al in acti3ate. slu.ge 0i;e. liBuor using rotoGoan isolates' at the en. of the incu6ation erio.' C was o6ser3e. to increase fro0 an a3erage of ?." to 8. (he fin.ings of this stu./ negate. the o6ser3ation of so0e other in3estigators. In the stu./ 6/ 4rishnaswa0/ et al.' (2#11!' a hos hate re0o3al of $2."D was o6taine. with a C change of the culture 0e.iu0 fro0 ?.2 to ".$. (he/ ostulate. that the re.uction in C 0a/ 6e .ue to the ro.uction of 3arious organic aci.s 6/ the hos hate re.ucers in the culture 0e.iu0. It is re orte. that aci.ic C is fa3oura6le for hos hate re0o3al' which is 6ase. on the suggestion that aci.ic C see0s to 6e fa3oura6le to aci. hos hatase' which ai.s in the re0o3al of hos hate (,ouBuet et al.' 1$8?@ 4rishnaswa0/ et al.' 2#11!. 5ulan an. co-wor+ers (2##2! ha3e also re orte. an enhance. hos hate u ta+e an. ol/ hos hate accu0ulation in %urkholderia ce#acia un.er aci.ic con.itions. It is re orte. that .uring nutrient re0o3al' the initial C at neutral an. aci.ic con.itions is fa3oura6le for the o ti0u0 re0o3al of hos hate 6/ the in.i3i.ual strain of "seudomonas s . an. consortiu0 (4rishnaswa0/ et al.' 2#11!. So0e wor+ers ha3e also in.icate. si0ilar o6ser3ations of hos hate utiliGing 0icroorganis0s ro.ucing 3arious organics an. conseBuentl/ fall in C of the 0e.iu0 (4un.u' 1$84@ Satar an. Eaur 1$84!. None of the isolates use. in this stu./ showe. significantl/ hos hate re0o3al a6ilit/ .uring the e; eri0ental con.itions. (he 0aHorit/ of the isolates use. for this in3estigation showe. significant nitrate re0o3al efficienc/. It is howe3er in.icate. that .uring .enitrification rocess' al+alinit/ is ro.uce. (,itton' 1$$$!. (he resent stu./ re3eale. significant nitrate re0o3al in the resence of "seudomonas, Klebsiella an. Sta#hylococcus. *ccor.ing to )ie an. 8uc' (2#13!' "seudomonas stut&eri is sai. to ha3e e;cellent .enitrif/ing a6ilit/ un.er aero6ic an. anaero6ic con.itions. *ll the isolates use. in stu./ were in3estigate. un.er aero6ic con.ition. In the stu./ 6/ &/0aGal' (2##?!' .i3erse 6acteria grou s ha3e 6een o6ser3e. to 6e in3ol3e. in nitrogen 0ineralisation' such as nitrification an. .enitrification. *lthough .enitrif/ing 6acteria are sai. to inclu.e organotro hs' lithotro hs' hototro hs an. .iaGotro hs' the 0aHorit/ of the0 are sai. to 6e che0otro hs.

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.11' 2#13

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In aBuatic en3iron0ents' the 0ost i0 ortant .enitrif/ing 6acteria are i0 licate. to 6e "seudomonas, Aeromonas and 'ibrio. %ther 6acteria' such as Arthrobacter globiformis, Aerobacter aerogenes, (ycobacterium #hlei, Stre#tomyces griseus, Thios#haera an. Corynebacterium ha3e also 6een i0 licate. for nitrogen 0ineralisation (&/0aGal ' 2##?@ )ie an. 8uc' 2#13!. . :! (oncl"sion (his stu./ re3eale. that three of the isolates ("seudomonas, Klebsiella an. Sta#hylococcus! ha3e significant nitrate re0o3al a6ilit/ un.er the e; eri0ental con.itions in3estigate.. In a..ition' although there were onl/ slight .ecreases in hos hate concentration in the wastewater in resence of the "seudomonas s ., Klebsiella s an. Sta#hylococcus s . (hese o6ser3ations were irres ecti3e of the res ecti3e initial 6io0asses use. for inoculation. No re0ar+a6le hos hate or nitrate re0o3al a6ilit/ was o6ser3e. in the resence of the Lysinibacillus s . (his stu./ also o6ser3e. consistent 6ut slight increases in C .uring nutrient re0o3al stu.ies. (his tren. was also irres ecti3e of the initial 6io0ass use. for inoculation. <inall/' the stu./ .i. not o6ser3e an/ relationshi 6etween nutrient re0o3al an. the initial 6io0asses use. for inoculation. Ac;nowledgements (he authors wish to than+ the 0anage0ent of 1an.0ar+ 2ni3ersit/' %0u *ran' 4wara State' Nigeria for ro3i.ing the resources an. facilities to carr/ out this in3estigation. (he contri6ution of the 5icro6iolog/ )e art0ent of the 7+iti State 2ni3ersit/' *.o 7+iti' Nigeria in the ro3ision of the isolates use. for the stu./ is also a reciate.. References *+ or' %.,.' 5o06a' 5.N.,. K %+on+wo J. (2##?!. Phos horus an. nitrate re0o3al 6/ selecte. wastewater rotoGoa isolates. "akistan )ournal of %iological Sciences' 1#(22!' 4##8-4#14 *PC* (2#12!' Standard (ethods for the E*amination of +ater and +aste ater' 22n. e.n. *0erican Pu6lic Cealth *ssociation' Fashington )8 ,itton E. (1$$4!. +aste ater (icrobiology. John File/ K Sons' Inc.' Pu6lication@ 2S* 8he3alier' R.' Proul;' ).' 1essar.' P.' &incent' F.<.K )e 1a Noue J. (2###!. Nitrogen an. hos horus re0o3al 6/ high latitu.e 0at-for0ing c/ano6acteria for otential use in tertiar/ wastewater treat0ent. )ournal of A##lied "hycology' 12' 1#"-112 )e6Ro/' S.' )as' S.' Ehosh' S. K ,anerHee' S. (2#12!. Isolation of nitrate an. hos hate re0o3ing 6acteria fro0 3arious en3iron0ental sites. Online )ournal of %iological Sciences' 12(2!' 62-?1 )ie ' 8.N. K 8uc' N.(. (2#13!' Ceterotro hic nitrogen re0o3al 6acteria in se.i0entar/an. water of stri e. catfish on.s in the 5e+ong )elta'&ietna0. American )ournal of Life Sciences' 1(1!' 6-13 7P*. (2##2!' %nsite wastewater treat0ent s/ste0s 0anual. E"A,-./,0122,223,.22.. htt 9>> www.e a.go3>ow0itnet>0t6fact.ht0 Era/' <.N. (2##2!. +ater Technology4 An 5ntroduction for Environmental Scientists and Engineers. ,utterworthCeine0ann' %;for. 3"-8# Ca00er' %.' Car er' ).*.(. K R/an' P.). (2##1!. P*S(9 Paleontological statistics software ac+age for e.ucation an. .ata anal/sis. "alaeontologia Electronica' 4(1!' $. htt 9>> alaeoelectronica.org>2##1L1> ast>issue1L#1.ht0 Jalal' 4.8.*.' Mahangir *la0' 5.).' 5atin' F.*.' 4a0aruGGa0an' ,.N.' *+6ar' J. K (offaGel' C. (2#11!. Re0o3al of Nitrate an. Phos hate fro0 5unici al Fastewater Slu.ge 6/ Chlorella vulgaris' S#irulina #latensis an. Scenedesmus 6uadricauda$ 55U( Engineering )ournal' 12(4!' 12"-132 4rishnaswa0/' 2.' 5uthucha0/' 5. K Peru0alsa0/' 1. (2#11!. ,iological re0o3al of hos hate fro0 s/nthetic wastewater using 6acterial consortiu0. 5ranian )ournal of %iotechnology' $(1!' 3?-4$ 4un.u' ,.S. K Eaur' *.8. (1$84!. Rice res onse to inoculation with N-fi;ing an. -solu6iliGing 0icroorganis0. "lant Soil, ?$' 22?-234 56wele' 1. (2##6!. 5icro6ial hos horus re0o3al in wastewater sta6iliGation on.. A Licentiate Thesis from the School of %iotechnology9 * Ro/al Institute of (echnolog/' *l6ano3a' Stoc+hol0' Swe.en 5o06a' 5.N.,. K 8loete' (.7. (1$$6!. ,io0ass relationshi togrowth an. hos hate u ta+e of "seudomonas fluorescens'Escherichia coli an. Acinetobacter radioresistens in 0i;e. liBuor 0e.iu0. )ournal of 5ndustrial (icrobiology, 16' 364O36$ 5ullan' *.' Puinn' J.P. K 5cErath J.F. (2##2!' 7nhance. hos hate u ta+e an. ol/ hos hate accu0ulation in %urkholderia ce#acia grown un.er low- C con.itions. (icrobial Ecology. 44' 6$-?? Ra0otho+ang' (.R.' Si0elane' S.8. K ,u;' <. (2##6!. ,iological nitrogen an. hos horus re0o3al 6/ fila0entous 6acteria in ure culture. +ater 5nstitute of South Africa 7+5SA8 %iennial Conference' )ur6an' South *frica

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Satar' 5.*. K Eaur' *.8. (1$84!. Solu6iliGation of insolu6le an. fi;e. hos hates in cro rhiGo6iu0 6/ hos ho0icroorganis0s. Proc. Int. S/0 osiu0 on soil test cro res onses correlation stu.ies. 5nternational Congress (eeting of Soil Science' <e6. ?-1#' )ha+a' . 23?-2"1 2SES. (1$$6!' National Fater-Pualit/ *ssess0ent (NFP*! Progra0. US 9eological Survey :act Sheet' <S218-$6 &/0aGal' J. (2##?!. Re0o3al of nutrients in 3arious t/ es of constructe. wetlan.s. Science of the (otal 7n3iron0ent' 38#9 48O6" Far.' 5.C.' .e4o+' (.5.' 1e3allois' P.' ,ren.er' J.' Eulis' E..' Nolan' ,.(. K &an)erslice' J. (2##"!. For+grou re ort9 .rin+ing-water nitrate an. health-recent fin.ings an. research nee.s. 7n3iron0ental Cealth Pers ecti3es' 113(11!' 16#?-14

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