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87
Journal ofEngineering, National Chung Hsing University, Vol. 17, No.2, pp. 87-104 (2006)
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ISOLATION AND PHYSIOLOGICAL
CHARACTERIZATION OF THE ACRYLAMIDE
DENITRIFYING BACTERIA
Chun-Chin Wang
I
1
Chi-Mei Lee
2
Department ofEnvironmental Engineering,
Hung Kuang University,
Taichung 433, Taiwan,R.o.C.
2
Department ofEnvironmental Engineering,
National Chung Hsing University,
Taichung 402, Taiwan,R.o.C.
Key words: acrylamide, biodegradation, denitrification, nitrate.
1 5L7I::f4.f;Z*~~mIf~*J.i}].f!j.!~B't
2
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E-mail: [email protected]
!lift
88
ABSTRACT
Acrylamide is widely used in industry and due to its carcinogenicity and toxicity,
discharge of acrylamide to the natural water and soil systems may lead to an adverse
environmental impact on water quality and thus endanger public health and welfare. This
aim of the study attempts to isolate and identify the denitrifying bacteria from the
acrylonitrile - butadiene - styrene (ABS) resin manufactured wastewater treatment system
and polyacrylonitrile (PAN) fiber manufactured wastewater treatment system. The bacteria
can utilize acrylamide for denitrification. The aim is also to understand the performance of
isolated pure strain and mixed bacteria culture for treating acrylamide from synthetic
wastewater. Finally, phylogenetic trees will be generated to understand the relationship of
bacteria which can utilize acrylamide for denitrification by methods based on 16S rONA
gene sequence. The results are: Both of the ABS and PAN mixed bacteria culture could
utilize acrylamide up to 1400 mg/l for denitrification from synthetic wastewater. Besides, the
suppl y of enough electron acceptor (nitrate) was necessary for the complete acrylamide
removal. The removal efficiency of acrylamide by the ABS mixed bacteria culture was better
than that of the PAN mixed bacteria culture. Strain P stutzeri and R. eutropha were isolated
from the ASS resin manufactured wastewater treatment system and the PAN fiber
manufactured wastewater treatment system, respectively. Both
strains could utilize
acrylamide up to 1700 mg/l for denitrification and the suppl y of enough nitrate was
necessary for the complete acrylamide removal. Strain P stutzeri and other denitrifying
bacteria did not have closely relationship based on the phylogenetic trees . But strain R.
eutropha and Stenotrophomonas sp. BO had intimate relationship based on the phylogenetic
trees. Although strain P stutzeri and R. eutropha could utilize acrylamide for denitrification,
both strains did not have closely relationship based on the phylogenetic trees.
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~~ :1§ - li~Ml§mffjj~$M~ pqMJWU!Z,~H~7El~ , itZ
~ 91.1 INl~¥}~nO 1444.4 mg/I Z lifjMl§m ¥} ~
noZlifjMmmtn 109.8 IN!~*~~ 231.9 mg /l - ~W
.~*~lt 219.8 INl~~9i Wl 1032.2 mg/I 2lifj• •m
tn 92.1 1N!~E. ~i~U/f ¥U ' 1l~ ~iRU~ rs~*~ 7 (2 19.8
INf,J) YJ!r:t -@:~ 0 mg/I (If!] 8 (b)) , J1:tJ*IZ9PJfig~
m:f* R. eutropha ~lifjM.m~ JJ¥:m2lifj • •mZ
I!: ~YnO.L2lifjM.mx1j(~f~~JJ¥:mm~ , itZtnw
ID ' ~~W~~*~rr~~~m~ l o rn ~ ep PJm ,
m ~7.kep
1445.8 mg/I
11 83.4 mg/I
pq~9~3(W
0
0
0
0
"@':f*
P. stutzeri Ztt1:~~$lliipq~9~ilr:t:ij}no
ffijfj 8}j mi---r~ZJm~ 'MiGm:f* P. stutzeri Z1:~~
"@':f* R. eutropha Ztt1:~
~$tnpq~§~ilr:t/J\tn 784.3 mg/I ~lliIpq~ eij3(
iI~:ij}tJoiffi .L7t ' ~t~iitRYmt.ll:ij}tJofjWJtn1¥.&1: ~:ij}
7& ' 19&,pqMJ9ij'tiJ!r:tr\§J1.i~ 784.3 mg/I ~ , tt1:~
~$flIJJllpq~eij'tYJ!r:t~tJDffij~'l:i ---r~ ,J1:t PJfig~ pq
~9~3(YIlr:t i&1 ~ , ~i&1m:f*pfTHg ~ W Z1J!r:t ' jiffjHfj]
*!J1¥.&1:~Z1: ~ ~Rffij § 'iiit* R. eutropha ~ P.
pq~§~iJ!r:t~Il!Jm*
0
0
stutzeri /f~pq~eij3(iJ!r:t~~
0
0
J1:tji-' ffili!l 5- 8
ep 'QJ ~D ' "@'t* P. s tutzeri
W R.
eutropha UiWPJtJpq~9D3(~ ~.ji1Tmm, ll ~{£
0
0
,l~~fjep*~iRU¥ U pqMJ9~3(~1ifj.l§m~nO{&Mz*
0
8Ami*~Wf ' ll tnW,~ *~lt
pq~9~~~~Il;J; '~£Z~-=f~~:1§(OO.l§m)
~~~~ o/f~JII~pq~M~.llmr\§J,m:f*~pq~
e~3(~£.ji1TmmZfigtJ~~ 0 m:f*
P. stutzeri §t
pq1$e~. r:t~W ~ ~r:t*~iii:f* R. eutropha
~m :f*
0
!J!i
R. eutropha §t pq 1$ § ~iJ! r:t z ~ Wfjr:t/J\
' 1ft
~1ii:f* R. eutropha tn&H!~fjep :5t5J Ufj2li~ • •
mw§oo~.m ' ~~2mZ~~o §oo • • m~~
~oo:f* P. stutzeri ' llm:f*1:~i&1fjep~~tvlfM
219.8 INf,J ~*UT 434.5
mg/I ~mJ1:tfJ!~PJfigZJJ¥: IZ9 ~ (I) ~~.i~yw ep lifj
• •~YJ!r:t~ r\§Jtfj]*umt*1:~~m (2) pqMJe~'t*
Ji&Ulillltnz-
~~~ 1:mZ ep ~ ~ ~pqMJM'~~PJ~~ *m :f*
J1:t =t*mPJmWM~pq~eD3(~:ft!! ' ~ ~~~.t§t ep li~.
0
0
R. eutropha &'fFiitRYmtfljfflffjj1: ~
eutropha
, {El~fElmt*
fUffl ZW$/f &pq~9ij3(*,(lJ~ZW$ '2&
~fI~~~ilr:t{&!ftm:f*
R. eutropha tr5tt~mtm
*iJ ,jiffjj{£&ffjH~lt °m*,,~~iRiJ~rs~*~7
~) ~fI
R.
502 umol (~ 8 (b))
0
i&{;t~.t§t{*~ tm ~~ ffjj~#9
' m:f*
0
~milr:t~£,~~~fj§OOM~ mW~~2mz~
fI ffjj
0
ABS Wij~~~liil1F~Z~fj~* eppq~eij3(
illJJt~fj
65 mg/I >- 80 mg/I ' ~J1:t =:f*'@f-t~.~fj
~ 7.k~pq~e~Z~Il;J;&~®fj §tpq~9~~~Z
0
pq~eD't.r:t (mg/I)"
Jl[§
P. stutzeri
tt1:~~$
(hr -1 )
tt1:~ ~$
(hr -1)
R.
eutropha z~ffl 1!l100:/J\1.i~iii:f* P. stutzeri /f~rn1.i~
11l:±'!!:~fjWJ1itt
0
@f*
-i'rm
R. eutropha
(219.8 IJ\
~~~~Z ± ~ ' ffjj ~~2m~~m~~~~ ' ~ ~
391.2
(396.1)
774.8
(784.3)
1151.1
(1165.1)
1428.6
(1445.8)
1702.1
(1722.5)
0.042
0.0 14
0.005
0,005
0.002
0.011
0.01 7
O.oI5
0,0 12
0.011
99
~ 1500
'0
"
], 1350 .
Z
~ 1050
900
'2
750
~
)::
600
2
oJ
.",
450
S
300
~ro
150
'"
0.6
1200
5
§
0.7
0.5
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l:.
OA
c
§
0.3
c:i
0
····A
0.2
0.1
~=±=I==I==!~--"----'~"=*=±====,====,==,======,,---,, 0
20
40
60
80
100
120 140 160 180 200 220 240
time (hrs)
···"'···acrylamide·· o . nitrate -*-nitrite --+--N, ---'--0.0'600
(a)
24
~ 21
5
.~
18
a
15
§
9
.£t .. ·0--'
G-'D .. E!-.. --.-I3" .... D
12
9
6
2
- .... ~ ·····0···
<) .. ,
..
"0
.(> . . . . .
o '-~--'-~---'_~~--'------'----'_.L..-~~ 0
o 20 40 60 80 100 120 140 160 180 200 220 240
time (hrs)
DO
pH
... <)- .•
I
(b)
[II 8 @1'*
R. eutropha ~~~AII'5~~*r:p 1445.8
1183.4
mg/I
E~MlJIi~U&1fE
3.4 ~1~l!n~~~~MWJt{m~~M2JJU~~{~
7k 2 ~ffjffl NCB I ~M:tI~~EE:@G SDSC
Biology Workbench ~ Yb r:p Z Ndjinn Multiple
Database Search :t1~~Mt ' :t1~1~¥Utj,Aft!!~~ (:JF
~mM~)~~~~h~MZ@1'*~~*~~o~.
~@1'*'~~fflm@1'*WAft!!~M@~~@1'*~~~
~5t~ 15
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0
ffij1?r*JlZ@f**Jl~~D7k 3
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0
~9~.~@1'*'~~fflm@1'*WMAft!!~~
~£~Z~M@f*~m7MifM5t:fJT[II
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0
ffij1?r*Jlrs~Z@eM
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4 r:p~R~
~'~~SfM~~~._~Zpqmll~~M@
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2 (P. stutzeri) [email protected]§'i=f-$~@eM
litmi7f,@f* P. stutzeri WA1iQ~M@Z!fJ[
~~1*.Mz/fWt;7] ffij*!3 PAN A~~*I~~.Zpq
mM§'t( ~ M@ TDM - 3 (R. eutropha) W@t*
Stenotrophomonas sp. BO (pq=~=EtJ~~JmM@) Z
(~ 0.16)'
0
mg/I pqmll§'t(R.
r:p , 1?rrj~11!U1i!1L:L~1t
M ~ lli ' lit mi 7f, @1'* R. eutropha W@1'*
Stenotrophomonas sp. BO Zm~M1*~lli mf*@M
@§
0
1*~lliZ~fm@:l2SI~lIt=1'*@fflJ;)JmMj:~Z~1!i
*E_~t§31I (p;;jmI£H~Z 1t~A~
pq=M=Ef3~~z1t~A~
CH2CHCONH2 '
CH2(COOCH3)2) ' i!&fflJ;)j£:
j:~~~~®~~ZM*B"J~I2SI~t§1J;I,Z~i!&
@f* MDM -
2 (P. stutzeri)
eutropha) !ME.I§'~pqmll§'t(~M@
0
W TDM - 3 (R.
, 19~1PJm~Z!fJ[
~M1*Mz*1Hlli (:f§!M@eM~ 0.18) ~~~~@:I2SI~
@t* P. stutzeri W R. eutropha *§/f[i'1JB"J~~.
(@f* P. stutzeri *§ ABS fMm:;~~. ' @f* R.
eutropha *§ PAN A~~*l~~.)o~~ ABS fM
Hg~~.Jl:j:Z~*~1)jW PAN A~~*l~~.
j:~~~*~1)jMz/f:f§1J;I, , ilt!x:~[FfMj:ff1J~/f[i'1J~ij[
Zffi1'*@ , ~7~~~pJT~~~ij[r:pc":i1l~ , A:tmi=fZ
M*~MfpHm/f[i'1J , ~ffij~~!ME.I§'~J;)pqmM§'t(~1T
JmM ' 19Mz~Wt;7]Z!fJ[*~~m1*
0
100
~;g
A
~rJffi
?ir;m
Bacteria; .Proteobacteria;
:$:Ej3M
Rhodocyc1aceae; Thauera.
B
lID
C
~rtD!E
0
rs'=
Ej3:$:
E
F
G
Rhodocyc1ales;
Bacteria; Proteobacteria; Alphaproteobacteria; Rhodospirillales;
Rhodospirillaceae; Magnetospirillum.
Bacteria;
Proteobacteria;
Betaproteobacteria;
Rhodocyc1ales;
Betaproteobacteria;
Rhodocyc1ales;
Rhodocyclaceae; Azoarcus .
Bacteria;
Proteobacteria;
Rhodocyc1aceae; Azoarcus.
= Ej3 rlli
Oxalobacteraceae; Herbaspirillum.
~i3
Bacteria; Proteobacteria; Betaproteobacteria; Burkholderiales;
pg~
Comamonadaceae; Comamonas.
~i3
Bacteria; Proteobacteria; Betaproteobacteria; Burkholderiales;
pg~
Comamonadaceae; Acidovorax.
I
Ej3 ~
J
Ej3~'
K
Ej3~'
N
Betaproteobacteria;
Bacteria; Proteobacteria; Betaproteobacteria; Burkholderiales;
M~liI
M
x litJ:
pq =~
H
L
~~
r~ M ~ fi~
*
*
*
Bacteria;
Proteobacteria;
Gammaproteobacteria;
Xanthomonadales; Xanthomonadaceae; Stenotrophomonas.
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Rhizobiales;
Methylocystaceae; Methylosinus,
[13]
[14]
[IS]
[16]
[17]
[18]
[18]
[ 19]
[20]
Bacteria; Proteobacteria; Alphaproteobacteria; Rhodobacterales;
[21]
Rhodobacteraceae; Paracoccus.
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Rhizobiales;
Hyphomicrobiaceae; Hyphomicrobium.
Bacteria; Proteobacteria; Betaproteobacteria; Burkholderiales;
Burkholderiaceae; Burkholderia; Burkholderia cepacia complex.
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Rhizobiales;
Alphaproteobacteria;
Rhizobiales;
Brucellaceae; Ochrobactrum.
Bacteria;
Proteobacteria;
[21]
[22]
[22,23]
[22,23]
Phyllobacteriaceae; Mesorhizobium.
A: Thauera chlorobenzoica strain 3BB 1; B: Magnetospirillum sp. CC-26; C: Azoarcus sp. pF6 ; 0: Azoarcus spo T; E:
Herba spirillum sp . G8AI ; F: Comamonas sp . 153S ; G : Acidovorax sp . PD-IO; H : Stenotrophomonas sp. BO ;
I: Methylosinus pucelana; 1: Paracoccus denitrificans ; K: Hyphomicrobium zavarzinii; L: Burkholderia cepacia; M :
Ochrobactrum anthropi; N : Mesorhizobium sp. NH-14
*
[email protected]*~tIf~Jml~Uii (aerobic denitrification) & JV~tUif'Hl::.@ (heterotrophic nitrifying bacteria)
:fljfflEj3MHy~~
, M1~ , jijffigl ~ :SiHi~~fN&{j~~fN ; :1)\PJ~tIf~fl*{tf:~ , ~
&7(r 5 ~* (L - asparagine)) 9J ~~
, gl±~1t2M
(NzO) &M~ (Nz)
0
0
~@f*Q]'
GN t~.~ (pg?i1$~~1~
101
@f*~~
*J3.3U
1
Thauera chlorobenzoica strain 3BB 1
2
Azoarcus sp. pF6; Azoarcus sp. T
3
Burkholderia cepacia
4
Herbaspirillum sp. G8Al
5
TDM-3; Ralstonia sp. 1278a; Ralstonia eutropha
6
Comamonas sp. 153S
7
Acidovorax sp. PD-l 0
8
Stenotrophomonas sp. BO
9
MDM-2; Pseudomonas sp. RNA-Ill; Pseudomonas stutzeri strain ATCC 17685
10
Magnetospirillum sp. CC-26
11
Paracoccus denitrificans
12
Ochrobactrum anthropi
13
Mesorhizobium sp. NH-14
14
Hyphomicrobium zavarzinii
15
Methylosinus pucelana
01
102
r-.
I
2
3
4
5
6
7
8
9
10
II
12
13
14
IS
I
-
0.06
0.15
0.11
0.11
0.13
0.12
0.16
0.18
0.19
0.22
0.19
0.20
0.19
0.20
2
3
0.15
0.06
0.14
0.14 0.10 0.11
0.10 0.13
0.12 0.16
0.11 0.15
0.15 0.20
0.17 0.22
0.18 0.23
0.21 0.26
0.18 0.24
0.19 0.24
0.18 0.24
0.19 0.24
4
5
6
7
0.11 0.11 0.13 0.12
0.10 0.10 0.12 0.11
0.11 0.13 0.16 0.15
0.09 0.12 0.11
0.12 0.12
0.09 0.05
0.12 0.12 0.11 0.12 0.05 0.16 0.17 0.18 0.17
0.18 0.18 0.19 0.19
0.19 0.19 0.21 0.20
0.22 0.22 0.24 0.23
0.20 0.20 0.21 0.21
0.20 0.20 0.22 0.21
0.20 0.20 0.21 0.21
0.20 0.20 0.22 0.21
8
0.16
0.15
0.20
0.16
0.17
0.18
0.17
-
0.16
0.17
0.20
0.18
0.18
0.18
0.18
9
0.18
0.17
0.22
0.18
0.18
0.19
0.19
0.16
-
0.17
0.21
0.18
0.19
0.18
0.19
10
0.19
0.18
0.23
0.19
0.19
0.21
0.20
0.17
0.17
-
0.15
0.13
0.13
0.13
0.13
II
0.22
0.21
0.26
0.22
0.22
0.24
0.23
0.20
0.21
0.15
-
12
0.19
0.18
0.24
0.20
0.20
0.21
0.21
0.18
0.18
0.13
0.10
13
0.20
0.19
0.24
0.20
0.20
0.22
0.21
0.18
0.19
0.13
0.11
0.06
14
0.19
0.18
0.24
0.20
0.20
0.21
0.21
0.18
0.18
0.13
0.15
0.12
0.13
0.10 0.11 0.06 0.15 0.12 0.13 0.15 0.13 0.13 0.09
IS
0.20
0.19
0.24
0.20
0.20
0.22
0.21
0.18
0.19
0.13
0.15
0.13
0.13
0.09
-
6.riif* P. stutzeri W R. eutropha 9ll~~~mMijy!Jft
1B1£i ' 19jjz*1'f1JU~lliz~~ruJ{*
1./f1jif!l ABS 1~-E;-~Wf~ PAN 1~-E;-~Wf~PJfljm
0
1400 mg/I j;JTz~mimijYJ1HJ}ffi;~, lL1EJEzliJ!j~
.~~&\~i¥J
'
tzDJ!:t:t/f¥!£f~~~.1~1flep~W~
~m~~~w~m:~~~
mMijyo
2.PAN 1~-E;-~MD~~mMijY1~~/J\D~ 784.3 mg/I
~'XM.~mM~z~$W~S~-E;-~Mm
(IT~~~:
NSC89-2317-B005-008 , NSC89-2317-B005-013)' :JJ1~
j;JII~flj5G~ , !ffJ!:tfW[!Y:~tt
0
lli ; m~mMijY1~~~~ 1538.5 mg/I B~ , ~tlt~
mimijYZ,':tt:!~$/J\~ ABS 1~-E;-~M
ll:B~~7~W~meijy
0
'
li&mM~
tj( ABS 1~€i~Mj;J~mim
ijy}ffi;~zfm:trf1fD~ PAN 1~-E;-~M
1. Cherry, A. B., Gabbacia, A. F. and Senn, H. W.,
"The Assimilation Behaviour of Certain Toxic
0
3.riif* P. stutzeri W R. eutropha PJL-) 1700 mg/I j;J
Organic Compounds in Natural Waters," Sewage
Tz~mMW~h}ffi;~'li~~~mMW~~~
~'~liZliJ!jM.M~&\~i¥J
and Industrial Wastes, Vol. 28, pp. 1137 (1956).
2.
0
4.15iffi'f~mMijY¥~Ilm:~ , riif*fljm~meijy}ffi;~z
~~j]~E:
' liriif* P. stutzeri
B-jfEM*D~1£if* R. eutropha
5.001'*
K.
S't~mMijY1~1l~.
and
Kuroiwa,
Y.,
"Acrylamide
Enceph-
aloneuropathy due to well Water Pollution," Journal
ofNeurology, Neurosurgery and Psychiatry, Vol. 38,
0
P. stutzeri W~@!Jft1BooZ~J[~~1~ttttz/fi$
fJ] , ffffriif* R. eutropha Wriif* Stenotrophomonas
sp. BO (~=M=Ej3~~}ffi;~1£i) z~J[~ruJf*~lli
Igisu, H., Goto, 1., Kawamura, Y., Kato, M., Izmui,
0
pp. 581-584 (1975).
3.
Tilson, H. A., "The Neurotoxicity of Acrylamide, an
Overview,"
Neurobehavioral
Toxicology
and
103
Teratology, Vol. 3, pp . 455-461 (1981).
"Isolation
and
of
Characterization
Diverse
4. Shanker, R. and Seth, P. K., "Toxic Effects of
Halobenzoate-Degrading Denitrifying Bacteria from
Acrylamide in a Fresh Water Fish, Heteropneustes
Soils and Sediments," Applied and Environmental
Fossilis," Bulletin of Environmental Contamination
Microbiology, Vol. 66 , No .8, pp. 3446-3453 (2000).
and Toxicology, Vol. 37 , pp. 274-280 (1986).
14. Shinoda, Y., Sakai, Y., Ue, M., Hiraishi, A. and
5. Shairashi, Y., "Chromosome Aberrations Induced
Kato, N., "Isolation and Characterization of a new
by Monomeric Acrylarnide in Bone Marrow and
Denitrifying
Germ Cells of Mice," Mutation Research, Vol. 57,
Degradation of Phenol," Applied and Environmental
pp. 313-324 (1978).
Microbiology, Vol. 66, No .4, pp. 1286-1291 (2000).
Spirillum
Capable
of
Anaerobic
6. Shanker, R., Chauhan, L. K. S. and Seth, P. K., "The
IS. Rhee, S. K., Lee, G. M., Yoon, J, H., Park, Y. H.,
Toxic Effects of Acrylarnide on Root Tip Cells of
Bae, H. S. and Lee, S. T., "Anaerobic and Aerobic
Allium Cepa," Cytologia, Vol. 52, pp. 895-899
Degradation of Pyridine by a newly Isolated
(1987) .
Denitrifying
7. Nozawa, T. and Maruyama, Y., "Denitrification by a
Soil Bacterium with Phthalate and other Aromatic
Compounds as Substrates," Journal ofBacteriology,
Applied
Bacterium,"
and
Environmental Microbiology, Vol. 63, No.7, pp.
2578 -2585 (1997).
16. Krieger, C. J., Beller, H. R., Reinhard, M. and
Spormann, A. M ., " Initial Reactions in Anaerobic
Vol. 170, No.6, pp. 2501-2505 (1988).
8. Jeter, R. M. and Ingraham, 1. L., "The Denitrifying
Oxidation
of
M-Xylene
by
the
Denitrifying
Prokaryotes," In: Starr, M. P., et al. (Eds) , The
Bacterium Azoarcus sp. Strain T," Journal of
Prokaryotes , Vol. I , Springer-Verlag, New York, pp.
Bacteriology, Vol. 181, No. 20, pp . 6403-6410
913-925 (1981).
( 1999).
9. Nawaz, M. S., Franklin, W., Campbell, W. L.,
17. Kniemeyer, 0., Probian,
c., Mora,
R. R. and Harder,
Heinze, T. M. and Cerniglia, C. E., "Metabolism of
J., "Anaerobic Mineralization of Quaternary Carbon
Acrylonitrile by Klebsiella Pneurnoniae," Archives
Atoms:
of Microbiology, Vol. 156, pp. 231-238 (1991).
Dimethylmalonate," Appli ed and Environmental
10. White , 1. M., Jones, D. D., Huang, D. and Gauthier,
J. J., "Conversion of Cyanide to Formate and
Ammonia
by
a
Pseudomonad
Industrial
Wastewater,"
Obtained
Journal
from
of Industrial
Microbiology , Vol. 3, pp. 263-272 (1988).
II. !i~{t~Sj ,
I"""
itffJif Pseudomonas sp . P90 ii± Z sli:
~*6ii tt ~ B ~:5t JW ~i¥J£~~ 7@
, Mlft!M ¥-ftt
:5t* J' ~±~~ ' ~~$ w*~~~~m~M '
5$
(2000)
of Denitrifying
Bacteria
on
Microbiology, Vol. 65, No.8 , pp. 3319-3324 (1999) .
18. Horiba,
Y.,
Khan,
S.
T.
and
Hiraishi,
A.,
"Characterization of the Microbial Community and
Culturable
Denitrifying
Phase-Denitrification
Bacteria
Process
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Solid-
Uusing
Poly
(Epsilon-Caprolactone) as the Carbon and Energy
Source," Microbes Environ , Vol. 20 , pp. 25-33
(2005) .
19. Costa,
0
Isolation
c.,
Dijkema,
c.,
Friedrich, M., Garcia-
12. Keeney, D. R. and Nelson, D. W., "Indophenol
Encina, P., Fernandez-Polanco, F. and Starns , A. J.,
-Blue Method," In: Page , A. L., Miller, R. H.,
"Denitrification with Methane as Electron Donor in
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