ISBN: 978-93-7143-285-6
174
Chap e - 10
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
p-Ni ophenol
D . Ning houjam Sho a ani
Dep . o En i onmen al Sciences,
Y.K. College, Wangjing-795148, Thoubal Dis ic , Manipu
Co esponding Email: [email p o ec ed]
ABSTRACT:
A bac e ium wi h hizoid pa e n o g ow h deg ading p-
ni ophenol (PNP) was isola ed om local ga den soil. This
s ain was iden i ied as a membe o he genus Bacillus based on
mo phological, biochemical, physiological and 16S DNA
sequence analyses. The s ain BGSC1 is a g am-posi i e od
a anged in agmen ed chain o ms. I is posi i e in ca alase,
oxidase, casein hyd olysis, s a ch hyd olysis, and y osine
clea ing es s. The s ain p oduces acid om glucose and
suc ose and shows posi i e esul s in ni a e educ ion, gela in
lique ac ion, Tween 80 hyd olysis, and glucose O/F es s. 16S
DNA sequence analysis es ablished s ain BGSC1 as mos
closely ela ed o Bacillus an h acis and designa ed as Bacillus sp.
s ain BGSC1. Neu al pH (7.0) was ound o be he op imal pH
o deg ada ion o PNP. PNP conc. was measu ed a 400nm and
ni i e was eleased almos s oichiome ically along wi h
deple ion o PNP du ing he biodeg ada ion p ocess. To ou
knowledge BGSC1 s ain may be a po en agen o
biodeg ada ion o ni oa oma ic xenobio ics as well as a good
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
175
p omising addi ion o he epe oi e o PNP deg ading
mic obial isola es.
Keywo ds: Bacillus an h acis, P-ni ophenol, Biodeg ada ion,
Ni oa oma ic compound, Xenobio ics
1. In oduc ion
Ni oa oma ic compounds exis in na u e due o hei ex ensi e
use in he p oduc ion o dyes, plas ics and explosi es and also
as in e media e p oduc s om pa ially ans o med
insec icides, o ganophospha e pes icides and he bicides
(Qu eshi e al., 2001). They exhibi high oxici y and/o
mu agenici y o plan s, animals and mic obes and pose heal h
and en i onmen al isks, ei he di ec ly o h ough some o
hei ca abolic me aboli es (Rehman e al., 2007). Se e al
ni oa oma ic compounds a e powe ul ca cinogens (Kulka ni
and Chaudha i, 2007) and se e al o hem a e lis ed as p io i y
pollu an s (US EPA, 2007). p-Ni ophenol (PNP) is a
ni oa oma ic compound and US EPA (2007) has lis ed PNP
along wi h se e al o he ni oa oma ics as p io i y pollu an s.
I is also a b eakdown p oduc o pa a hion and me hyl
pa a hion, o ganophospha e pes icides widely used in
de eloping coun ies including India. Thus, biodeg ada ion
s udies o p-ni ophenol (PNP) a e o p ime impo ance
(Debananda and Ning houjam, 2008).
Recen esea ch had epo ed se e al bac e ia capable o
deg ading PNP including he s ains o A h obac e and
Noca dia (Hanne e al., 1993), Bacillus sphae icus (Kadiyala and
Spain, 1998), B e ibac e ium linens (Ning houjam, 2005),
D . Ning houjam Sho a ani
176
Mo axella (Spain e al., 1979), Noca diodes ni ophenolicus (Yoon e
al., 1999), Rhodobac e capsula us (Roldan e al., 1998),
Sphingomonas sp. (Zabla owicz e al., 1999), Pseudomonas
ae uginosa s ain DN1 (Ning houjam and Sho a ani, 2008). We
ha e ecen ly isola ed a new p-ni ophenol deg ading Bacillus
sp. s ain BGSC1 by selec i e en ichmen in M63 medium. The
p esen s udy deals wi h he isola ion, cha ac e iza ion and
biodeg ada ion s udy o his PNP deg ading s ain.
2. Ma e ials and Me hods
2.1 Media and cul u al condi ions
M63 medium had he ollowing composi ion (pe li e): 5.8g
Na2HPO4, 3.0g KH2PO4, 0.5g NaCl, 1.0g NH4Cl
(Gunaseka an,2000) and was used o en ichmen s udies. Basal
Sal s Medium (BSM) had he composi ion (pe li e): 0.25g
FeCl3.6H2O, 22.5g MgSO4.7H2O, 27.5g CaCl2, 40.0g (NH4)2SO4
and phospha e bu e (pH 7.0) and was used o deg ada ion
s udies in liquid as well as solid media. The sole ca bon sou ce
used in cul i a ion media was p-ni ophenol (20mg/L). The
cul u e empe a u e was kep a ambien condi ions wi h
shaking a 170 pm and pH 7.0.
2.2 En ichmen and Isola ion
A local ga den soil sample was used as an inoculum o
en ichmen o PNP-deg ading mic oo ganisms. Ae obic shake
lask cul u es we e se up in M63 medium (pH, 7.0) and
inocula ed wi h il e ed soil suspension (10% / ) de i ed om
10g ga den soil mixed wi h 100ml dis illed wa e and shaken
o 1 hou a ambien empe a u e. A e 2 o 3 mon hs o
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
177
incuba ion, 0.1mL aliquo om he en ichmen lask was aken
and sp ead on s e ile Nu ien Aga (NA) pla es and incuba ed
a 300C (24-48 h). Visible colonies we e picked up and hen sub-
cul u ed on NA pla es and slan s. We ob ained ou di e en
isola es g owing as bluish g een, whi e (wi h hizoid pa e n),
an colo and whi ish colonies. On u he in es iga ion, wo
s ains we e ound o be p omising PNP deg ade s i.e. bluish
g een s ain now epo e d as Pseudomonas ae uginosa DN1
(Ning houjam and Sho a ani, 2008) and whi e s ain wi h
hizoid pa e n o g ow h (BGSC1). Deg ada ion was
moni o ed by isible u bidi y and/o disappea ance o
cha ac e is ic yellow colo o PNP. PNP deple ion was also
moni o ed by ollowing he abso bance o alkalinized cul u e
supe na an s a 400 nm. The biodeg ading s ains we e hen
sub-cul u ed o se e al gene a ions in BSM con aining a ious
concen a ions o PNP. I was main ained on NA slan s and
BSM + PNP slan s.
2.3 Cha ac e iza ion o BGSC1
2.3.1: Pheno ypic cha ac e iza ion
Pheno ypic cha ac e is ics include all hose ea u es o a s ain
such as mo phological, biochemical and physiological
p ope ies. Pheno ypic cha ac e iza ion o he s ain BGSC1
was done acco ding o s anda d p o ocols (Halebian e al., 1981;
Gunaseka an, 2000; Cappuccino and She man, 2004; A las,
1997, A las, 2010.G oss mo phology was obse ed by isual
inspec ion. G ow h was obse ed a di e en pH, empe a u es
and sal concen a ions on NA pla es as well as on a ious
g ow h media such as pep one wa e aga , ap wa e aga , 0.1%
D . Ning houjam Sho a ani
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yeas aga , ace amide aga e c. All he o he es s, when no
speci ically men ioned, a e as pe Be gey’s Manual o
De e mina i e Bac e iology and o he s anda d p ocedu es.
Liquid cul u es o BGSC1 we e analyzed wi h he Bio Me ieux
API 50CH es s ips as pe manu ac u e ’s guidelines o
ca bohyd a e u iliza ion.
2.3.2 Geno ypic cha ac e iza ion
2.3.2.1. 16S DNA sequencing
The 16S RNA gene sequence o he s ain BGSC1 was
submi ed o EzTaxon se e (Chun e al., 2007) and aligned
wi h he 16S RNA gene sequences o o he Bacillus species
using Clus al X2 e sion 2.1 (La kin e al., 2007). Phylogene ic
analysis was done using he so wa e package MEGA e sion
4.0 (Tamu a e al., 2007). Dis ances (using dis ance op ions
acco ding o Kimu a’s wo-pa ame e model; Kimu a, 1983)
we e calcula ed and clus e ing was pe o med wi h he
neighbou joining me hod (Sai ou and Nei, 1987). To de e mine
he suppo o each clade, boo s ap analysis was pe o med
wi h 1000 esamplings (Felsens ein, 1985). The 16S DNA
sequence o BGSC1 was pe o med wi h he help o Ins i u e o
Mic obial Technology (IMTECH) Chandiga h, India.
2.4. Biodeg ada ion s udies o p-ni ophenol
2.4.1. Iden i ica ion o maximum abso bance o PNP
Fo he iden i ica ion o maximum abso bance o PNP, 50
mg/L was used as he s anda d. I was hen scanned h ough
UV-VIS spec opho ome e a a ious wa eleng hs and he
maximum abso bance o PNP was de e mined.
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
179
2.4.2. Op imiza ion o medium
Op imiza ion o medium is necessa y o e ec i e deg ada ion
o xenobio ics. Va ious minimal media we e examined o PNP
deg ada ion a pH 7.0 such as BSM (Ning houjam, 1998),
Minimal Sal s Medium (MSM) (Liu e al., 2007) and Minimal
Medium (MM) (Li e al., 2008). 5% inoculum om he cell
suspension o PNP induced cells (A600 = 1) we e inocula ed in
he di e en media and he deg ada ion imes we e measu ed.
Among he media es ed he medium in which deg ada ion
occu ed in minimum ime was selec ed as he op imal medium
o u he deg ada ion s udies.
2.4.3. E ec o pH on deg ada ion
Op imiza ion o di e en en i onmen al and cul u al
pa ame e s is necessa y along wi h medium op imiza ion o
e icien biodeg ada ion. pH, empe a u e, sal concen a ion
e c. a e some o he pa ame e s in luencing biodeg ada ion.
Va ious pH alues (pH 5.5-12) we e checked o hei e ec s on
deg ada ion. Op imum pH o deg ada ion o PNP (50 mg/L)
was hen de e mined.
2.4.4. E ec o di e en inoculum sizes on deg ada ion
Inoculum densi y is also one o he cul u al condi ions ha
a ec biodeg ada ion. Su icien amoun o inoculum is
necessa y o success ul deg ada ion. E ec o di e en
inoculum sizes on deg ada ion o PNP was s udied o iden i y
he op imum inoculum size o e ec i e PNP deg ada ion.
D . Ning houjam Sho a ani
180
2.4.5. PNP deple ion assay
To de e mine he ime cou se o PNP biodeg ada ion, samples
we e collec ed a a ious ime in e als. The samples we e hen
cen i uged a 8,000 pm o 15 minu es and he supe na an s
we e collec ed. The pH o he supe na an s was b ough o 10.0
using 1N NaOH. The supe na an s we e checked o PNP
deple ion by measu ing he abso bance a 400 nm.
2.4.6. Ni i e libe a ion assay
Ni i e libe a ion du ing PNP deg ada ion was measu ed
spec opho ome ically acco ding o s anda d p o ocol
(Mon gome y and Dymock, 1961: Ning houjam 1998). I was
de e mined h ough he o ma ion o a eddish pu ple dye
p oduced by coupling sulphanilic acid and α- nap hylamine
and used by compa ing alues wi h hose o s anda d cu e
p epa ed using sodium ni i e. Highe concen a ion o NO2-
can be de e mined by dilu ing he samples.
3. Resul s
3.1. Isola ion o BGSC1
F om en ichmen cul u e, ou di e en isola es we e ob ained
exhibi ing bluish g een, di y whi e (wi h hizoid pa e n o
g ow h), pi ch colou and whi ish colonies. Th ee isola es we e
ound o be p omising PNP deg ade s. O hese, BGSC1 is he
PNP deg ading isola e which o med hizoid pa e n o g ow h
(Fig A).
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
181
Fig A: BGSC1 g owing on pep one wa e aga
3.2. Pheno ypic cha ac e iza ion o BGSC1
3.2.1. Mo phological cha ac e iza ion
BGSC1 colony was whi ish wi h d y su ace. The colonies we e
la and hizoid (ha ing ma gins wi h oo -like ilamen s). The
o ganism was g am posi i e, exhibi ing ods wi h agmen ed
chain a angemen . The o ganism was non-mo ile wi h
ellipsoidal endospo es loca ed cen ally. The agmen ed chain
a angemen was u he con i med by mic oscopic
obse a ions using me hylene blue, c ys al iole and nega i e
s aining p ocedu es (Table 1a). I showed luxu ian ilamen ous
and agmen ing mycelia on poo nu ien media such as 0.1%
yeas aga , pep one wa e aga and ap wa e aga and
luxu ian ilamen ous g ow h on Mulle Hin on aga , Nu ien
aga , Sabou aud aga and S ep omyces aga (Table 1b).
Table 1a: Mo phological cha ac e is ics o BGSC1
Sl.No.
Tes s
Resul s
1.
Colony Mo phology
i) Ele a ion
Fla
D . Ning houjam Sho a ani
182
ii) Ma gin`
Rhizoid pa e n
iii) Su ace
D y
i ) Colou
Di y whi e
2.
G am’s Reac ion
+ e
i) Shape
Rod
ii) A angemen
F agmen ed chain
3.
Spo e P oduc ion
Ellipsoidal
endospo e
4.
Mo ili y
- e
5.
S ing es (3% KOH)
- e
6.
Capsule S ain
- e
7.
Mo ili y
- e
8.
Me hylene blue s ain,
c ys al iole s ain,
nega i e s ain
Rods in agmen ed
chains
Table 1b: G ow h cha ac e is ics o BGSC1 on a ious media
Sl.
no.
G ow h
media
G ow h esul
1.
Pep one
wa e aga
G ow h wi h b anching ib es ( hizoid
o m) wi hin 24h .
2.
Tap wa e
aga
Thin g ow h wi h ligh b anching ib es
( hizoid o m)
3.
Yeas (0.1%)
aga
Thin g ow h wi h b anching ib es
( hizoid o m) wi hin 24 h .
4.
Nu ien
aga
G ow h wi h b anching ib es ( hizoid
o m) wi hin 24 h .
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
189
D . Ning houjam Sho a ani
190
Fig. B: Phylogene ic ee based on 16S RNA gene sequences
showing he posi ion o s ain BGSC1 among he ype
species o he genus Bacillus. Boo s ap alues (in
pe cen age) a e shown a he node o each b anch
3.4 Biodeg ada ion s udies o p-ni ophenol
3.4.1. Op imiza ion o medium
Among a ious cul u e media es ed such as MSM, MM and
BSM. BSM was ound o be he op imal medium o
deg ada ion o PNP by BGSC1 (Table 2a). Among he a ious
concen a ions o BSM media es ed, no mal s eng h BSM was
obse ed o be op imal o deg ada ion o PNP and i was used
as he s anda d medium o u he deg ada ion s udies (Table
2b).
Table 2a: Deg ada ion o PNP (50 mg/L) in a ious media a
pH 7.0.
Sl.
no.
Medium
Deg ada ion ime
(h)
Final OD
(A600) nm
1
BSM
39. 8 ± 0.115
0.083 ± 0.007
2
MSM
42.3± 0.347
0.066 ± 0.024
3
MM
42.3 ± 0.824
0.085 ± 0.006
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
191
(No e: Ini ial OD a 600nm = 0.064- 0.070)
Table 2b. Deg ada ion o PNP (20 mg/L) in a ious
concen a ions o BSM a pH 7.0.
(No e: Ini ial OD a 600nm = 0.065-0.070.)
3.4.2. E ec o pH on deg ada ion
E ec o pH on deg ada ion o PNP (20 mg/L) is shown in Table
3. Deg ada ion o PNP occu ed be ween pH 6 - 10 bu no
deg ada ion was obse ed a pH 5.5 and pH 11.0. Op imal pH
o deg ada ion o PNP by BGSC1was ound o be pH 7.0.
Table 3. E ec o pH on deg ada ion o PNP (20 mg/L).
pH
alues
Deg ada ion
ime
(h)
Final OD
(A600) nm
Ra e o PNP
deg ada ion
mg L-1 h-1
5.5
No deg ada ion
ND
ND
6.0
37.4 ± 0.108
0.065 ±
0.010
0.53
7.0
19.3 ± 0.108
0.086 ±
0.002
1.04
8.0
20.3 ± 0.216
0.087 ±
0.004
0.99
Sl.
no.
Medium
Deg ada ion ime
(h)
Final OD
(A600) nm
1
BSM
24.1 ± 0.414
0.080 ± 0.010
2
5X BSM
24.5 ± 0.374
0.085 ±0.008
3
10x BSM
31.0 ± 0.148
0.072 ±0.015
D . Ning houjam Sho a ani
192
9.0
21.1 ± 0.674
0.088 ±
0.007
0.95
10.0
24.7 ± 0.738
0.071 ±
0.008
0.81
11.0
No
deg ada ion
ND
ND
(No e: Ini ial OD a 600 nm = 0.072-0.077; ND= No
De e mined.)
3.4.3. E ec o di e en inoculum sizes on deg ada ion
5% inoculum was ound o be op imal o deg ada ion o PNP
(20 mg/L). De ailed indings on he e ec s o inoculum sizes on
PNP biodeg ada ion a e shown in Table 4 and Fig C.
Table 4. E ec o di e en inoculum sizes on deg ada ion o
PNP (20 mg/L)
Inoculum
sizes (%)
Deg ada ion
ime (h)
Ini ial OD
(A600) nm
Final
OD
(A600)
nm
Ra e o PNP
deg ada ion
mgL-1 h-1
1
32.6± 0.071
0.014±0.005
0.014
±
0.001
0.613
2
27.4 ± 0.492
0.036±
0.010
0.023
±
0.006
0.729
4
21.6 ± 0.070
0.058±0.007
0.043
±
0.012
0.925
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
193
5
19.8 ± 0.247
0.065±0.004
0.060
±
0.001
1.010
6
19.9 ± 0.105
0.068±0.002
0.070
±
0.005
1.005
7
19.9 ± 0.050
0.075±0.005
0.075
±
0.003
1.005
Fig. C: E ec o di e en inoculum sizes on deg ada ion o
PNP (20 mg/L).
D . Ning houjam Sho a ani
194
3.4.4. PNP deple ion assay
The ime cou se o deg ada ion o PNP (20 mg/L) wi h and
wi hou ni ogen sou ce a e shown in Figs D(a) and D(b). The
comple e deg ada ion o PNP (20 mg/L) was obse ed a 22 h in
BSM wi h ni ogen sou ce (Table 5 and Fig Da) and a 20 h in
BSM wi hou ni ogen sou ce (Table 5 and Fig Db).
Fig.D(a): Abso p ion spec a
o PNP (20mg/L) in
BSM medium
wi hou N-sou ce a
a ious ime
pe iods
Fig. D(b): Abso p ion spec a
o PNP (20 mg/L) in
BSM medium wi h
N-sou ce a a ious
ime pe iods.
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
195
3.4.5. Ni i e Libe a ion
Ni i e was eleased almos s oichiome ically du ing
deg ada ion o 20 mg/L o PNP. 98.4% o ni i e was eleased a
he end o deg ada ion (Table 6a and Fig Fa) in p esence o
ni ogen sou ce and 91.1% in absence o ni ogen sou ce (Table
6b and Fig Fb). The concen a ion o ni i e was calcula ed based
on he s anda d cu e as shown in Fig E.
Table 6a: Ni i e libe a ion in BSM wi h n-sou ce
Time (h)
A543 (nm)
NO2-(µmoles/L)
Pe cen age (%)
0
0.012
0.6
0.42
4
0.122
7.2
5.00
8
0.411
24.2
16.83
12
0.705
41.4
28.79
16
0.834
48.9
34.01
18
0.867
51.6
35.89
19
0.880
52
36.16
20
0.988
58.4
40.62
22
1.068
63
43.81
24
1.180
70
48.68
26
1.760
104
72.33
28
2.070
122
84.85
30
2.185
129
89.72
32
2.270
134
93.20
34
2.310
136.5
94.94
36
2.400
141.5
98.42
D . Ning houjam Sho a ani
196
Fig. F(a): PNP deple ion
and ni i e libe a ion in BSM
wi h N-sou ce
Fig. F(b): PNP deple ion and
ni i e libe a ion in BSM
wi hou N-sou ce
Table 6b: Ni i e libe a ion in BSM wi hou N-sou ce
Time (h)
A543 (nm)
NO2-
(µ moles/L)
Pe cen age (%)
0
.004
0.2
0.14
4
.247
14.6
10.15
8
.671
39.6
27.54
12
1.280
75
52.16
16
1.335
78
54.25
18
1.360
81
56.33
19
1.385
81.5
56.68
20
1.410
83.5
58.07
22
1.475
86.5
60.16
A Bacillus Sp. S ain Bgsc1 Tha E icien ly Deg ades
197
24
1.550
91
63.29
26
1.565
92
63.99
28
1.605
95
66.07
30
1.695
100
69.55
32
2.044
119.7
83.25
34
2.060
122
84.85
36
2.220
131
91.11
Fig. E: S anda d g aph o NO2 – assay.
4. Discussion
BGSC1 is an ae obic, g am posi i e, od-shaped, spo e o ming
and non-mo ile o ganism. The colonies show hizoid g ow h
on aga pla es. Simila mo pho ypes o hizoidal g ow h
exhibi ed by some Bacillus spp. ha e been epo ed in he
D . Ning houjam Sho a ani
198
li e a u e (Flugge, 1886; Gause, 1939; Nakamu a and Jackson,
1995; Nakamu a, 1998; Aa hi and Ramana, 2011). Cells a e
a anged in agmen ed chains. The s ain showed luxu ian
ilamen ous g ow h on poo nu ien media such as pep one
wa e aga , yeas ex ac aga and ap wa e aga . The
o ganism showed posi i e esul s in ca alase, oxidase, casein
and s a ch hyd olysis es s. Acid was p oduced om
ca bohyd a es such as glucose and suc ose bu no om
lac ose and mal ose. BGSC1 did no hyd olyze lipid and could
no u ilize ci a e. Aa hi and Ramana (2011) epo ed Bacillus
mycoides and Bacillus ce eus s ains which showed biochemical
and physiological cha ac e is ics simila o hose o BGSC1.
Nakamu a (1998) also epo ed a s ain o Bacillus
pseudomycoides which showed cha ac e is ics simila o ha o
BGSC1. O e all, mos o he biochemical cha ac e is ics o
BGSC1 esembled hose o Bacillus ce eus g oup as epo ed by
Claus and Be keley (1986). The op imum empe a u e o
g ow h o BGSC1 was 300C. Simila ly, Aji hkuma e al. (2002)
had also epo ed ha he op imum empe a u e o g ow h o
Bacillus uniculus was 300C bu in con as a s ain o Bacillus
pseudomycoides showed op imum empe a u e o g ow h a
280C (Nakamu a, 1998). BGSC1 could g ow a pH 5.0 – 9.0
wi h op imum a pH 7.0. Aji hkuma e al. (2001) and Saman
e al. (2010) ha e also epo ed s ains o ilamen ous Bacillus
and Bacillus ce eus g oup C1 which could g ow op imally a pH
7.0. BGSC1 could ole a e mode a e concen a ions o NaCl
(0.5% o 6.0%). I was ound o be sensi i e o se e al
an ibio ics es ed. API 50CH es s showed ha he s ain
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