Relationship between population dynamics of nitrifiers in biofilms and reactor performance at various C:N ratios

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Per9am0n 0043-1354(95)00321-5

Wat. Re5. V01. 30, N0. 7, pp. 1563-1572, 1996 C0pyr19ht • 1996 E15ev1er5c1enceLtd Pr1nted 1n 6reat 8r1ta1n. A11r19ht5 re5erved 0043-1354/96 $15.00 + 0.00

RELA710N5H1P 8 E 7 W E E N P 0 P U L A 7 1 0 N DYNAM1C5 0 F N17R1F1ER5 1N 810F1LM5 A N D R E A C 7 0 R PERF0RMANCE A7 VAR10U5 C:N RA7105 5. 0 K A 8 E 1•*, Y. 0 0 2 A W A 2, K. H 1 R A 7 A 3 and Y. W A 7 A N A 8 E 1e ~Department 0f 5an1tary and Env1r0nmenta1 En91neer1n9, H0kka1d0 Un1ver51ty 5app0r0, 060, 2Department 0f C1v11 and Env1r0nmenta1 En91neer1n9, M1ya2ak1 Un1ver51ty M1ya2ak1, 889-21 and 3Pac1f1c C0n5u1tant5 C0. Ltd, 4-3-24 N15h1naka21ma, Y0d09awa-ku, 05aka, Japan (F1r5t rece1ved Apr11 1995; accepted 1n rev15ed f0rm Decem6er 1995) A1~tract--7he re1at10n5h1p 6etween t1me dependent p0pu1at10n dynam1c5 0f n1tr1f1er5 and heter0tr0ph5 1n undef1ned m1xed-p0pu1at10n 610f11m5 and the1r n1tr1f1cat10n eff1c1encywa5 exper1menta11y 1nve5t19ated at var10u5 C:N rat105 0f feed 501ut10n5. F1ve type5 0f 610f11m5 were cu1tured 1n part1a11y 5u6mer9ed r0tat1n9 6101091ca1 c0ntact0r5 (R8C•5) at d1fferent C: N rat105 and were u5ed a5 te5t mater1a15.7he re5u1t5 1nd1cated that 1n1t1a1 m1cr061a1 c0mp051t10n 1n the 610f11m5 and 5u65trate c0mp051t10n (e.9. C:N rat10) 5tr0n91y 1nf1uenced the 1ater p0pu1at10n dynam1c5 and the n1tr1f1cat10n eff1c1ency. H19her 1nf1uent C:N rat10 retarded accumu1at10n 0f n1tr1fy1n9 6acter1a, e5pec1a11y N02-0x1d12er5, re5u1t1n9 1n a c0n51dera61y 10n9 5tart-up per10d f0r c0mp1ete and 5ta61e n1tr1f1cat10n due t0 c0mpet1t10n f0r d15501ved 0xy9en and 5pace 1n the 610f11m. Furtherm0re, a 5tart-up 1n0cu1um wa5 very 1mp0rtant t0 keep 5tart-up t1me 0f n1tr1f1cat10n t0 a m1n1mum. 71me-dependent p0pu1at10n dynam1c5 1n the 610f11m5 ref1ected we11 the 6u1k water 4ua11ty and m1cr061a1 c0mmun1ty 5tructure 1n the 6u1k 114u1d. 7he5e re5u1t5 5u99e5t that the 5tructure 0f m1cr061a1 c0mmun1ty 1n the 610f11m can 6e pred1cted fr0m m0n1t0r1n9 the water 4ua11ty and m1cr0610109y 0f the 6u1k 114u1d. Phy5101091ca11y 1nact1ve ce1151n the 610f11m were determ1ned 6y an 1N7 dehydr09enea5e a55ay. 7he5e ceU5 9radua11y accumu1ated up t0 a60ut 30% 0f the t0ta1 6acter1a1 p0pu1at10n w1th1n the 610f11m5. 7he re5u1t5 0f th15 5tudy w111pr0v1de a rat10na1 6a515 f0r deve10p1n9 and c0ntr0111n9 de51red 610f11mp0pu1at10n dynam1c5 t0 max1m12e n1tr1f1cat10n eff1c1ency0f wa5tewater 610f11m5 C0pyr19ht • 1996 E15ev1er 5c1ence Ltd. Key w0rd5--610f11m, n1tr1f1cat10n, p0pu1at10n dynam1c5, heter0tr0ph5, n1tr1f1er5, 1N7 dehydr09ena5e a55ay

1N7R0DUC710N 810f11m 5y5tem5 have 6een u5ed f0r n1tr09en rem0va1 6ecau5e they a110w a 5uff1c1ent1y 10n9 610ma55 retent10n t1me t0 ach1eve re11a61e n1tr1f1cat10n (Watana6e et a1., 1992). U5ua11y, 5uch 610f11m5 are mu1t1-5pec1e5 610f11m5, d15p1ay1n9 c0n51dera61e heter09ene1ty, 60th w1th re5pect t0 the m1cr00r9an15m5 pre5ent (Ma5uda et a1., 1991; 2 h a n 9 et a1., 1993) and the1r phy51c0chem1ca1 m1cr0env1r0nment5 (de8eer et a1., 1994a, 6; 5t00d1ey et a1., 1994; 2 h a n 9 and 815h0p, 1994). 7he m1cr061a1 c0mp051t10n 0f aer061c n1tr1fy1n9 610f11m5 15 n0t fu11y under5t00d even th0u9h f0ur maj0r tr0ph1c 9r0up5 are c0mm0n1y c0n51dered a5 6e1n9 re5p0n5161e f0r c0mm0n d0me5t1c wa5tewater treatment (Ma5uda et a1., 1991; 2 h a n 9 et a1., 1993). 7he5e 9r0up5 are aer061c heter0tr0ph5, NH4-0x1d12er5, N02-0x1d12er5 and den1tr1f1er5. L1kew15e, mu1t1p1e e1ectr0n-d0n0r5 (e.9. 0r9an1c matter5 and amm0n1um) and, 50met1me5, e1ectr0n-accept0r5 (0xy9en and n1trate) are pre5ent 1n the wa5tewater5 treated 6y *Auth0r t0 wh0ma11 c0rre5p0ndence 5h0u1d 6e addre55ed (Fax: 011 706 7890).

6101091ca1 pr0ce55e5. 7he pre5ence 0f 0r9an1c 5u65trate5 1n the wa5tewater re5u1t5 1n c0mpet1t10n f0r d15501ved 0xy9en and 5pace 1n the 610f11m 6etween the n1tr1fy1n9 6acter1a and heter0tr0ph1c 6acter1a (Wanner and 6ujer, 1985, 1986; Furuma1 and R1ttmann, 1992; R1ttmann and Manem, 1992; 0 k a 6 e et a1., 1995). 1n th15 c0mpet1t10n, the m05t cr1t1ca1 tr0ph1c 9r0up5 are NH4-0x1d12er5 and N02-0x1d12er5 6ecau5e the1r 9r0wth rate and 9r0wth y1e1d are very 10w a5 c0mpared t0 th05e 0f 0ther 9r0up5. 7heref0re, even when n1tr1f1cat10n 15 de51red 1n a pr0ce55, the n1tr1fy1n9 6acter1a are u5ua11y 0utc0mpeted 6y the heter0tr0ph1c 6acter1a. 1nh161t10n 0r e11m1nat10n 0f n1tr1fy1n9 6acter1a 6y the5e 1nter5pec1e5 c0mpet1t10n u5ua11y 1ead5 t0 a decrea5e 1n n1tr1f1cat10n eff1c1ency 0r even t0 a fa11ure 0f the pr0ce55. 1n add1t10n, 0ne 0f the ma1n draw6ack5 c0mm0n t0 6101091ca1 n1tr1f1cat10n pr0ce55e5 15 the re4u1rement 0f a 10n9 5tart-up per10d. 7h15 15 pr06a61y 6ecau5e 0f 10w 9r0wth rate and 9r0wth y1e1d 0f n1tr1fy1n9 6acter1a and 1nter5pec1e5 c0mpet1t10n f0r d15501ved 0xy9en and 5pace 1n 610f11m5. 51ud9e fr0m mun1c1pa1 wa5tewater treatment p1ant5 15 c0mm0n1y u5ed t0 5tart up new 610f11m pr0ce55e5 a5 a 5eed1n9 mater1a1.

1563

5. 0ka6e et a1.

1564

EXPER1MEN7AL ME7H0D5

Rem0Va61e 511de 4 ~ .~6~Cm~.~1~

16m 0.5cm

1a,f11,,1,n11

[HU111H1UHU1[ Nt~6er 0f d15k :10 D15k d1ameter :16 crn D15k 5urface area :4019 Cm 2 React0r v01ume :2.65 L

1 ~I Re5erv01r

F19. 1. A 5chemat1c d1a9ram 0f a part1a11y 5u6mer9ed R8C 5y5tem.

7 0 0verc0me 5uch a 10n9 5tart-up per10d, def1ned 5tarter cu1ture5 mu5t 6e needed f0r n1tr1f1cat10n 1n wa5tewater treatment p1ant5. 5uch cu1ture5 are ava11a61e f0r var10u5 f00d pr0ce55e5 u5ed 1n the da1ry 1ndu5try (72en9, 1985), h0wever, they are 5carce f0r wa5tewater treatment (J0hn50n et a1., 1985). Rat10na1 610f11m cu1tur1n9 pr0cedure f0r de51red pr0ce55e5 (e.9. n1tr1f1cat10n) ha5 n0t 6een deve10ped yet due ma1n1y t0 a 1ack 0 f under5tand1n9 0 f m1cr061a1 p0pu1at10n dynam1c5 1n wa5tewater 610f11m5. 1t 15 de51ra61e t0 0pt1m12e the n1tr1f1cat10n pr0ce55 and, theref0re, 1mp0rtant t0 4uant1fy the p0pu1at10n dynam1c5 0f n1tr1fy1n9 and heter0tr0ph1c 6acter1a 1n the 610f11m under d1fferent nutr1ent c0nd1t10n5 (1.e. 5u65trate C : N rat10). 7 h e m1cr061a1 p0pu1at10n dynam1c5 1n 610f11m5 w111 6e h19h1y 1nf1uenced 6y the 1n1t1a1 m1cr061a1 c0mp051t10n and 5u65trate c0mp051t10n5. H0wever, n1tr1fy1n9 610f11m5 have 6een treated a5 ••a 61ack 60x•• 1n m05t 0f the prev10u5 re5earch 1n wa5tewater treatment f1e1d5, wh1ch ha5 h a m p e r e d the under5tand1n9 0f the c0rre1at10n 6etween p0pu1at10n dynam1c5 and react0r perf0rmance. 7 h e 90a1 0f th15 re5earch 15 t0 ev01ve the mean5 t0 under5tand pr0ce55e5 c0ntr0111n9 p0pu1at10n dynam1c5 0 f n1tr1f1er5 dur1n9 610f11m accumu1at10n t0 max1m12e the n1tr1f1cat10n eff1c1ency. 7 h e 5pec1f1c 06ject1ve5 0f the pre5ent w0rk are: (1) t0 def1ne the m1cr061a1 p0pu1at10n 1n n1tr1fy1n9 610f11m5 1n re1at10n t0 the m1cr061a1 p0pu1at10n and water 4ua11ty 1n the 6u1k 114u1d; (2) t0 e1uc1date the effect 0f car60n:n1tr09en rat10 ( C : N rat10) 1n cu1ture med1a 0n the m1cr061a1 p0pu1at10n dynam1c5; and (3) t0 determ1ne the effect 0f 5tart-up 1n0cu1um 0n the m1cr061a1 p0pu1at10n dynam1c5 and n1tr1f1cat10n eff1c1ency. 1nf0rmat10n 0n 610f11m p0pu1at10n dynam1c5 and fact0r5 affect1n9 p0pu1at10n dynam1c5 w111 pr0v1de a mean5 t0 deve10p and c0ntr01 de51red 610f11rn5 1n var1ety 0f wa5tewater treatment app11cat10n5.

AND M A 7 E R 1 A L 5

810f11m react0r A part1a11y 5u6mer9ed r0tat1n9 6101091ca1 c0ntact0r (R8C) u5ed 1n th15 5tudy wa5 a C0mp1ete1y m1xed react0r and c0n515ted 0f 10 p01y-methy1-methaCry1ate d15k5 (d1a = 16 Cm). A 5Chemat1C d1a9ram 0f the R8C react0r 15 111u5trated 1n F19. 1.7w0 1dent1Ca1 react0r5 were 0perated 1n para11e1 at d1fferent C : N rat105. F0ur rem0va61e 511de5were 1n5ta11ed 1n each d15k f0r 5amp11n9 610f11m5. 7emperature wa5 ma1nta1ned at 20°C. D15k r0tat10na1 5peed wa5 f1xed at 14rev m1n -~. A 6a5a1 nutr1ent med1um (C0mp051t10n 15 de5Cr16ed 6e10w) wa5 fed u51n9 a per15ta1t1C pump and 5peed c0ntr011er. A11 R8C react0r5 were 0perated at a c0n5tant d11ut10n rate 0f0.2 h -~ and a NH4-N hydrau11c 10ad1n9 rate 0f 0.91 9 NH4-N m -2 d 1. Exper1menta1 pr0cedure F1ve d1fferent type5 0f 610f11m5 were cu1tured w1th d1fferent 5tarter cu1ture5 and at var10u5 5u65trate C : N rat105.7he 610f11m cu1t1vat10n c0nd1t10n5 are 5ummar12ed 1n 7a61e 1. Pr1mary 5ett11n9 tank eff1uent (P57E) fr0m the K16ana wa5tewater treatment p1ant 1n M1ya2ak1, Japan, and enr1chment cu1ture 0f n1tr1f1er5 were u5ed a5 5tarter cu1ture5.7he enr1chment cu1ture 0f n1tr1fer5 wa5 06ta1ned 6y 5u6cu1tur1n9 the pr1mary 5ett11n9 tank eff1uent 1n a 6a5a1 nutr1ent med1um c0nta1n1n9 n0 0r9an1c 5u65trate u51n9 the f111 and draw meth0d f0r a60ut 2 m0nth5.7he m1cr061a1 character15t1c5 0f 60th 5tarter cu1ture5 are 115ted 1n 7a61e 2. 7he P57E wa5 d0m1nated w1th aer061c heter0tr0ph5 and c0nta1ned a 5ma11 fract10n 0f NH4-0x1d12er5 and N02-0x1d12er5. 1n c0ntra5t, the enr1chment cu1ture 0f n1tr1f1er5 c0nta1ned h19her N020x1d12er5 (1.3 +0.4 x 106MpN m1-t) and re1at1ve1y 10w heter0tr0ph1c 6acter1a. 7he R8C react0r5 were 1n0cu1ated w1th the5e 5tarter cu1ture5 and 1n1t1a11y 0perated a5 a fed6atch m0de f0r a60ut 1 wk t0 pr0v1de 1n1t1a1 c010n12at10n and accumu1at10n. Dur1n9 the fed-6atch cu1t1vat10n per10d, a60ut ha1f 0f the 6u1k 114u1d wa5 w1thdrawn and a fre5h 6a5a1 nutr1ent med1a wa5 ref111ed 0nce every 2 d. 5u65e4uent1y, the react0r wa5 0perated at a c0nt1nu0u5 f10w w1th a 6a5a1 nutr1ent med1um. 7he 6a5a1 med1um c0nta1ned the f0110w1n95 (1n m9 1-t): NH4C1, 76.4; NaHC03, 600; K2HP04, 70; M9504.7H20, 100; NaC1, 71; 50d1um acetate, 0, 17.1 and 102.5 (f0r C:N =0, 0.25 and 1.5, re5pect1ve1y). Run-4 wa5 fed w1th 50% 5tren9th pr1mary 5ett11n9 tank eff1uent (P57E) 5upp1emented w1th acetate and NH4C1 t0 06ta1n 60th 7 0 C and NH4-N c0ncentrat10n5 0f 20 m9 1 1, re5pect1ve1y, c0rre5p0nd1n9 t0 C:N rat10 = 1.0. Chan9e5 1n eff1uent water 4ua11ty: (1) t0ta1 0r9an1c car60n (70C); (2) NH~-N; (3) N02-N; and (4) N 0 3 - N and p0pu1at10n dynam1c5 0f m1cr061a15pec1e51n the 610f11m5 and 1n the 6u1k 114u1d were per10d1ca11y mea5ured. Enumerat10n 0f m1cr061a1 5pec1e5 Rem0va61e 511ce5 w1th attached 610f11m5 were rem0ved carefu11y and r1n5ed tw1ce w1th ph05phate 6uffer 5a11ne

Run 1 2 3 4 5

7a61e 1. 5ummary 0f 610f11mcu1t1vat10nc0nd1t10n5 C :N rat10 1n0cu1um Med1um 0f the med1um Enr1chment cu1ture 5ynthet1c 0 0f n1tr1f1er5 Enr1chment cu1ture 5ynthet1c 0.25 0f n1tr1f1er5 Enr1chment cu1ture 5ynthet1c 1.5 P57Ea 5ynthet1c 0 P57E~ 50% 5tren9th 1.0 P57E6

~Pr1mary 5ett11n9tank eff1uent(P57E) at K16ana wa5tewater treatment p1ant, Japan. ~P57E wa5 d11uted w1th tap water and 5upp1ementedw1th acetate and NH4-N t0 meet the C:N rat10 = 1.0.

P0pu1at10n dynam1c5 1n a m1xed-p0pu1at10n 610f11m 7a61e 2. M1cr061a1character15t1c50f 5tarter cu1ture5 Enr1chment Pr1mary 5ett11n9 cu1ture tank eff1uent 0f n1tr1f1er5 (n = 2) (n = 3) M1cr061a1 tr0ph1c 9r0up5 70ta1 6acter1a1c0unt (ce115/m1) 5.1 + 0.9 x 107 9.9 + 2.4 x 10 6 Heter0tr0ph5 (CFU/m1) 2.1 + 0.5 x 10 7 8.7•+ 1.6x 104 NH4-0x1d12er5 (MPN/m1) 6.7 + 5.3 x 103 1.0 • 0.4 x 104 N02-0x1d12er5 (MPN/m1) 3.4 + 3.0 x 103 1.3 + 0.4 x 10 6 Den1tr1f1er5(MPN/m1) 3.3 •+ 2.2 x 105 2.6+2.1x103

(P85). 7he 610f11m5were 5craped fr0m a kn0wn 5urface area (A = 6 cm 2) 1nt0 25 m1 0f P85. 7he 5crapped 610f11m5were h0m09en12ed f0r 1 m1n at 2000 rev m1n-• u51n9 a P01ytr0n t155uen12er (P01ytr0n P7 2000). 5er1a1 d11ut10n5 were made 0f the h0m09en12ed m1xture, wh1ch were u5ed f0r v1a61e and t0ta1 ce11 c0unt5. V1a61e aer061c heter0tr0ph5 were enumerated 6y p1ate c0unt 0n R2A a9ar (D1fc0). NH4-0x1d12er5, N02-0x1d12er5 and den1tr1f1er5 were enumerated 6y the f1ve tu6e mu1t1p1e d11ut10n m05t pr06a61e num6er (MPN) meth0d (APHA et a1., 1989). C0mp051t10n5 0f med1a u5ed f0r enumerat10n 0f NH4-0x1d12er5 and N02-0x1d12er5 have 6een de5cr16ed e15ewhere (A1exander and C1ark, 1965). 611tay•5 med1um (A1exander, 1965) wa5 u5ed f0r the enumerat10n 0f den1tr1f1er5. 70ta1 ce11c0unt wa5 determ1ned 6y d1rect c0unt1n9 (H0661e et a1., 1977). Ce115were 5ta1ned and f1xed w1th an 0.02% acr1d1ne 0ran9e 501ut10n 1n a 2% f0rma1dehyde 501ut10n, and 1ncu6ated 1n the dark f0r 2-4 h. 7he m1xture wa5 f11tered thr0u9h a 25 mm, 0.2 •am p0re 512e nuc1e0p0re p01ycar60nate f11ter (C05tar 5c1ent1f1c). 7he f11ter5 were exam1ned w1th an ep1f1u0re5cence m1cr05c0pe (N1K0N X2F-EFD2). Enumerated ce11 num6er5 6y the MPN meth0d5 and the ep1f1u0re5cence d1rect c0unt were c0nverted t0 v01umetr1c 6acter1a1 den51ty d1v1d1n9 6y a 5urface area (A = 6cm 2) where the 610f11mwa5 5crapped fr0m and an avera9e 610f11m th1ckne55. 810f11m th1ckne55 810f11m th1ckne55 wa5 determ1ned u51n9 a M1cr0511cer7 M (D,5.K. M0de1 D 7 K 1000). 7he 610f11m5 attached t0 the rem0va61e 511de5 were f1xed 0n a 5pec1men 5ta9e w1th a d0u61e 51ded tape. 7he 5pec1men 5ta9e wa5 f111ed w1th 25-30 m1 0f P85. 7he 610f11m5 were then 5ect10ned 1n th1n 1ayer5 (30-70 •am) para11e1 t0 the 5u65tratum u51n9 a ra20r 61ade unt11 the ra20r 61ade wa5 at the 5u65tratum. 7he 610f11m th1ckne55 wa5 determ1ned 6y add1n9 the th1ckne55 0f each 0f the 1ayer5. 7h15 meth0d 15 u5efu1 f0r mea5ur1n9 610f11m th1ckne55 6ecau5e the 610f11m 15 fu11y hydrated e11m1nat1n9 art1fact5 fr0m f1x1n9 and dry1n9. 1N7-dehydr09ena5e act1v1ty a55ay 8acter1a were c1a551f1eda5 act1ve 0r 1nact1ve acc0rd1n9 t0 the1r a6111tyt0 reduce 2-(p-10d0pheny1)-3-(p-n1tr0pheny1)-5pheny1 tetra2011um ch10r1de (1N7) under 5pec1f1ed 1ncu6at10n c0nd1t10n5 (21mmermann et a1., 1978; R1ttmann et a1., 1992). 7he act1ve ce11 fract10n 0f t0ta1 d15per5ed 610f11m 5amp1e5 wa5 a55ayed dur1n9 610f11m accumu1at10n. 7he 610f11mwa5 5craped fr0m a 511de 1nt0 30 m1 0f P85 and h0m09en12ed 6y a P01ytr0n t155uen12er f0r 1 m1n at 2000 rev m1n- ~. 7hen, 7 m1 0f a 5u5pen510n 0f the 610f11m wa5 m1xed w1th 1 m1 0f 6a5a1 nutr1ent med1um w1th yea5t extract [0.03% (w/v)] and 1 m1 0f0,2% (w/v) 0f 1N7 501ut10n. 7he m1xture wa5 1ncu6ated 1n the dark f0r 1 h at 25°C. After the 1ncu6at10n per10d, 1 m1 0f 37% f0rma1dehyde wa5 added t0 5t0p a11 react10n5. Act1ve (re5p1r1n9) 6acter1a were 1dent1f1ed 6y the f0rmat10n 0f a reduced 1N7 cry5ta1 (redd15h-purp1e under ep1f1u0re5cence m1cr05c0py) 1n51de the ce115(21mmermann et a1., 1978). A fract10n 0f the m1xture wa5 c0unter5ta1ned w1th the 0.02% (w/v) acr1d1ne 0ran9e 501ut10n, 1ncu6ated 1n the dark f0r 2-4 h, and f11tered thr0u9h a 0.2•am nuc1e0p0re f11ter t0 enumerate the t0ta1 num6er 0f

1565

6acter1a. 7he percenta9e rat10 0f act1ve 6acter1a c0unt5 t0 t0ta1 6acter1a c0unt5 91ve5 the fract10n 0f act1ve 610ma55

(1N7%). Chem1ca1 ana1y5e5 A11 6u1k 114u1d 5amp1e5 except th05e f0r 7 0 C ana1y515 were pref11tered w1th 0.45 •am mem6rane f11ter5. 5amp1e5 f0r 7 0 C ana1y515were h0m09en12ed 6y an u1tra50n1c h0m09en12er and ana1y2ed 6y a 5h1ma2u 7 0 C ana1y2er (70C-5000). NH4-N and N03-N c0ncentrat10n5 were determ1ned u51n9 an 10n chr0mat09raphy (D10NEX Q1C Ana1y2er) w1th 10nPac C53 and HP1C A54A c01umn5, re5pect1ve1y. N02-N wa5 ana1y2ed 6y a c010r1metr1c meth0d (APHA et a1., 1989). RE5UL75 React0r p e r f 0 r m a n c e F19ure5 2 ( A - E ) 5h0w typ1ca1 react0r p e r f 0 r m a n c e at var10u5 5u65trate C : N rat105. C : N = 0 (run5-1 and-4). 7 h e f1r5t 5ta9e 0 f n1tr1f1cat10n, 0x1dat10n 0f N H : N t0 N 0 2 - N , 0ccurred 1mmed1ate1y after a c0nt1nu0u5 feed 0 f med1um wa5 5tarted 1n 6 0 t h run-1 a n d run-4. Even t h 0 u 9 h the 0x1dat10n 0 f NH4-N t00k p1ace 5m00th1y at run-4, the 0x1dat10n 0f N 0 : N d1d n 0 t pr09re55 rap1d1y. 7h15 wa5 6ecau5e the accumu1at10n 0 f N02-0x1d12er5 wa5 c0n51dera61y 510w due t0 a 10w 1n1t1a1 den51ty [5ee F19.4(D)]. 7 h e c0mp1ete 0x1dat10n 0 f N 0 2 - N wa5 n0t ach1eved even after 2 m0nth5. After 40 day5 1n run-1, the eff1uent NH4-N a n d N 0 2 - N c0ncentrat10n5 were 6e10w 0.4 m 9 1-1, re5pect1ve1y, wherea5 th05e c0ncentrat10n5 1n run-4 were a 6 0 u t 0.5 a n d 1 0 m 9 1-~, re5pect1ve1y, C : N = 0.25 (run-2). 7 h e 5h1ft 0 f n1tr1f1cat10n t0 the 5ec0nd 5ta9e, 0x1dat10n 0f N 0 2 - N t0 N 0 : N , wa5 c0mp1eted w1th1n 2 wk, wh1ch wa5 even 4u1cker t h a n at C : N = 0 (run-1) (appr0x. 23 d). 7 h e eff1uent 7 0 C c0ncentrat10n wa5 re1at1ve1y c0n5tant 1n the ran9e 2-3 m9-C 1-1. 7 h e eff1uent NH4-N c0ncentrat10n wa5 6e10w 0 . 2 m 9 1-1 after a 6 0 u t 30 d. C : N = 1.5 (run-3). 7 h e accumu1at10n 0 f N 0 2 - N peaked after a 6 0 u t 1 m 0 n t h , thereafter the pr0ce55 9radua11y 5h1fted t0 the 5ec0nd 5ta9e 0f n1tr1f1cat10n. 7 h e eff1uent NH4-N a n d N 0 2 - N c0ncentrat10n5 were 5t111 1-2 a n d 2-3 m 9 1-1, re5pect1ve1y, a n d un5ta61e even after 1.5 m0nth5. 7 h e eff1uent 7 0 C c0ncentrat10n decrea5ed 1mmed1ate1y after the c0nt1nu0u5 0perat10n a n d 9radua11y decrea5ed t0 a 6 0 u t 2.0 m9-C 1-1 after a 6 0 u t 30 d. C : N = 1.0 (run-5). A c0mp1ete 0x1dat10n 0f NH4N wa5 ach1eved w1th1n a 6 0 u t 20 d, wh1ch wa5 1n the 6etween 10 d at C : N = 0 (run-1) a n d 30 d at C : N = 1.5. 7 h e eff1uent NH4-N c0ncentrat10n wa5 6e10w 0.4 m 9 1-1. A 519n1f1cant accumu1at10n 0f N02N wa5 n0t 065erved 1n th15 r u n [F19. 2(E)]. P0pu1at10n dynam1c5 1n 610f11m 810f11m th1ckne55. 810f11m accumu1at10n w1th t1me 15 pre5ented 1n F19. 3 . 7 h e 1n1t1a1 610f11m th1ckne55e5 0f 4 0 ~ 5 0 / t m were a1ready deve10ped 1n 6 a t c h m 0 d e at each C : N rat10, thereafter the react0r5 were 5w1tched t0 a c0nt1nu0u5 cu1ture w1th a 114u1d

1566

5. 0ka6e et a1.

re51dence t1me 0f 5 h. 7he 610f11m th1ckne55e5 reached 5teady 5tate th1ckne55e5 0f a60ut 300 # m after a60ut 1 m0nth at C : N = 0 (run-1 and run-2), 0.25 and 1.0. 7he 610f11m th1ckne55 wa5 h19h1y dependent 0n the 5u65trate C : N rat10. At C : N = 1.5, the 610f11m th1ckne55 1ncrea5ed t0 a 5teady 5tate th1ckne55 0f a60ut 1100/~m after 30 d. F19ure5 4(A-E) repre5ent the chan9e51n v01umetr1c ce11 den51t1e5 0f m1cr061a1 tr0ph1c 9r0up5 attached 0n the d15k 511de5, wh1ch ref1ected we11 the chan9e5 1n the eff1uent water 4ua11ty a5 pre5ented 1n F195 2(A-E). C : N Rat10 = 0 (run-1). 7he 1n1t1a1 m1cr061a1 c0n50rt1a ( 7 = 0) wa5 d0m1nated 6y heter0tr0ph5. 7he den51t1e5 0f NH4-0x1d12er5, N02-0x1d12er5 and den1tr1fer5 were 1n the 1eve15 0f 1 x105 , 5 × 102 and 7 x 106 M P N cm 3 re5pect1ve1y. Wh11e the den51t1e5 0f heter0tr0ph5 and den1tr1f1er5 decrea5ed 0ver t1me, th05e 0f NH4- and N02-0x1d12er5 1ncrea5ed t0 107-108 M P N c m - L

25

C : N Rat10 = 0.25 (run-2). 5ta61e n1tr1f1cat10n wa5 ach1eved m0re eff1c1ent1y than at C : N = 0 (run-1) 6ecau5e 0f a h19her 1n1t1a1 den51ty 0f N02-0x1d12er (5 x 103 M P N cm -3) f0110wed 6y a rap1d accumu1at10n rate. 7he den51t1e5 0f NH4- and N0r0x1d12er5 1ncrea5ed t0 107-108 M P N cm -3. 5teady 5tate den51t1e5 f0r heter0tr0ph5 and den1tr1f1er5 were a60ut tw0 0rder5 0f ma9n1tude h19her than th05e at run-1, wherea5 th05e f0r the n1tr1f1er5 were the 5ame 0rder. C : N Rat10 = 1.5 (run-3). 7he den51t1e5 0f heter0tr0ph5 and den1tr1f1er5 rema1ned re1at1ve1y c0n5tant 1n the ran9e 109-101° and 107-108 M P N cm -3, re5pect1ve1y, dur1n9 the c0ur5e 0f the exper1ment. 7he den51ty 0f N02-0x1d12er5 510w1y 1ncrea5ed a5 c0mpared t0 that at C: N = 0.25, a1th0u9h the f1na1 den51ty wa5 the 5ame 0rder. 7he accumu1at10n rate 0f NH4-0x1d12er5 wa5 e55ent1a11y 1dent1ca1 t0 th05e at C : N = 0 and 0.25.

25

(8)Run-2

20 15, 10 5 A

25

70""

30

A A

A

4~) -50- 60

A

70 %

10

20

30

40

50

25 (C) Run-3

E 20

20

0 15•

t 10F 5P

0

06

10

20

30

40

50

25

(E)Run-5

10

20

30

40

50

60

70

71me(day)

20• 15 10 5 00

10

20 30 71me (day)

40

50

F19. 2. Chan9e5 1n the eff1uent water 4ua11ty: (*) 70C; (A) NH4-N; ( 0 ) N02-N; (•) N03-N; w1th t1me at each run. 1nf1uent NH4-N c0ncentrat10n wa5 20 m9 1-~. 1nf1uent 7 0 C c0ncentrat10n5 were: (A) 0 m9 1-~ (C:N = 0); (8) 5 m9 1-~ (C:N = 0.25); (C) 30 m9 1-~ (C:N = 1.5); (D) 0 m9 1-~ w1th P57E (C:N = 0); and (E) 20 m9 1-~ w1th P57E and 50% 5tren9th P57E (C:N = 1.0), re5pect1ve1y.

P0pu1at10n dynam1c5 1n a m1xed-p0pu1at10n610f11m

C0ncentrat10n d15tr16ut10n 0 f m1er061a1 5pec1e5 1n the 114u1d pha5e

•c- 1,400 ¢1

~ 1,200 9 .0 1,000 E

800

,.0

600

~1--

400

E

200

m

0

.w

0

1567

10

20

30

40

50

60

71me (day)

F19. 3. Pr09re5510n5 0f 610f11m th1ckne55 at each run: (*) run-f; (•) run-2; (11) run-3; (0) run-4; (A) run-5. Err0r 6ar5 repre5ent the 5tandard dev1at10n5 0f dup11cate mea5urement5 (n = 2). 7he err0r 6ar wa5 n0t 91ven f0r 50me data p01nt5 6ecau5e the1r 5tandard dev1at10n5are 50 5ma11.

C : N Rat10 = 0 (run-4). 7he pr1mary 5ett11n9 tank eff1uent wa5 u5ed a5 a 5eed1n9 mater1a1 and the 5ynthet1c med1um wa5 fed. 7he 1n1t1a1 den51ty 0f N0:0x1d12er5 wa5 6e10w the detect10n 11m1t. 7he 1n1t1a1 6acter1a1 den51t1e5 except f0r N02-0x1d12er5 were 51m11ar t0 th05e at run-1. 7he N02-0x1d12er5 9radua11y accumu1ated and reached 3 x 106MpN cm -3 at 44 d, wh1ch wa5 a60ut tw0 0rder5 0f ma9n1tude 10wer than that at run-1. Heter0tr0ph5 rema1ned at a60ut 10 9 MPN cm -3 dur1n9 the c0ur5e 0f the exper1ment (the1r num6er5 dec11ned w1th t1me 1n run-1). 7h15 15 pr06a61y a ma1n rea50n f0r the retardat10n 0f the 0x1dat10n 0f N02-N a5 5h0wn 1n F19. 2(D). C : N Rat10 = 1.0 (run-5). 7he P57E wa5 u5ed a5 a 5eed1n9 mater1a1 and 50% 5tren9th P57E wa5 fed. 7he 1n1t1a1 den51ty 0f N02-0x1d12er5 wa5 under the detect10n 11m1t 6ecau5e the P57E c0nta1ned a 10w c0ncentrat10n 0f n1tr1fy1n9 6acter1a. H0wever, the den51ty 0f N02-0x1d12er5 rap1d1y 1ncrea5ed t0 4 × 106 MPN cm -3 w1th1n a week and rema1ned at that 1eve1 dur1n9 the c0ur5e 0f the exper1ment. 7h15 wa5 pr06a61y cau5ed 6y the c0nt1nu0u5 feed1n9 0f 50% 5tren9th P57E wh1ch c0nta1ned 107 MPN m1-~ 0f N02-0x1d12er5.7heref0re, a c0nc0m1tant 0x1dat10n 0f N02-N w1th the 0x1dat10n 0f NH4-N t00k p1ace w1th1n 3 wk [F19. 2(E)]. 70ta1 ce11 den51ty. F19ure 5 dep1ct5 chan9e5 1n avera9e t0ta1 ce11 den51t1e5 w1th t1me at var10u5 run5. 7he 1n1t1a1 avera9e t0ta1 ce11 den51t1e5 were a60ut 7 × 109 ce115cm -3 at each run. 7he avera9e t0ta1 ce11 den51t1e5 at C : N rat105 0f 1.0 (run-5) and 1.5 (run-3) decrea5ed t0 4-5 × 109 ce115 cm -3 dur1n9 the 1n1t1a1 accumu1at10n pha5e, wherea5 th05e at C : N = 0 (run1 and run-4) and 0.25 (run-2) 1ncrea5ed t0 12-13.5 × 10 9 ce115 cm -3. 7he avera9e t0ta1 ce11 den51ty at C : N = 0 wa5 a60ut 3 4 t1me5 h19her than that at C : N = 1.5.

7he c0ncentrat10n d15tr16ut10n5 0f m1cr061a1 tr0ph1c 9r0up5 1n the 114u1dpha5e were determ1ned at dur1n9 the c0ur5e 0f the exper1ment [F195 6(A-E)]. 7he mea5ured m1cr061a1 c0mmun1ty 1n the 114u1d pha5e were dependent 0n the m1cr061a1 c0n50rt1a 1n the 610f11m at each run except at run-5 w1th 50% 5tren9th P57E. C : N = 0 (run-1). 7he c0ncentrat10n 0f N02-0x1d12er5 wa5 under the detect10n 11m1t (~<101MPN m1-1) unt11 10 day5 and thereafter rap1d1y 1ncrea5ed a5 the den51ty 0f N02-0x1d12er5 1n the 610f11m5tead11y 1ncrea5ed. 7he c0ncentrat10n5 0f heter0tr0ph5 and den1tr1f1er5 9radua11y decrea5ed w1th t1me and the c0ncentrat10n 0f NH4-0x1d12er5 rema1ned re1at1ve1y c0n5tant (103-104 MPN m1-~) [F19. 6(A)]. C : N = 0.25 (run-2). 7he c0ncentrat10n 0f N020x1d12er5 1ncrea5ed rap1d1y fr0m 101 t0 105 MPN m1w1th1n a60ut 10 d, wh1ch wa5 fa5ter than at C : N = 0 (run-1). 7h15 rap1d 1ncrea5e c0rre5p0nded we11 t0 the chan9e 1n the den51ty 0f N01-0x1d12er5 1n the 610f11m [F19. 4(8)] and the chan9e 1n the water 4ua11ty [F19. 2(8)]. C : N = 1.5 (run-3). 7he c0ncentrat10n 0f N020x1d12er5 510w1y 1ncrea5ed after 20 d a5 c0mpared w1th at C : N = 0 (run-1) and 0.25, wherea5 the f1na1 c0ncentrat10n wa5 c0mpara61e. 7he c0ncentrat10n ran9e5 0f 0ther tr0ph1c 9r0up5 were 1n the 5ame 1eve15 a5 th05e at C : N = 0 (run-1) and 0.25. C : N = 0 (run-4). 7he c0ncentrat10n 0f N02-0x1d12er5 wa5 6e10w 102 MPN m1-1 unt11 30 d and then 1ncrea5ed t0 104MpN m1-~ at 43 d. 5uch a 510w accumu1at10n 0f N02-0x1d12er5 wa5 attr16uted t0 the 1nh161t10n 0f n1tr1f1cat10n. C : N = 1.0 (run-5). 51nce 50% 5tren9th P57E wh1ch c0nta1n5 var10u5 m1cr00r9an15m5 a5 115ted 1n 7a61e 2 wa5 fed 1n th15 exper1ment, the c0ncentrat10n 0f each m1cr061a1 tr0ph1c 9r0up wa5 51m11ar t0 the 1eve15 115ted 1n 7a61e 2. 70ta1 num6er5 0f 5u5pended m1cr061a1 5pec1e5 1n the 114u1d pha5e [x: V, where x~ = c0ncentrat10n 0f 5pec1e5 1 1n the 6u1k 114u1d (M L -3) and V = react0r v01ume (L -3) (M = ma55, L = d15tance, 7 = t1me)] were p10tted a5 a funct10n 0f t0ta1 num6er5 0f m1cr061a1 5pec1e5 1n the 610f11m5 [A•p:L:, where A = t0ta1 610f11m area (L-2), P1 = den51ty 0f 5pec1e5 1 1n 610f11m (M L -3) and L: = 610f11m th1ckne55 (L)] at C : N = 0 (run-1) and C : N = 1.5 1n F195 7(A) and (8). F19ure5 7(A) and (8) 1nd1cated that x~ 1ncrea5ed w1th 1ncrea51n9 the t0ta1 ce11 num6er 1n the 610f11m re9ard1e55 0f m1cr061a1 tr0ph1c 9r0up. Act1ve ce11fract10n 1n 610f11m

70 determ1ne the act1ve ce11 fract10n 1n the 610f11m, an 1N7 dehydr09ena5e act1v1ty a55ay wa5 c0nducted f0r d15per5ed 610f11m6acter1a 9r0wn 0n 5 0 0 5tren9th P57E 5upp1emented w1th NH4C1 and acetate (run-4). 7w0 1ndependent exper1ment5 were c0nducted 1n

5. 0ka6e et a1.

1568

para11e1. Appr0x1mate1y 90% 0f t0ta1 ce11 c0unted 6y the ep1f1u0re5cence techn14ue were p0tent1a11y act1ve (••re5p1r1n9••) 1n th1n 610f11m5 1n1t1a11y(7a61e 3). 7he act1ve ce11fract10n (% 1N7) 9radua11y decrea5ed fr0m m0re than 90 t0 70% a5 the 610f11m5 6ecame th1cker 1n 60th run5. D15CU5510N

React0r perf0rmance and p0pu1at10n dynam1c5 7h15 paper pr0v1ded under5tand1n9 0f 6101091ca1 pr0ce55e5 0ccurr1n91n a wa5tewater treatment 610f11m and c0rre1ated the m1cr061a1 c0mmun1ty 5tructure5 1n the 610f11m and 1n the 6u1k 114u1d w1th the react0r perf0rmance (1.e. n1tr1f1cat10n). 7he re5u1t5 06ta1ned 1n th15 5tudy 1nd1cated that the m1cr061a1 c0mmun1ty 5tructure 1n the 610f11m and act1v1ty (1.e. n1tr1f1cat10n eff1c1ency) 1n the react0r were dependent 0n C : N

12

t[•

12

(A) Run-1

101 8 6

4

8, 6,

4,

2 "

1

00 12

10

=

20

0

50

(C) Run-3

16

26

1

1

a6

12 10

2

40

30

2 10

0

(8) Run-2

10

4

%

rat10 1n the feed med1um. 1ncrea51n9 acetate c0ncentrat10n 1n the feed 501ut10n re5u1ted 1n retardat10n 0f accumu1at10n 0f N02-0x1d12er5 1n the 610f11m at C : N = 1.5 [F19. 4(C)]. 7h15 wa5 p055161y 6ecau5e heter0tr0ph5 0utc0mpeted f0r 0xy9en and 5pace w1th N02-0x1d12er5 1n the 610f11m (0ka6e et a1., 1995). C0n5e4uent1y, the n1tr1f1cat10n wa5 very 510w and un5ta61e even th0u9h 5uff1c1ent num6er5 0f n1tr1f1er5 (107-105 MPN Cm-3) Were pre5ent after 30 d. 7he eff1Uent NH4-N C0nCentrat10n after 30 d at C : N = 1.5 Wa5 1n the ran9e 1.0-2.0 m9 1-1, Wh1Ch Wa5 h19her than th05e at C : N = 0 (rUn-1) and 0.25 [F195 2(A) and (8)]. 7h15 1nCrea5e 1n eff1Uent NH4-N c0ncentrat10n wa5 pre5uma61y attr16uted t0" (1) an 1ncrea5e 1n the 1nterna1 ma55 tran5fer re515tance f0r NH4-N; and (2) 0xy9en d1ffu510n 11m1tat10n fr0m the accumu1at10n 0f heter0tr0ph5 1n the 0uter 610f11m5 (0ka6e et a1., 1995). 51m11ar 1ncrea5e5 1n eff1uent

4•0

16

00

26

30•

0 0

50

(D) Run-4

10

50

40•

,

,

1

10

1

1

20 30 71me (day)

40

50

(E)Run-5 e~e~--e---,e~.~

u

8 6

4 2 00

1

10

1

1

20 30 71me (day)

1

40

50

F19.4. P0pu1at10ndynam1c50f."( . ) heter0tr0ph5; (A) den1tr1f1er5;(C)) NH4-0x1d12er5;(0) N02-0x1d12er5 1n the 610f11m59r0wn at: (A) C: N = 0; (8) C:N = 0.25; (C) C:N = 1.5; (D) C:N = 0 w1th P57E; and (E) C:N = 1.0 w1th P57E and 50% 5tren9th P57E, re5pect1ve1y.

P0pu1at10n dynam1c5 1n a m1xed-p0pu1at10n 610f11m

1569

cumu1at10n, the 0x1dat10n 0f N02-N d1d n0t take p1ace unt11 a60ut 40 d [F19. 2(D)]. 7he5e re5u1t5 14 1nd1cated that the 1n1t1a1 m1cr061a1 c0mp051t10n 5tr0n91y 1nf1uence5 the m1cr061a1 p0pu1at10n dynam12 1c5 and n1tr1f1cat10n eff1c1ency, e5pec1a11y f0r m1cr00r9an15m5 (1.e. N02-0x1d12er5) w1th a 10wer 9r0wth rate and 9r0wth y1e1d wh1ch w0u1d take a 10n9 t1me t0 1ncrea5e the p0pu1at10n 1n the 610f11m. 7heref0re, enr1chment 1n0cu1a 0f n1tr1f1er5 are nece55ary t0 keep 5tart-up t1me t0 a m1n1mum. W1th a 5ter1112ed 5ynthet1c med1um (1.e. n0 m1cr00r9an15m5), the accumu1at10n 0f N02-0x1d12er5 wa5 0 determ1ned 6y 0n1y the 1n1t1a1 p0pu1at10n 0f N020 10 20 30 40 50 60 0x1d12er5 and the1r 9r0wth. 7he 9r0wth 0f n1tr1f1er5 1n 71me (day) the m1xed p0pu1at10n 610f11m 15 h19h1y 1nf1uenced 6y F19. 5. Chan9e5 1n avera9e ce11 den51ty w1th t1me at each 1nter5pec1e5 c0mpet1t10n5 f0r 0xy9en 1n the 610f11m run: (,) run-1; (0) run-2; (11) run-3; (0) run-4; (A) run-5. and the 5u65trate C : N rat10 (0ka6e et a1., 1995). 70ta1 ce11 c0unt wa5 determ1ned d1rect m1cr05c0p1c N1tr1f1er5 wh0 have 10wer 9r0wth rate5 and 9r0wth c0unt1n9. y1e1d5 are u5ua11y 0utc0mpeted 6y heter0tr0ph51n the m1xed p0pu1at10n 610f11m. 7he raw wa5tewater 1n NH4-N c0ncentrat10n5 have 6een rep0rted prev10u51y 9enera1, h0wever, c0nta1n5 n1tr1f1er5 1n a certa1n 1eve1 6y R1ttmann and Manem (1992), h0wever, they have (e.9. 103-104 MPN m1-1 0f N02-0x1d12er5). 7he c0nn0t d1rect1y dem0n5trated the c0rre1at10n 6etween the t1nu0u5 feed 0f the wa5tewater a110wed a rap1d acpr0ce55 perf0rmance w1th the m1cr061a1 p0pu1at10n 1n cumu1at10n 0f N02-0x1d12er5 and c0n5e4uent1y 5ta61e th15 610f11m5. n1tr1f1cat10n eff1c1ency w1th1n a 5h0rt t1me per10d a5 7he t0ta1 ce11 den51t1e5 0f the 610f11m5 9r0wn at dem0n5trated 1n run-5. 1n th15 run, the fract10n 0f C : N = 0 (run-1 and run-4) and 0.25 (1.e. n1tr1fy1n9 t0ta1 ce11 num6er5 0f each m1cr061a1 5pec1e5 1n the 610f11m5) were a60ut three t1me5 h19her than th05e 0f 114u1d pha5e wa5 1e55 than 10% 0f th05e 1n the the 610f11m5 9r0wn at C : N = 1.0 and 1.5 (1.e. heter0- 610f11m5, 1nd1cat1n9 that c0ntr16ut10n 0f 5u5pended tr0ph1c 610f11m5) a5 5h0wn 1n F19. 5.7h15 15 pr06a61y 610ma55 t0 the n1tr1f1cat10n eff1c1ency 15 m1n0r. 6ecau5e that heter0tr0ph1c 6acter1a pr0duce a 1005er 610f11m5 a5 a c0n5e4uence 0f pr0duct10n 0f m0re M1cr061a1 c0mmun1ty 5tructure 1n the 114u1dpha5e extrace11u1ar p01ymer1c 5u65trate5 (EP5) and m0re 7he 5tructure 0f the m1cr061a1 c0mmun1ty 1n the 5pace than the n1tr1fy1n9 6acter1a. 1t 15 5pecu1ated that 610f11m wa5 1ar9e1y 1nf1uenced 6y the 5u5pended c0mth15 d1fference 15 attr16uted t0 the d1fference 0f ener9y mun1t1e5 5tructure 0r v1ce ver5a. 7he m1cr061a1 c0m9a1n5 6etween fr0m 0x1dat10n 0f 0r9an1c car60n5 and mun1ty 1n the 610f11m and 1n the 114u1d pha5e were fr0m 0x1dat10n 0f 1n0r9an1c n1tr09en (1.e. NH~- and mea5ured at var10u5 C : N rat105. 6 0 0 d c0rre1at10n5 N0~). were f0und 6etween t0ta1 num6er5 0f 5u5pended

81

Effect 0f 5tarter cu1ture5 0 n e 0f the ma1n draw6ack5 c0mm0n t0 6101091ca1 n1tr1f1cat10n pr0ce55e5 15 the re4u1rement 0f a 10n9 5tart-up per10d. 7he 1mp0rtance 0f the 1n1t1a1 m1cr061a1 c0mp051t10n 1n the 610f11m 5h0u1d 6e empha512ed. 7he re5u1t5 0f th15 5tudy 5u99e5ted that a key tr0ph1c m1cr061a1 9r0up f0r 5ta61e n1tr1f1cat10n 5eemed t0 6e N02-0x1d12er5. 7he accumu1at10n and 9r0wth 0f N02-0x1d12er5 1n the 610f11m determ1ned the 0vera11 n1tr1f1cat10n eff1c1ency. 70 eva1uate the effect 0f the 1n1t1a1 m1cr061a1 c0mp051t10n 1n the 610f11m 0n f0110w1n9 p0pu1at10n dynam1c5 and react0r perf0rmance, tw0 d1fferent 5tart-up 1n0cu1a were u5ed at C : N = 0 (run-1 and run-4). 7he enr1chment cu1ture 0f n1tr1f1er5 c0nta1ned a60ut three 0rder5 0f ma9n1tude h19her c0ncentrat10n 0f N02-0x1d12er5 than the P57E. 7he 1n1t1a1 den51ty 0f N02-0x1d12er5 1n the 610f11m 1n0cu1ated w1th the P57E (run-4) wa5 a60ut tw0 0rder5 0f ma9n1tude 10wer than that w1th the enr1chment cu1ture 0f n1tr1f1er5 (run-1). A5 a re5u1t 0f the 10wer 1n1t1a1 den51ty 0f N02-0x1d12er5 and the1r 510w ac-

m1cr061a1 5pec1e5 1n the 114u1d pha5e (x; V) and th05e 1n the 610f11m5 (A •P1•L1) a5 pre5ented 1n F195 7(A) and (8). 7he fract10n5 0f t0ta1 num6er 0f 5u5pended m1cr061a1 5pec1e5 1n the 114u1d pha5e were 0n1y a60ut 10 and 1% 0f t0ta1 num6er5 1n the 610f11m5 at C : N = 0 and C : N = 1.5, re5pect1ve1y. 51nce the d11ut10n rate 0f 0.2 h - J wa5 app11ed f0r a11 exper1ment5 1n th15 5tudy, 5u5pended 9r0wth 0f e5pec1a11y heter0tr0ph5 may n0t 6e ne91ected. 7h15 mean5 the m1cr061a1 c0mmun1ty 1n the 114u1d pha5e 15 a net re5u1t 0f 610f11m detachment pr0ce55 and 5u5pended 9r0wth. 7heref0re, e5pec1a11y, the data 0f heter0tr0ph5 5h0u1d 6e 5een a5 a 9enera1 1nd1cat10n. H0wever, the 9enera1 trend 0f heter0tr0ph5 a9reed we11 w1th the trend 0f 0ther type5 0f m1cr061a1 5pec1e5 [F19ure5 7(A) and (8)], 1nd1cat1n9 that detachment 0f attached 6acter1a1 ce115 ma1n1y pr0v1ded the m1cr061a1 c0mmun1ty 5tructure 1n the 6u1k 114u1d and the var1at10n 1n the detachment fre4uency am0n9 d1fferent tr0ph1c 9r0up5 wa5 1n519n1f1cant. 810f11m 5y5tem5 are 0ften a550c1ated w1th 510u9h1n9 event5, wh1ch have 5tr0n9er 1nf1uence 0n m1cr061a1

1570

5. 0ka6e et a1.

c0mmun1ty 5tructure5 60th 1n the 610f11m and 1n the 6u1k 114u1d. N0 510u9h1n9 event wa5, h0wever, 065erved dur1n9 any 0f the exper1ment5. 1n add1t10n, m1cr05c0p1c exam1nat10n and 512e d15tr16ut10n 0f detached 610ma55 1nd1cated that the maj0r1ty 0f part1c1e5 were re1at1ve1y 5ma11 f10c5 0f a few ce115. 7hu5, 610f11m detachment 1n th15 5tudy 5eemed t0 6e ma1n1y due t0 ce11 d1v1510n5 (1.e. ce11 9r0wth) at the f1u1d 1nterface re1ea51n9 0ne 0f the dau9hter ce1151nt0 the 114u1d pha5e (5tewart, 1993). 7he re5u1t5 0f th15 5tudy 1nd1cated that the m1cr061a1 c0mmun1ty 1n the 114u1d pha5e ref1ected we11 that 1n the 610f11m5.7h15 1mp11e5that the m1cr061a1 p0pu1at10n dynam1c5 c0u1d 6e eva1uated 6y m0n1t0r1n9 the m1cr061a1 c0mmun1ty 1n the 6u1k 114u1d. 7h15 15 very 1mp0rtant 6ecau5e an ana1y515 0f m1cr061a1 p0pu1at10n dynam1c5 1n the 610f11m 15 t1me-c0n5um1n9 and

8 ~

-E0 m

0

x

10

(A)Run-1

20

a0

2

-x------ ~

1N7-dehydr09ena5e act1v1ty 7he t0ta1 num6er 0f v1a61e ce115 f0r the f0ur d1fferent 5pec1e5 never exceed5 40% 0f t0ta1 ce11c0unt determ1ned 6y d1rect c0unt1n9, wh1ch 15 c0mm0n 1n env1r0nmenta1 5amp1e5 (1.e. m1xed-p0pu1at10n 610f11m5). Var1a61e c0unt5 tend t0 undere5t1mate 6acter1a1 p0pu1at10n 6ecau5e 0f 6acter1a1 c1ump1n9 and the pre5ence 0f n0n-cu1tura61e 5pec1e5 1n art1f1c1a1 med1a a5 we11 a5 n0nv1a61e 6acter1a. D15t1nct10n 6etween act1ve and 1nact1ve (dead) ce11515e55ent1a1 t0 c0rrect1y eva1uate 610f11m p0pu1at10n dynam1c5 and act1v1ty. 7he data 0n the act1ve ce11 fract10n 5u99e5t that m05t 0f the m0rph01091ca11y d15t1nct 6acter1a1 ce1151n the 610f11m5were phy5101091ca11yact1ve dur1n9

8

40~50

2

8

d1ff1cu1t 1n pract1ce due t0 a 1ack 0f pr0per 610f11m 5amp11n9 techn14ue5.

x.-

°0

ke

(8)Run-2 x 4•-)(•

16•26

a6

46

1~

30

40

50

8•

(C) Run-3 ,~, -x

6

~4

~2

2

r-

0 0

;0~:/0

:/0~10~50

°7

•

20

1

50

71me (day)

(E) Run-5

8 6

2 0 0

1~0 ~ :/0

~0~10

1

50

71me (day) F19.6. P0pu1at10ndynam1c50f: (*) t0ta1 ce11c0unt; ( . ) heter0tr0ph5; (A) den1tr1f1er5;(C)) NH4-0x1d12er5;

(Q) N02-0x1d12er51nthe 114u1dpha5e at: (A) C:N = 0; (8) C :N = 0.25; (C) C :N = 1.5; (D) C: N = 0 w1th P57E; and (E) C:N = 1.0 w1th P57E and 50% 5tren9th P57E, re5pect1ve1y.

P0pu1at10n dynam1c5 1n a m1xed-p0pu1at10n610f11m the 1n1t1a1 610f11m accumu1at10n pha5e, then 1nact1ve ce115 9radua11y accumu1ated 1n the 610f11m5w1th t1me. 7h15 re5u1t 5u99e5t5 that app1y1n9 0n1y the d1rect ce11 c0unt 15 n0t 5uff1c1ent t0 accurate1y eva1uate and pred1ct 610f11m act1v1ty. Furtherm0re a5 an 1mp11cat10n f0r m0de11n9 5tud1e5, the 6acter1a1 death rate 5h0u1d 6e 1nc0rp0rated, 0therw15e the pred1ct10n5 0f p0pu1at10n dynam1c5 and act1v1ty 1n the 610f11mc0u1d 6e 5u6jected t0 err0ne0u5. 7he dead ce115 c0u1d aut01y515 and 116erate 5ec0ndary 5u65trate5.1n add1t10n, act1ve n1tr1f1er5reduce 1n0r9an1c car60n t0 f0rm 610ma55 and 501u61e m1cr061a1 pr0duct5 (5MP), wh1ch 5upp0rt5 the 9r0wth 0f heter0tr0ph5 (Namkun9 and R1ttmann, 1986; Furuma1 and R1ttmann, 1992). 7h15 c0u1d exp1a1n why

1E+11 (A) Run-1 1E+10 D LL c.)

C0NCLU510N5

M1cr061a1 5tructure and act1v1ty 1n the 610f11m5 vary 9reat1y 1n t1me and are 5tr0n91y dependent 0n chan9e5 1n the env1r0nment (1.e. water 4ua11ty). 8a5ed 0n the re5u1t5 0f th15 re5earch, the f0110w1n9 c0nc1u510n5 can 6e drawn:

00 0

1E+7

0 0

>

~ - 1E+6

AA0 A A •

1 E+5 1E+4 4-

w ~-

,,,

,,,

~

~--

w 7-

~ 1J.1

+ 111

¥ W

7-

7-~

MPN0rCFU)

A.p1.Lf 1E+11 (8) RLrt-3 1E+10 1E+9

AA

¢0 0.. ~ >

heter0tr0ph5 5t111 d0m1nated the 610f11m even at C: N = 0. 7he act1ve ce11 fract10n 1n the m1xed-p0pu1at10n 610f11m5determ1ned 1n th15 5tudy wa5 c0mpara61e w1th the va1ue5 rep0rted 1n the 11terature (C05tert0n, 1977). 1t 15 a150 1ntere5t1n9 t0 determ1ne a pr0f11e 0f meta6011ca11y act1ve ce11 fract10n 1n the 610f11m, 6ecau5e the 6acter1a 1n the deeper 610f11m 5uffer 5u65trate 11m1tat10n, wh1ch m19ht re5u1t 1n a h19her death rate.

A

~ 1E+8

2

7a61e 3. chan9e 1n act1vece11fract10n(% 1N7) 1n 610f11m59r0wn 0n 500 5tren9th P57E 5upp1ementedw1th acetate and NH4c1 (c:N = 1.0, run-5).7w0 1dent1ca1exper1ment5were c0nducted 1n para11e1 E1ap5ed t1me L~ L~ % 1N7, % 1N72 (day5) (#m) (,um) (%) (%) 0 26+8 38••+13 88-1-10 91+5 7 55+13 70_+20 89•+6 94+4 14 164_+22 144+14 73•+15 89•+5 24 152••.21 178_+49 80-1-5 70•-1-8 35 206-1-57 211 +75 68-1-7 73_+ 13

1E+9

2

LL

1571

0 0 2~ 0

1E+8 1E+7

0

(]D

>~- 1E+6 1 E+5 1E+4

....... • ........•

........

~

........

•

........

J

........

~

........

J

+ 1J.1

+ 111

+ 1Jd

+ W

+ 111

+ L1J

r-+

~-+

,,--

r.-

~--

~--

.,.-.

v. -

U.1

1JJ

........

W

(1) 71me dependent p0pu1at10n dynam1c5 1n the 610f11m and act1v1ty (610chem1ca1 tran5f0rmat10n) 1n the react0r were dependent 0n the 6u1k water 4ua11ty. 7he h19her 0r9an1c 10ad (C: N rat10) retarded accumu1at10n 0f n1tr1f1er5, re5u1t1n9 1n a c0n51dera61y 10n9 5tart-up per10d (0ver 45 d) f0r c0mp1ete n1tr1f1cat10n. (2) 7here wa5 a 900d c0rre1at10n 6etween the e5ta6115hment 0f m1cr061a1 c0mmun1ty 1n the 610f11m w1th that 0f the 114u1dpha5e, 5u99e5t1n9 that the 5tructure 0f m1cr061a1 c0mmun1ty 1n the 610f11mcan 6e pred1cted 6y m0n1t0r1n9 the 0ne 1n the 114u1d pha5e. (3) 5tart-up 1n0cu1um ha5 a 519n1f1cant 1nf1uence 0n the 1n1t1a1 react0r perf0rmance. (4) 1nact1ve ce115 9radua11y accumu1ated up t0 20-30% 0f t0ta1 ce11 p0pu1at10n 1n the 610f11m, wh1ch mu5t 6e c0n51dered t0 c0rrect1y eva1uate p0pu1at10n dynam1c5 and act1v1ty 1n the 610f11m. An exper1menta1 ana1y515 0f 610f11m p0pu1at10n dynam1c5 c0rre1at1n9 t0 the 610f11m perf0rmance pr0v1de5 a rat10na1 6a515 f0r deve10p1n9 and c0ntr0111n9 a de51red 610f11m t0 max1m12e the react0r perf0rmance.

A . P1" Lf (MPN 0r CFU)

auth0r5 w15ht0 thank 7. Y05h1da and Y. Yamada, Department 0f An1ma1, 6ra551and and F15her1e5 5c1ence5, M1ya2ak1 Un1ver51ty, f0r pr0v1d1n9 m1cr05c0p1ce4u1pment5and f0r the1r a5515tancew1th m1cr05c0p1c ana1y5e5. 7he auth0r5 are a150 9ratefu1 t0 Pau1 5t00d1ey, 7he Center f0r 810f11m En91neer1n9, M0ntana

Ackn0w1ed9ement5--7he

F19. 7. C0rre1at10n5 6etween t0ta1 ce11 num6er5 0f each m1cr061a1 5pec1e5 1n the 114u1d pha5e and th05e 1n the 610f11m5 at: (A) C : N = 0 (run-1); and (8) C : N = 1.5; ( • ) heter0tr0ph5; (/k) den1tr1f1er5;(0) NH4-0x1d12er5;(0) N020x1d12er5.

5 . 0 k a 6 e et a1.

1572

5tate Un1ver51ty, f0r h15 va1ua61e c0mment5 1n preparat10n 0f th15 manu5cr1pt and 5. Ma5uda, Department 0f C1v11and Env1r0nmenta1 En91neer1n9, M1ya2ak1 Un1ver51ty, f0r 5har1n9 w1th u5 h15 expert kn0w1ed9e a60ut m1cr0511cer techn14ue5.

REFERENCE5

APHA, AWWA and WPCF (1989) 5tandard Meth0d5 f0r the Exam1nat10n 0f Water and Wa5tewater, 17th edn. Amer1can Pu611c Hea1th A550c1at10n, Amer1can Water W0rk5 A550c1at10n and Water P011ut10n C0ntr01 Federat10n, New Y0rk. A1exander M. (1965) Den1tr1fy1n9 6acter1a. 1n Meth0d5 0f 5011 Ana1y515, Part 2 (ed1ted 6y C. A. 81ack et a1.), Amer1can 50c1ety 0f A9r0n0my 1nc., Mad150n, W1. A1exander M. and C1ark F. E. (1965) 1n Meth0d5 0f 5011 Ana1y515, Part 2 (ed1ted 6y C. A. 81ack et a1.), pp. 1477-1483. Amer1can 50c1ety 0f A9r0n0my 1nc., Mad150n, W1. C05tert0n J. W. (1977) 1n Nat1ve A4uat1c 8acter1a: Enumerat10n, Act1v1ty, and Ec0109y (ed1ted 6y J. W. C05tert0n and R. R. C01we11.). Amer1can 50c1ety f0r 7e5t1n9 and Mater1a15, Ph11ade1ph1a. de8eer D., 5t00d1ey P. and Lewand0w5k1 2. (1994a) L14u1d f10w 1n heter09ene0u5 610f11m5. 810techn01, 810en9n9 44, 636-641. de8eer D., 5t00d1ey P., R0e F. and Lewand0w5k1 2. (19946) Effect5 0f 610f11m 5tructure5 0n 0xy9en d15tr16ut10n and ma55 tran5p0rt. 810techn01. 810en9n9 43, 1131-1138. Furuma1 H. and R1ttmann 8. E. (1992) Advanced m0de11n9 0f m1xed p0pu1at10n5 0f heter0tr0ph5 and n1tr1f1er5 c0n51der1n9 the f0rmat10n and exchan9e 0f 501u61e m1cr061a1 pr0duct5. 1•Vat. 5c1. 7echn01. 26, 493-502. H0661e J. E., Da1ey R. J. and Ja5per 5. (1977) U5e 0f nuc1e0p0re f11ter5 f0r c0unt1n9 6acter1a 6y f1u0re5cence m1cr05c0py. App1. Env1r0n. M1cr06101. 33, 1225-1228. J0hn50n L. M., McD0we1 C. 5. and Krupka M. (1985) M1cr0610109y 1n p011ut10n c0ntr01: fr0m 6u95 t0 610techn0109y. Dev. 1nd. M1cr06101. 47, 1-6. Ma5uda 5., Watana6e Y. and 15h19ur0 M. (1991) 810f11m pr0pert1e5 and 51mu1tane0u5 n1tr1f1cat10n and den1tr1f1ca-

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