World Journal of Microbiology and Biotechnology

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World Journal of Microbiology and Biotechnology
Volume 21, Number 3
Production of single cell protein through fermentation
of a perennial grass grown on saline lands with
Cellulomonas biazotea
(207 - 211)
M. Ibrahim Rajoka
DOI: 10.1007/s11274-004-2889-6
Chromate reduction by Bacillus megaterium TKW3
isolated from marine sediments
(213 - 219)
K. H. Cheung and Ji-Dong Gu
DOI: 10.1007/s11274-004-3619-9
Optimization of redox reactions employing whole cell
biocatalysis
(221 - 227)
Abhishek A. Chakraborty, Ravindra P. Phadke, Fauzia A. Chaudhary, Prakash S. Shete,
Bhalchandra S. Rao, Kushan D. Jasani
DOI: 10.1007/s11274-004-3620-3
Formation of Yersinia pseudotuberculosis biofilms on
multiple surfaces on Caenorhabditis elegans
(229 - 231)
Marc A. Nascarella and Steven M. Presley
DOI: 10.1007/s11274-004-5299-x
Xylanase production under solid-state fermentation and
its characterization by an isolated strain of Aspergillus
foetidus in India
(233 - 243)
Amita R Shah and Datta Madamwar
DOI: 10.1007/s11274-004-3622-1
Use of RAPD to investigate the epidemiology of
Staphylococcus aureus infection in Malaysian hospitals
(245 - 251)
V. Neela, N. S. Mariana, S. Radu, S. Zamberi, A. R. Raha, R. Rosli
DOI: 10.1007/s11274-004-3624-z
Isolation of Enterobacteria able to degrade simple
aromatic compounds from the wastewater from olive oil
extraction
(253 - 259)
Emna Ammar, Moncef Nasri, Khaled Medhioub
DOI: 10.1007/s11274-004-3625-y
Effects of Zn supplementation on the growth, amino acid
composition, polysaccharide yields and anti-tumour
activity of Agaricus brasiliensis
(261 - 264)
Xiang Zou
DOI: 10.1007/s11274-004-2614-5
Isolation and characterization of a new carbendazim-
degrading Ralstonia sp. strain
(265 - 269)
Gui-Shan Zhang, Xiao-Ming Jia, Tian-Fan Cheng, Xiao-Hang Ma, Yu-Hua Zhao
DOI: 10.1007/s11274-004-3628-8
Survey of plasmid profiles of Shigella species isolated in
Malaysia during 1994–2000
(271 - 278)
C. H. Hoe, R. M. Yassin, Y. T. Koh, K. L. Thong
DOI: 10.1007/s11274-004-3631-0
Physico-chemical characterization of exomannan from
Rhodotorula acheniorum MC
(279 - 283)
K. Pavlova, I. Panchev, Ts. Hristozova
DOI: 10.1007/s11274-004-3632-z
Isolation and characterization of Bacillus thuringiensis
strains from different grain habitats in Turkey
(285 - 292)
Özgür Apaydin, A. Fazil Yenidünya, Şebnem Harsa, Hatice Güneş
DOI: 10.1007/s11274-004-3633-y
Preliminary studies on chorote – a traditional Mexican
fermented product
(293 - 296)
Marisol Castillo-Morales, María del Carmen Wacher-Rodarte, Humberto Hernández-
Sánchez
DOI: 10.1007/s11274-004-3634-x
Biodegradation of pentachorophenol by tropical
basidiomycetes in soils contaminated with industrial
residues
(297 - 301)
Kátia Maria Gomes Machado, Dácio Roberto Matheus, Regina Teresa Rosim Monteiro,
Vera Lúcia Ramos Bononi
DOI: 10.1007/s11274-004-3693-z
Solid-state fermentation of wood residues by Streptomyces
griseus B1, a soil isolate, and solubilization of lignins
(303 - 308)
Anju Arora, Lata Nain, J. K. Gupta
DOI: 10.1007/s11274-004-3827-3
Detecting the heavy metal tolerance level in ectomycorrhizal
fungi in vitro
(309 - 315)
Prasun Ray, Richa Tiwari, U. Gangi Reddy, Alok Adholeya
DOI: 10.1007/s11274-004-3572-7
Potential of Rhodococcus erythropolis as a bioremediation
organism
(317 - 321)
Alena Čejková, Jan Masák, Vladimír Jirků, Martin Veselý, Miroslav Pátek, Jan Nešvera
DOI: 10.1007/s11274-004-2152-1
Production of the ligninolytic enzymes by immobilized
Phanerochaete chrysosporium in an air atmosphere
(323 - 327)
Guoce Yu, Xianghua Wen, Yi Qian
DOI: 10.1007/s11274-004-3571-8
Use of cereals as basal medium for the formulation of
alternative culture media for fungi
(329 - 336)
A. O. Adesemoye and C. O. Adedire
DOI: 10.1007/s11274-004-3907-4
Investigation of the active site of the extracellular β-
D
-
glucosidase from Aspergillus carbonarius
(337 - 343)
Szilvia Jäger and László Kiss
DOI: 10.1007/s11274-004-2609-2
Effect of frozen storage and aging on the Kashkaval cheese starter
culture
(345 - 350)
Zh. I Simov and G. Y. Ivanov
Characteristics of the bacteriocin produced by Lactococcus lactis
subsp. cremoris CTC 204 and the effect of this compound on the
mesophilic bacteria associated with raw beef
(351 - 358)
R. Bromberg, I. Moreno, R. R. Delboni, H. C. Cintra, P. T. V Oliveira
DOI: 10.1007/s11274-004-2610-9
The effect of salinity on trichloroethylene co-metabolism by mixed
cultures enriched on phenol
(359 - 365)
Chi-Yuan Lee, Yu-Chia Chan, Chin-Lung Lin
DOI: 10.1007/s11274-004-2611-8
Microbial decolorization of reactive azo dyes under aerobic
conditions
(367 - 370)
K. M. Kodam, I. Soojhawon, P. D. Lokhande, K. R. Gawai
DOI: 10.1007/s11274-004-5957-z
Rapid method for the affinity purification of thermostable α-amylase
from Bacillus licheniformis
(371 - 375)
M. Damodara Rao, B. V. V. Ratnam, Dasari VenkataRamesh, C. Ayyanna
DOI: 10.1007/s11274-004-3908-3

Production of single cell protein through fermentation of a perennial grass grown on
saline lands with
Cellulomonas biazotea
M.Ibrahim Rajoka
National Institute for Biotechnology and Genetic Engineering,P.O.Box 577,Faisalabad,Pakistan
(Tel.:+92-41-651475,+92-041-550815,Fax:+92-41-651472,E-mail:mirajoka@nibge.org)
Received 12 May 2004;accepted 30 June 2004
Keywords:Cellulomonas biazotea
,growth of bacteria,kinetics of production,
Leptochloas fusca
Summary
Microbial protein from alkali-treated
Leptochloa fusca
(kaller grass) was produced by growing
Cellulomonas
biazotea
in shake flasks and in an aerated 6-l fermentor.Single cell protein,produced in the fermentor contained
56.10 ±4.64,60.00 ± 5.04,11.50 ±1.34,12.95 ± 1.24,3.50 ± 0.24 and 1.00 ± 0.44% true protein,crude
protein,crude fibre,ash,cellulose and RNA content respectively.Maximum values compared favourably with
published data.The biomass contained all desired amino acids with isoleucine as limiting acid.The dried biomass
showed a gross metabolizable energy value of 3500 kcal kg
)
1
and indicated that it might serve as energy as well as a
protein source particularly when fed to poultry.
Introduction
Single cell protein (SCP) has attracted commercial
interest due to the possible substitution of microbial
protein for the conventional protein supplements cur-
rently being used in the dairy and poultry industries
(Rosenberg 1993;Chanda & Chakrabarti 1996).Micro-
organisms have the ability to upgrade low protein plant
material to high protein feed.Large-scale fermentation
of methanol,starch and molasses-based media have
proved economically viable for the production of animal
feed and human food (Rosenberg 1993;Hongpattarak-
ere & H-Kittikun 1995;Paul
et al.
2002).
Production of SCP from lignocellulosic (LC) agricul-
tural biomass is of immense importance to get high
quality protein in developing countries (Rajoka 1990;
Bajpai &Bajpai 1991).The product can be fed to poultry
and livestock as cheaper rations for the production of
eggs,milk and meat (Rosenberg 1993).Moreover,SCP
will save cereals for human consumption in third world
countries (Hongpattarakere & H-Kittikun 1995).At the
moment,cereals are being used in livestock and poultry
feeds.Efforts are still being made to develop a process to
produce low cost SCP through fermentation of abun-
dantly available agro-industrial wastes (Rosenberg 1993;
Chanda & Chakrabarti 1996).
Many workers have used pure
Cellulomonas
strains or
mixed culture of bacteria for production of SCP from
cellulose-based raw materials (Hitchner & Leatherwood
1980;Enriquez & Rodriquez 1983;Rodriguez-Vesquez
et al
.1992;Pece
et al.
1994).
Leptochloa fusca
(kallar
grass) can be inexpensively raised on highly saline lands
(one-third of cultivable land in Pakistan) without
application of fertilizer and can produce biomass (up
to 50 metric tonnes ha
)
1
year
)
1
) for biotechnical appli-
cations (Latif
et al
.1994;Rajoka & Malik 1997).The
cost of pre-treatment is also very low ($0.03 kg
)
1
).
C.
biazotea
effectively utilized this substrate and produced
more cell mass than that produced on wheat straw,
bagasse,
Sesbania aculeate
,
Panicum maximum
,cotton
Nomenclature
X
cell mass (g l
)
1
)
S
substrate (g l
)
1
)
Q
x
rate of cell mass formation (g cells l
)
1
h
)
1
)
Q
s
rate of substrate consumption (g l
)
1
h
)
1
)
Y
X/S
cell yield coefficient (g cells g
)
1
substrate
utilized)
q
s
specific rate of substrate consumption
(g g
)
1
cells h
)
1
)
l
specific growth rate (h
)
1
)
Q
CP
rate of crude protein formation (g l
)
1
h
)
1
)
Y
CP/X
specific yield of crude protein (g crude
protein g
)
1
cells)
SCP single cell protein:
Leptochloa fusca
(kallar
grass) grown cell mass
DO dissolved oxygen (%)
v/v/m air flow rate (volume of air per volume of
fermentation medium per min.)
q
CP
specific rate of crude protein productivity
(g g
)
1
cells h
)
1
)
LC lignocellulosic
World Journal of Microbiology & Biotechnology (2005)
21
:207–211

Springer 2005
DOI 10.1007/s11274-004-2889-6
stalks and
Atriplex lentiformis
(Rajoka & Malik 1997).
SCP produced on kallar grass is expected to possess a
high nutritive value as reported earlier (Enriquez &
Rodriquez 1983;Rajoka 1990;Zayed & Mostafa 1992;
Pece
et al
.1994).
Cell mass and product formation kinetic parameters
need to be studied for scaling-up to large bioreactors
(Tobajas & Garcia-Calvo 1999).Use of kallar grass for
enzyme production has already been studied (Rajoka &
Malik 1997;Rajoka
et al.
1997) but no detailed
information is available on its use for production of
single cell protein.Moreover,information on the
detailed kinetics of SCP production from LC substrates
is not available.In this work,it was planned to study the
kinetics of SCP production in greater detail by culturing
C.biazotea
on alkali-pre-treated kallar grass which
contained 6.5 ± 0.45,11.82 ± 1.04,57.37 ± 4.13,
55 ± 4.54 and 23.00 ± 2.00% crude protein,crude
fibre,nitrogen-free extract,cellulose and hemicellulose
respectively and evaluate it for crude protein,RNA,
metabolizable energy and amino acid content,so as to
work out the suitability of the single cell protein product
as a poultry and livestock feed supplement.
Materials and methods
Organism
Cellulomonas biazotea
NIAB 442 was maintained on
Dubos-Sigmacell 100-agar slants.The inoculum was
prepared by transferring a loopful of cells to 50 ml seed
culture mediumcontaining (g l
)
1
)KH
2
PO
4
1.0,NaNO
3
0.5,KCl 1.0,MgSO
4
,0.5,yeast extract 0.2 (pH
7.0 ± 0.1) (Rajoka & Malik 1997) and grown at
30

C on an orbital shaker (150 rev min
)
1
for 24 h).
Dry cell mass was estimated using the pre-determined
conversion factor of 1 g dry cell weight per l for unit
absorbance at 610 nm.The conversion factor was
calculated from the relationship between absorbance
and dry cell mass (g per l).Concentration of the culture
was adjusted to contain 2.5 ± 0.14 g dry cells l
)
1
of the
inoculum medium.
Substrate
Leptochloa fusca
(kallar grass) was collected from
Lahore Biosaline Research Substation of Nuclear Insti-
tute for Agriculture and Biology (NIAB),Faisalabad,
where it is grown as an energy crop from highly saline
waste lands in Pakistan (Latif
et al
.1994).It contained
55 ± 1.2 and 23 ±2.4% cellulose and hemicellulose
respectively and were determined as described previ-
ously (Latif
et al
.1994).
Shake flask studies
The ability of the organism to produce cell mass and
protein from pre-treated kallar grass straw as a sole
carbon and energy source was examined in optimized
salt mediumcontaining (g l
)
1
):KH
2
PO
4
1.0,NaNO
3
4.0
(instead of 0.5),KCl 1.0,MgSO
4
0.5,FeSO
4
0.1;yeast
extract 2.0 and alkali-treated straw (1.5 g total carbo-
hydrates).All media were adjusted to pH 7.3 with 1 M
NaOH or 1 M HCl.The data of batch fermentation
were gained by performing experiments (three runs) on
above media under optimized conditions in shake flasks
(Rajoka
et al
.1997).Sample flasks in triplicate were
withdrawn periodically to follow the assay of dry cell
mass,crude protein,true protein and RNA.
Fermentor studies
Fermentation was carried out in 6-l Eyela fermentor
(Model M-160 Eyela,Tokyo,Japan) fitted with auto-
matic pH,temperature,dissolved oxygen tension
(DOT),agitation and airflow rate controls.The opti-
mized medium (4 l) was steam sterilized in an auto-
clave.The medium was inoculated with seed culture
(10% v/v inoculum) prepared as above.Standard
temperature,agitation speed,airflow rate and pH were
30

C,400 rev min
)
1
,2 v/v/m and 7.3 respectively.
Silicone oil was used as an antifoaming agent.Samples
in triplicate were collected periodically to follow the
assay of dry cell mass,crude protein,true protein and
RNA.
Analytical methods
Samples in triplicate were taken at different time
intervals (h) to assay concentration of cell,solid
substrate,crude protein,true protein,crude fibre,
nitrogen free extract and RNA (Pacheco
et al.
1997;
Paul
et al.
2002).Culture samples (100 ml) were
centrifuged (7000
·
g
at 10

C for 10 min) to remove
substrate.The substrate was washed twice with saline
and dried to estimate unutilized solid material.The
supernatant (100 ml) was also centrifuged (10,000
·
g
,
10 min).The cell mass was washed twice with saline,
suspended in 10 ml distilled water and dried at 70

C.
The remaining 100 ml sample,containing cell mass and
unutilized substrate was also dried (called dry biomass).
It was routinely analysed for crude protein,true protein,
and RNA as described previously (Pacheco
et al.
1997;
Paul
et al.
2002).The micro-Kjeldahl nitrogen of dry
biomass was multiplied by 6.25 to calculate crude
protein.For determination of true protein,0.5 g
homogenized and dry biomass was treated with 20 ml
(5% v/v) trichloroacetic acid for 5 min with shaking,
placed at 90

C (in an oven) for 15 min and shaken
occasionally.It was filtered while hot and the residue
was rinsed with hot water three times and dried to
constant weight.Its nitrogen content was determined by
the micro-Kjeldahl method and true protein was calcu-
lated as above.Soluble proteins in fermentation broth
were determined by the Lowry method.Total RNA
content was determined using orcinol-HCl reagent as
described previously (Pacheco
et al.
1997;Paul
et al.
208
M.Ibrahim Rajoka
2002) and its content was reduced by heat-treatment as
mentioned earlier (Abouzeid
et al
.1995).
The calorific value of dry biomass was determined
using the Parr method(Hill &Anderson1958) witha Parr
oxygen bomb calorimeter.The calorific value was calcu-
lated by the amount of heat generated by the combustion
of a known weight of the sample in the presence of 20 atm
oxygen.Moisture,dry matter,ash,ether extract,crude
fibre,nitrogen free extract,carbon and cellulose were
analysed according to AOAC (1984) methods.
Amino acid analysis and chemical score
The amino acid profile of the SCP was determined by
automatic amino acid analyser (Evans Electroselenium
Limited,UK) on an HCl-hydrolysate of SCP.The
chemical score of the biomass product was calculated
following method of FAO/WHO (1957).
Determination of kinetic parameters
All kinetic parameters were determined as described
earlier (Aiba
et al
.1973;Zayed & Mostafa 1992).
Volumetric rate of crude and true protein production
(Q
P
) was determined from a plot between protein (g l
)
1
)
and time of fermentation (h).Process product yield (Y
P/S
)
was determined fromd
P
/d
S
,specific product yield (
Y
P/x
,
gg
)
1
cells) was determined using relationship d
P
/d
X
,
and volumetric rate of substrate consumption was
determined from a plot between solid substrate (g l
)
1
)
present in the fermentation medium and time of
fermentation (h).Cell mass productivity expressed as
g dry cells l
)
1
h
)
1
was determined from a plot of g dry
cells l
)
1
and time of fermentation.Specific growth rate
was determined from the relationship
l
t
¼
ln
X
t
/
X
o
while specific productivity was a multiple of
l
and
Y
P/X.
Results and discussion
Cultural conditions in shake flask
To maximize the total crude or true protein productiv-
ity,the cultural conditions for the batch culture process
were optimized in shake flasks.Among concentrations
of kallar grass and nitrogen sources employed,1.9%
kallar grass and NaNO
3
(0.40 g 100 ml
)
1
) favoured
maximum crude protein productivity,crude protein
yield,crude protein’s specific yield,cell mass yield and
RNA accumulation in the cells respectively at 30

C.
The cell yield (Table 1) obtained following growth on
kallar grass was higher than that reported by Hitchner &
Leatherwood (1980),Meyer
et al
.(1993) and Pece
et al
.
(1994).
Cultural conditions in 6-l fermentor
Various experiments were conducted in a 6-l fermentor
to test the above-optimized cultural conditions.They
were found to hold good to synthesize more crude
protein,true protein,cell mass productivities and cell
yield and were adopted for detailed studies.There was
enhanced substrate consumption and product formation
in fermentor when the organism was grown on 1.9%
kallar grass in optimized Dubos salts medium (con-
trolled pH 7.3) inoculated with 10%seed culture.When
agitation and aeration rates were investigated in the 6-l
fermentor it was found that
C.biazotea
grew faster with
Table 1.
Comparative kinetic parameters of
C.biazotea
for kallar
grass consumption and protein production following growth under
optimized cultural conditions in shake flask and 6-l fermentor studies.
Kinetic parameter Shake flask 6-l fermentor
l
max
(h
)
1
) 0.12 ±0.011 0.21 ±0.014
Y
x/s
(g cells g
)
1
) 0.48 ±0.04 0.56 ±0.05
Q
s
(g l
)
1
h
)
1
) 0.51 ±0.05 0.59 ±0.071
Q
x
(g cells l
)
1
h
)
1
) 0.31 ±0.06 0.36 ±0.082
Q
CP
(g l
)
1
h
)
1
) 0.48 ±0.03 0.56 ±0.090
Q
TP
(g l
)
1
h
)
1
) 0.45 ±0.02 0.52 ±0.025
q
CP
(g g
)
1
cell h
)
1
) 0.11 ±0.01 0.20 ±0.010
q
TP
(g g
)
1
cell h
)
1
) 0.085 ±0.01 0.16 ±0.011
Y
CP/S
(g g
)
1
substrate) 0.35 ±0.02 0.36 ±0.02
Y
CP/X
(g g
)
1
cells) 0.88 ±0.05 1.00 ±0.05
Y
TP/X
(g g
)
1
cells) 0.71 ±0.04 0.86 ±0.06
RNA (after heat treatment) 1.12 ±0.12 1.0 ±0.08
Each value is a mean of three independent experiments.±Stands
for standard deviation among replicates.
Figure 1.
Kinetics of crude protein (CP) (
s
),true protein (TP) (
n
),
cell mass (X)
ð
(
Þ
and solid kallar grass (
,
) present in the fermentation
broth in shake flask (a) and continuously stirred tank fermentor (b).
The initial pH of the medium containing 1.9% (w/v) substrate and
inoculated with 10% (v/v inoculum size) was regulated at 7.3,and
temperature 30

C.Error bars show standard deviation among three
replicates.
Production of SCP from a grass
209
a high aeration rate.Maximum growth rate and protein
productivities were realized at an agitation speed of 400
rev min
)
1
and an aeration rate of 2.0 v/v min at 30

C
throughout the fermentation period of 3 days.
For comparative studies of both fermentation pro-
cesses,C.
biazotea
was permitted to grow (in triplicate)
in shake flasks,and 6-l fermentor respectively in
optimized fermentation conditions.The kinetics of
crude protein production,cell mass formation and solid
substrate in the medium in shake flasks (a),and 6 l
fermentor (b) is shown in Figure 1.All values of
fermentation attributes were higher in the fermentor
than in the shake flasks (Table 1) and were significantly
higher than those reported by Nigam & Vogel (1991),
Lee & Kim (2000),Nigam (2000) and Paul
et al.
(2002).
There was enhanced substrate metabolism by the
aerobic pathway,resulting in build-up of high cell mass
(maximum cell mass is equal to 8.9 g l
)
1
) (Figure 1).
The values of the above process variables were higher
than those reported previously (Zayed & Mustafa 1992;
Nigam2000) and were attributed to the presence of high
cellulase and xylanase activities in the organism(Rajoka
& Malik 1997;Rajoka
et al.
1997) and in the case of the
fermentor,due to the higher aeration and mass transfer
rates.The improvement of crude protein from 6.3 to
60%with 56.1%as true protein indicated a great deal of
nitrogen source utilization by the cells.Single cell
protein product reported by Singh
et al.
(1991) con-
tained 30.4% crude protein and
Kluyveromyces fragilis
biomass grown on deproteinized when supplemented
with 0.8%diammonium hydrogen phosphate contained
37%crude protein (Paul
et al.
2002).Similarly yeast cell
mass reported by Chanda & Chakrabarti (1996) and
Meyer
et al
.(1993) contained 54.3%,and 47% crude
protein respectively.
Biochemical evaluation of SCP
Compositional analysis of single cell protein obtained
with
C.biazotea
in the fermentor (Table 2) revealed that
dry biomass was rich in crude protein,true protein and
low in RNA and was found superior to those reported
by Singh
et al.
(1991),Abouzeid
et al.
(1995),Nigam
(2002),and Paul
et al.
(2002).The RNA content was
found to be 1.0%which is significantly lower than those
reported by other workers (Nigam & Vogel 1991;Singh
et al.
1991;Paul
et al.
2002).The dry biomass showed a
Table 2.
Nutrient composition (percent) of alkali-treated kallar grass
and the
Cellulomonas biazotea
biomass.
Nutrients Kallar grass
C.biazotea
biomass
Moisture 2.50 ±0.15 0.2 ±0.04
Dry matter 97.50 ±0.04 100.00 ±8.65
Crude protein 6.5 ±0.45 60.0 ±5.04
True protein 0.2 ±0.0 56.1 ±4.64
Ether extract 3.01 ±0.24 3.5 ±0.24
Ash 17.8 ±1.14 12.95 ±1.24
Crude fibre 11.82 ±1.04 11.50 ±1.34
Nitrogen free extract 57.37 ±4.13 0.5 ±0.04
Cellulose 55.0 ±4.54 1.50 ±0.06
Hemicellulose 23.0 ±2.00 0.54 ±0.02
RNA 0.25 ±0.03 1.00 ±0.44
Calorific value (kcal kg
)
1
) 100 ±9.25 3500 ±21.23
Each value is a mean of three replicates.±Stands for standard
deviation among replicates.The product was rich in true protein and
had low cellulose,hemi cellulose and RNA content.
Table 3.
Chemical score of single cell protein obtained through fermentation of alkali-treated kallar grass with
C.biazotea
using FAO/WHO
(1957) amino acid pattern.
Amino Acid SCP Amino acid SCP Available
(AA) AA content pattern
a
AA (%)
(g 100 g
)
1
) (mg g
)
1
) (mg g
)
1
)
Lysine 3.7 ±0.21 42 36.59 87.10
Leucine 5.9 ±0.31 48 58.32 121.50
Isolecucine 3.2 ±0.21 42 31.25 74.40
Phenylalanine 3.6 ±0.24 28 35.72 127.58
Methionine 1.9 ±0.14 22 19.18 87.18
Threonine 2.5 ±0.15 28 24.98 89.21
Valine 4.5 ±0.24 42 45.19 107.59
Alanine 2.1 ±0.14 – – –
Arginine 1.5 ±0.20 – – –
Aspartic acid 3.4 ±0.25 – – –
Cystein/cystine 5.5 ±0.36 – – –
Glutamate 5.5 ±0.45 – – –
Glutamine 1.8 ±0.21 – – –
Histidine 1.6 ±0.12 – – –
Proline 4.8 ±0.34 – – –
Serine 1.6 ±0.15 – – –
Threonine 1.8 ±0.17 – – –
Tyrosine 1.5 ±0.21 – – –
Test samples were hydrolysed with HCl and analysed by automatic amino acid analyser in triplicate.Each value is a mean of three replicates.±
Stands for standard deviation among replicates.
a
FAO/WHO amino acid pattern.The protein product was deficient in isoleucine whose chemical score was 74.4.
210
M.Ibrahim Rajoka
gross metabolizable energy value of 3500 kcal kg
)
1
.The
calorific value clearly indicated that the biomass could
serve as energy source when it may be fed to poultry and
livestock.Single cell protein contained all the amino
acids and the chemical score of seven selected essential
amino acids (Table 3) indicated favourably comparative
values to FAO/WHO (1957) reference protein in mg g
)
1
and confirmed the findings of Nigam (2000).
Conclusions
Kallar grass has shown excellent potential as alternative
energy crop.It gives a massive biomass yield per ha of
saline land.On the basis of SCP production kinetic data
using
C.biazotea
,a yield of 1000 kg per ha could be
obtained.This product contains fairly good quality
protein.The NIAB Laboratories have already intro-
duced kallar grass in our province and is providing
assistance to farmers.More saline lands in the country
are being identified which are not suitable for other
crops and can be used for the cultivation of this
perennial grass for bulk production of SCP economi-
cally for fortification of livestock or poultry feed.
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Production of SCP from a grass
211
Chromate reduction by
Bacillus megaterium
TKW3 isolated from
marine sediments
K.H.Cheung
1
and Ji-Dong Gu
1,2,*
1
Laboratory of Environmental Toxicology,Department of Ecology & Biodiversity,The University of Hong Kong,
Pokfulam Road,Hong Kong SAR,PR China
2
The Swire Institute of Marine Science,The University of Hong Kong,Shek O,Cape d’Aguilar,Hong Kong SAR,
PR China
*
Author for correspondence:Tel.:+852-2299-0605,Fax:+852-2517-6082,E-mail:jdgu@hkucc.hku.hk
Received 8 April 2004;accepted 10 July 2004
Keywords:Bacillus megaterium
,bioremediation,chromate reduction,chromium,metal resistance,selenium
Summary
Bacillus megaterium
strain TKW3 was isolated from multiple-metal-contaminated marine sediments of Tokwawan,
Hong Kong SAR.This facultative aerobe utilized arabinose,mannitol,
N
-acetylglucosamine,maltose,caprate,
citrate,butyrate or lactate as the sole source of carbon and energy for growth.
B.megaterium
TKW3 reduced highly
toxic and soluble Cr
6+
(as CrO
2

4
) into almost non-toxic and insoluble Cr
3+
under aerobic conditions.Complete
reduction of 0.20 mMCr
6+
by
B.megaterium
TKW3 was achieved within 360 h.Initial Cr
6+
concentration below
0.90 mM or inoculum less than 10
7
cells ml
)
1
did not have significant effect on Cr
6+
reduction,while the residue
Cr
6+
concentration was the lowest at 10
7
cells ml
)
1
.Cr
6+
reduction by this strain was inhibited by high levels of
NaCl (55
&
).
B.megaterium
TKW3 was also resistant to other oxyanions including 0.34 mM Cr
2
O
2

7
0.32 mM
AsO
3

4
,0.58 mMSeO
2

3
and 0.53 mMSeO
2

4
,and reduced soluble Se
4+
(as SeO
2

3
) to insoluble red amorphous Se
0
.
B.megaterium
TKW3 might have potential application in bioremediation of Cr-laden sediments associated with
other oxyanions.
Introduction
Chromium (Cr) is used extensively in industries like
electroplating,stainless steel production and wood
preservation (Cheung & Gu 2002;Ryan
et al.
2002).
Although Cr is an essential trace metal to humans,its
potential teratogenicity,mutagenicity,carcinogenicity
and toxicity render it hazardous at very low concentra-
tions and it has been classified as priority pollutant by
the US Environmental Protection Agency (US EPA)
(1998) (Fernando & Flora 1977;Sharma
et al.
1995;
Gibb
et al.
2000).The valency state of Cr ranges from
)
2 to +6,with the hexavalent state (Cr
6+
) like
chromate (CrO
2

4
) and dichromate (Cr
2
O
2

7
) being the
dominant species in natural seawater,the trivalent state
(Cr
3+
) prevalent in wastewater inundated with organics
(Fukai 1967;Jan & Young 1978).In contrast to the
highly toxic and soluble Cr
6+
,Cr
3+
compounds,e.g.
Cr(OH)
3
are almost non-toxic and insoluble which can
be precipitated readily.In wastewater treatment,Cr
6+
reduction is traditionally being catalysed by physico-
chemical methods,which involve initial pH adjustment
with strong acid (e.g.H
2
SO
4
) followed by the addition
of reducing agent (e.g.SO
2
) and re-adjustment of pH
with alkali (e.g.NaOH) for precipitation (Mahajan
1985).
Bioremediation of Cr
6+
using microorganisms has
been studied,where periplasmic biosorption,intracellu-
lar bioaccumulation and biotransformation (mainly as
dissimilatory reduction) through direct enzymatic reac-
tion or indirectly with metabolites are the major
processes (Lovley 1993;Lee
et al.
2000;Valls
et al.
2000).Cr
6+
-reducing bacteria have been isolated,a
number of them belong to the group of anaerobic
sulphate-reducing bacteria (SRB),while studies on
species of
Pseudomonas
,
Bacillus
,
Escherichia
and
Ente-
robacter
have also been conducted (Lovley & Phillips
1994;McLean & Beveridge 2001;Cheung & Gu 2003).
Investigations on
Bacillus megaterium
were previously
focused on its endospore and plasmids,no reports on
multiple-metal resistance and reduction by this species
have been reported (Slepecky & Hemphill 1999).
In the present study,
B.megaterium
TKW3 has been
isolated from marine sediments and was further inves-
tigated for the reduction of Cr
6+
under laboratory
conditions.
World Journal of Microbiology & Biotechnology (2005)
21
:213–219

Springer 2005
DOI 10.1007/s11274-004-3619-9
Materials and methods
Enrichment and isolation
Sediments heavily polluted with various metals,includ-
ing Cr,were collected from a sheltered bay Tokwawan,
Hong Kong SAR in the vicinity of industrial effluent
outlets (HK EPD 2000).5 ml sediment slurry was
transferred into 50 ml nutrient broth (NB) (Difco
Laboratory,Detroit,USA) containing (g l
)
1
) 1.0 beef
extract,2.0 yeast extract,5.0 peptone and 5.0 NaCl,
amended with 1.0 mM Cr
6+
(as K
2
CrO
4
;Fisher Scien-
tific).The inoculated culture was incubated in a rotary
shaker (150 rev/min) at 30

C for 7 days,serving as the
initial enrichment culture.Subsequent enrichment trans-
fer cultures were established in a similar manner with
inoculum from the preceding culture.Pure culture was
obtained fromthe third enrichment transfer culture with
standard spreading plates and streaking techniques on
nutrient agar (Difco Laboratory,Detroit,Michigan)
plates with 1.5% agar.All the procedures above were
conducted aseptically.
Characterization and identification
After Gram staining of the pure bacterial isolate,API
20NE Multiple Kit (bioMerieux,Marcy l’Etoile,
France) was adopted to preliminarily characterize the
isolates following standard protocol according to the
manufacturer’s instructions (bioMerieux 07615C 1998).
The biochemical utilization of the strain was further
studied,5 ml pure culture was inoculated into 60 ml
minimal salt medium (MSM) supplemented with
20 mM of various carbon substrates.The MSM was
composed of (g l
)
1
) 0.8 K
2
HPO
4
,0.2 KH
2
PO
4
,0.05
CaSO
4
Æ
2H
2
O,0.5 MgSO
4
Æ
7H
2
O,0.01 FeSO
4
Æ
7H2O,
and 1.0 (NH
4
)
2
SO
4
(Gu
et al.
1998).The carbon sources
under investigation included sodium salts of succinate,
propionate,pyruvate,butyrate,caproate,formate,ace-
tate,citrate,fumarate,lactate and benzoate;
D
(+)-
glucose,phenol,indole,ethanol,propanol,butanol and
quinoline.
The optimum growing conditions of the isolate were
studied at temperatures of 10,20 and 30

C,and
salinities of 5,35 and 55
&
NaCl.Bacterial growth
was quantified by measuring the protein contents of
liquid culture with the modified Bradford assay (Daniels
et al.
1994) as follows.200
l
l sub-sample was added to
2.5 ml Coomassie Brilliant Blue reagent (100 mg Coo-
massie Brilliant Blue G250,50 ml 95%ethanol,100 ml
85% H
3
PO
4
,1 l distilled water).The absorbance at
wavelength 595 nm (
A
595
) was measured using spectro-
photometer (UV-1201V,Shimadzu,Kyoto) after 5-min
reaction.Concentration of standard protein (Albumin
fromSigma) showed linear relationship between 2.5 and
30
l
gml
)
1
.
For anaerobic growth of the strain,50 ml NB (Difco
Laboratory) in a serum bottle was flushed with pure N
2
for 30 min to purge out O
2
,0.25 g l
)
1
Na
2
S
Æ
9H
2
O was
added to reduce the residual amount of dissolved
oxygen,then capped with a
tert
-butyl rubber stopper
and crimp sealed with aluminium.Resazurin
(1.0
l
gm
)
1
) was added as the redox indicator showing
any potential contamination of O
2
.5 ml pure culture
was inoculated into the anaerobic NB and incubated
with shaking at 150 rev/min and 30

C for the assess-
ment of bacterial growth.All experiments above were
conducted aseptically in duplicate with uninoculated
controls.
Bacterial 16S rDNA extraction and analysis
One ml overnight culture of the bacterium grown in NB
(Difco Laboratory) was centrifuged and the cell pellet
was suspended in 200
l
l extraction buffer (100 mMTris,
100 mM EDTA,200 mM NaCl,1% polyvinylpyrroli-
done (w/v),2%CTAB pH 8.0).Then 200
l
l SDS buffer
consisting of 2%SDS (w/v),10 mMTris,and 200 mM
NaCl (pH8.0) was added.The suspension was extracted
with 400
l
l phenol/Tris-HCl,400
l
l phenol and chlo-
roform–isoamyl alcohol (25:24:1) and 400
l
l chloro-
form/isoamyl alcohol (24:1) with centrifugation at
16,000 rev/min for 5 min,respectively.Bacterial DNA
was precipitated in 400
l
l isopropanol at
)
20

C for 1 h
and centrifuged at 14,000 rev/min for 20 min.DNA was
vacuum-dried,dissolved in 50
l
l sterile distilled water
and stored at
)
20

C before further experiment.Con-
centration of DNA was determined using an Eppendorf
BioPhotometer.
The PCR mixture (50
l
l) contained the bacterial
DNA,0.5
l
M primers (for the first 527-bp fragment),
PCR buffer (10 mM Tris-HCl at pH 8.3,50 mM KCl,
2 mM MgCl
2
,0.01% gelatin),a 200
l
M concentration
of each dNTP,and 1.0 U of Taq polymerase.Primers
were 5
¢
-GAGAG TTTGA TCCTG GCTCA G-3
¢
(for-
ward) and 5
¢
-CTACG GCTAC CTTGT TACGA-3
¢
(reverse) (Cello
et al.
1997).The mixture was amplified
for 30 cycles at 95

C for 30 s,60

C for 30 s,and 72

C
for 45 s with a final extension at 72

C for 10 min,in an
automated thermal cycler.Purified PCR product (Qia-
gen PCR Purification Kit) was cloned in pGEM-T
vector and
E.coli
DH5
a
competent cells for sequencing.
DNA nucleotide sequence was analysed using the
database of GenBank.For the construction of a
phylogenetic tree,sequences of the 10 most closely
related organisms with that of
Escherichia coli
were
included in the comparison.The similarity values for
these sequences were transformed into phylogenetic
distance values that compensate for multiple substitu-
tions at any given site in the sequence.The phylogenetic
dendrogramwas constructed with the neighbour-joining
method using the PHYLIP package.Bacteria used in
construction of the phylogenetic tree with their Gen-
Bank accession numbers included
Bacillus megaterium
Strain C1 (AJ491841),
Bacillus flexus
(AY422986),
Bacillus asahii
(AB109209),
Bacillus psychrosaccharolyt-
icus
(AB021195),
B.circulans
(X60613),
Bacillus cereus
strain G3317 (AY138278),
Bacillus thuringiensis
serovar
214
K.H.Cheung and Ji-Dong Gu
israelensis
(AY461762),
B.simplex
(X60638),
Bacillus
subtilis
(AF545570),
Bacillus anthracis
(AB116124),
E.
coli
(J01859).16S rDNA sequence of isolate TKW was
deposited in GenBank with the accession number
AY524978.
Metal resistance and chromate reduction
To investigate heavy metal resistance of the isolate on
other environmentally important metals under aerobic
conditions,500
l
l pure culture was inoculated into
20 ml NB (Difco Laboratory) amended with 0.34 mM
dichromate (Cr
2
O
2

7
),0.32 mM arsenate (AsO
3

4
),
0.53 mMselenate (SeO
2

4
) or 0.58 mMselenite (SeO
2

3
).
One ml sample was extracted at regular time intervals
for the measurement of bacterial growth.For the effect
of initial cell density on Cr
6+
reduction,1 ml pure
culture with an initial cell density of 10
5
,10
6
or 10
7
cells ml
)
1
was inoculated into 100 ml NB (Difco Lab-
oratory) amended with 0.48 mM Cr
6+
(as CrO
2

4
).
Two ml samples were taken at regular time intervals for
the assessment of bacterial growth,and Cr
6+
concen-
tration with colorimetric diphenylcarbazide (DPC) anal-
ysis as described previously (Cheung & Gu 2003).300
l
l
sub-sample was pipetted into 9.7 ml 0.4 M H
2
SO
4
buffered with 25
l
lH
3
PO
4
,0.5 ml DPC reagent
(0.025 g 1,5-diphenylcarbazide dissolved in 10 ml acet-
one) was added for 5 min reaction before measuring the
A
540
.For the effect of initial concentration of Cr
6+
and
NaCl,inoculum (1 ml) with initial cell density of 10
5
cell ml
)
1
were added to NB (Difco Laboratory)
amended with different concentrations of Cr
6+
(0.05,
0.20,0.45 or 0.90 mMCrO
2

4
) and NaCl (5,35 or 55%,
plus 0.4 mM CrO
2

4
).Two ml samples were taken at
regular time intervals for the assessment of bacterial
growth and Cr
6+
concentration.All experiments above
were conducted in duplicate with non-inoculated con-
trols.
Results
Enrichment culture and the isolate
A pure culture capable of reducing Cr
6+
was isolated
frommarine sediments after enrichment.The isolate was
a Gram-positive facultative aerobe of size 4.0
·
1.0
l
m,
with limited growth under anaerobic condition (data not
shown).The phylogenetic identity of the isolate was
determined by 16S rDNA sequencing,which revealed
that it was most closely related to
B.megaterium
(designated as strain TKW3) of family Bacillaceae with
99.9% similarity (Figure 1).A broad biochemical
utilization profile was found for
B.megaterium
TKW3,
where arabinose,mannitol,
N
-acetylglucosamine,malt-
ose,caprate,citrate,butyrate and lactate could be
utilized as sole source of carbon and energy (Table 1)
(Slepecky & Hemphill 1999).Among the tested temper-
ature values,the highest growth for
B.megaterium
TKW3 was at 30

C with a rate of 0.067
l
g (protein)
s
)
1
,lower temperatures retarded bacterial growth
(deferred by 96 h at 10

C) yet the maximum growth
yield was similar at about 200
l
g (protein) ml
)
1
(after
188 h at 10

C,while 96 h at 20 and 30

C) (Figure 2).
At the tested salinity levels,
B.megaterium
TKW grew
best at 5
&
NaCl with a growth rate of 0.080
l
g
(protein) s
)
1
,higher salinity retarded both the growth
rate and maximum yield (Figure 3).
Cr
6+
reduction by
B.megaterium
TKW3 was retarded
by 35
&
NaCl,with Cr
6+
reduction rate of
5.26
·
10
)
12
l
mol (Cr
6+
) cell
)
1
h
)
1
compared with
1.39
·
10
)
11
l
mol (Cr
6+
) cell
)
1
h
)
1
for 5
&
NaCl.
Strain TKW3
Bacillus megaterium
Bacillus flexus
Bacillus circulans
Bacillus subtilis
Bacillus thuringiensi
s
Bacillus cereus
Bacillus anthracis
Bacillus asahii
Bacillus psychrosaccharolyticus
Bacillus simplex
Escherichia coli
74
100
96
100
79
60
55
29
55
0.05
Figure 1.
Phylogenetic relationships by a neighbouring-joining analysis of 16S rDNA sequences,showing the position of strain TKW3 isolated
from marine sediments.
E.coli
was used as the out-group.Scale bar =10 nucleotides substitutions per 100 nucleotides of 16S rDNA sequence.
Chromate reduction by
Bacillus sp.
215
Cr
6+
reduction by
B.megaterium
TKW3 ceased after
96 h with residue concentration of 0.28 mM (Cr
6+
)
(31.7% reduced) at 35
&
NaCl level,while complete
inhibition of Cr
6+
reduction was observed with 55
&
NaCl (Figure 3).
Metal resistance and chromate reduction
Protein analyses revealed that
B.megaterium
TKW3
maintained metabolic activity in 0.34 mM Cr
2
O
2

7
,
0.32 mMAsO
3

4
,0.58 mMSeO
2

3
and 0.53 mMSeO
2

4
,
while the magnitude of bacterial growth was in
descending order:SeO
2

4
,AsO
3

4
,SeO
2

3
and Cr
2
O
2

7
(Figure 4).Precipitates were found in cultures amended
with Cr
2
O
2

7
(white precipitates) and SeO
2

3
(red pre-
cipitates),while no visible precipitates were observed in
uninoculated or non-viable cell controls (data not
shown),illustrating that the transformation of soluble
metal ions to insoluble states was biologically mediated.
Although initial cell density did not significantly affect
the Cr
6+
reduction rate,the residual Cr
6+
(0.126 mM,
73.47%reduced) was the lowest at 10
7
cells ml
)
1
as the
Table 1.
Phenotypic (morphological and biochemical) expression of
B.megaterium
TKW3.
Test/Characteristic Result
Cell type Rod
Cell size 4.0
l
m
·
1.0
l
m
Gram staining +
Nitrate reductase
)
Oxidase
)
Tryptophan
)
Esculine hydrolase +
Gelatine +
p
-Nitrophenyl-D-galactopyranoside +
D-Glucose +
L-Arabinose +
D-Mannose +(weak)
D-Mannitol +
N-acetylglucosamine +
D-Maltose +
Gluconate +(weak)
Caprate +
Adipate +(weak)
Malate +
Citrate +
Phenylacetate +(weak)
Succinate
)
Propionate
)
Pyruvate +(weak)
Butyrate +
Formate
)
Acetate +(weak)
Fumarate +(weak)
Lactate +
Phenol
)
Indole
)
Benzoate
)
Ethanol
)
Butanol
)
Propanol
)
Quinoline +(weak)
Glucose
a
)
Arginine dihydrolase
a
)
Urease
a
)
a
Tested under anaerobic conditions.
0
50
100
150
200
250
0 40 80 120 160 200
Time (h)
Proteins(ug ml
-1
)
Figure 2.
Effect of temperature on the growth of
B.megaterium
TKW3.Bacterial growth was determined by measuring the protein
contents of the culture with Bradford assay.1 ml inoculum of
B.megaterium
TKW3 was added into 20 ml nutrient broth and
incubated in a rotary shaker (150 rev/min) at temperatures of 10

C
(
n
),20

C(
s
) and 30

C(
h
).
0
100
200
300
400
0 40 80 120 160
Time (h)
Proteins (ug ml
-1
)
0
0.1
0.2
0.3
0.4
0.5
Cr(VI) (mM)
Figure 3.
Effect of salinity on the growth of and Cr
6+
reduction by
B.
megaterium
TKW3.Bacterial growth was investigated with the
Bradford assay,while the reduction of Cr
6+
was quantified with
colorimetric diphenylcarbazide method.1 ml inoculum of
B.megate-
rium
TKW3 was added into 100 ml nutrient broth amended with
0.40 mMCr
6+
of salinity 5
&
(proteins:
n
,Cr
6+
:
h
);35
&
(proteins:
r
,
Cr
6+
:
e
) and 55
&
(proteins:
d
,Cr
6+
:
s
) NaCl,then incubated with
shaking (150 rev/min) at 30

C.Vertical bars indicate standard
deviation from duplicate from each treatment.
0
50
100
150
200
0 40 80 120 160 200 240
Time (h)
Proteins (ug ml
-1
)
Figure 4.
Growth of
B.megaterium
TKW3 in the presence of other
metals.Bacterial growth was determined by measuring the protein
contents of the culture with the Bradford assay.500
l
l culture of
B.megaterium
TKW3 was inoculated into 20 ml nutrient broth
amended with Cr
2
O
2

7
(0.34 mM;
e
),AsO
3

4
(0.32 mM;
·
),SeO
2

3
(0.58 mM;
h
) and SeO
2

4
(0.53 mM;
n
),then incubated with shaking
(150 rev/min) at 30

C.Vertical bars indicate standard deviation from
duplicate from each treatment.
216
K.H.Cheung and Ji-Dong Gu
inoculum,and the residual Cr
6+
was similar between
inocula of 10
5
and 10
6
cells ml
)
1
with 0.195 and
0.186 mM (Cr
6+
),respectively (Figure 5).The Cr
6+
reduction rate was similar with initial Cr
6+
concentra-
tions between 0.05 and 0.90 mMin our study.Complete
reduction of 0.05 and 0.20 mM(Cr
6+
)by
B.megaterium
TKW3 was observed within 24 and 360 h,respectively.
The residue Cr
6+
was 0.13 mM (71.1% reduced) and
0.58 mM (35.6% reduced) respectively for initial con-
centrations of 0.45 and 0.90 mM Cr
6+
after incubating
for 360 h (Figure 6).No significant Cr
6+
reduction was
detected in all uninoculated or non-viable controls.
Discussion
B.megaterium
TKW3 was isolated from marine sedi-
ments contaminated with high level of metals,such as
160 mg Cr kg
)
1
(HK EPD 2000).
B.megaterium
was
commonly found in soil and could utilize a range of
substrates;whereas arabinose,mannitol,
N
-acetylglu-
cosamine,maltose,caprate,citrate,butyrate and lactate
were firstly reported to be utilized by this species
(Table 1) (Slepecky & Hemphill 1999).The broadened
spectrum of utilizable substrates might enhance the
flexibility of culturing
B.megaterium
TKW3.Although
studies on the cytology and biochemistry of
B.megate-
rium
were conducted,strain TKW3 has other interesting
characteristics of multiple-metal resistance and Cr
6+
reduction compared with other strains (Hu & Boyer
1996;England
et al.
1997;Adam
et al.
2001;Tang
et al.
2001).Similar to
B.megaterium
TKW3,high NaCl level
of salinity at 58.5
&
caused a concentration-dependent
growth delay and decreased growth yield in
B.megate-
rium
strain 27,related to the transformation of stable
long-lived cell proteins (LLP) into quickly degraded
short-lived proteins (SLP) (Nekolny & Chaloupka
2000).Since Cr
6+
reduction by
B.megaterium
TKW3
was inhibited by a high level of NaCl,direct application
of this strain in Cr
6+
bioremediation at high salinity
over 55
&
NaCl is limited (Figure 3).
Cr
6+
reduction by
B.megaterium
TKW3 was depen-
dent on the initial cell density,while the initial Cr
6+
concentration did not have significant effect on Cr
reduction rate when below 0.9 mMin our study.Similar
phenomena were observed in
Microbacterium
sp.MP30
and
Enterobacter cloacae
HO1,where the Cr
6+
reduc-
tion rate was similar with initial Cr
6+
concentration
below 0.5 and 10 mM,respectively,illustrating the
presence of other limiting factors like the availability of
carbon substrates (Komori
et al.
1990;Ohtake
et al.
1990).Resistance and reduction of Cr
6+
were indepen-
dent processes in
Pseudomonas fluorescens
,where Cr
6+
reduction was mediated by a non-specific enzyme that
naturally consumes other substrate(s) (Cervantes &
silver 1992).The similar Cr
6+
reduction rate below 0.9
mM Cr
6+
by
B.megaterium
TKW3 reflected the normal
functioning of its detoxification mechanism(s) to Cr
6+
.
B.megaterium
TKW3 was found to be resistant to
Cr
2
O
2

7
,SeO
2

3
,SeO
2

4
and AsO
3

4
,so it is worthwhile to
investigate the interaction (neutral,synergistic or antag-
onistic) between these metals/metalloids on Cr
6+
reduc-
tion by this strain.The formation of precipitates only in
cultures amended with Cr
2
O
2

7
and SeO
2

3
illustrated the
transformation of these soluble oxyanions by
B.mega-
terium
TKW3.Cr
6+
was reduced to Cr
3+
that forms
insoluble hydroxide Cr(OH)
3
as a white precipitate
(Lovley & Phillips 1994;Chardin
et al.
2002).The
detection of Cr
5+
as a short-lived intermediate in Cr
6+
reduction to Cr
3+
by
Pseudomonas ambigua
G-1
indicated that the reduction involved at least two
reaction steps (Suzuki
et al.
1992).Aerobic Cr
6+
reduc-
tion involved a soluble reductase using NADH or
endogenous electron reserves as electron donor;while
under anaerobic conditions,Cr
6+
might serve as termi-
nal electron acceptor through respiratory chains
mediated by soluble reductase,membrane-bound reduc-
tase or both (Wang & Shen 1995).SeO
2

3
was reduced
by
B.megaterium
TKW3 to elemental Se
0
as insoluble
0
0.1
0.2
0.3
0.4
0.5
0.6
0 40 80 120 160 200
Time (h)
Cr(VI) (mM)
Figure 5.
Effect of initial cell density on Cr
6+
reduction by
B.megaterium
TKW3.The concentration of Cr
6+
was quantified
with the colorimetric diphenylcarbazide method.1 ml inoculum of
B.megaterium
TKW3 with initial cell density of 10
5
(
e
),10
6
(
h
) and
10
7
(
n
) cell ml
)
1
was added into 100 ml nutrient broth amended with
0.48 mM Cr
6+
,then incubated with shaking (150 rev/min) at 30

C.
Vertical bars indicate standard deviation from duplicate from each
treatment.
0
0.2
0.4
0.6
0.8
1
0 40 80 120 160 200 240 280 320 360
Time (h)
Cr(VI) (mM)
Figure 6.
Effect of initial Cr
6+
concentration on its reduction by
B.megaterium
TKW3.The concentration of Cr
6+
reduction was
quantified with the colorimetric diphenylcarbazide method.1 ml
inoculum (10
5
cell ml
)
1
)of
B.megaterium
TKW3 was added into
100 ml nutrient broth amended with Cr
6+
of concentration 0.05 (
s
),
0.20 (
e
),0.45 (
n
) and 0.90 (
h
) mM,then incubated with shaking
(150 rev/min) at 30

C.Vertical bars indicate standard deviation from
duplicate from each treatment.
Chromate reduction by
Bacillus sp.
217
red amorphous selenium,which was reported to be a
detoxification mechanism which is not associated with
energy conservation in bacteria (Blake II
et al.
1993;
White
et al.
1997).
B.megaterium
TKW3 reduced toxic and soluble Cr
6+
to non-toxic and insoluble Cr
3+
readily under moderate
conditions,with a wide range of simple carbon sub-
strates,and no supplement of chemicals like flocculants,
reducing agents,acid and alkali were required.Resis-
tance of this strain to other heavy metals is also of
advantage for
in situ
and
ex situ
Cr
6+
bioremediation,
which might be associated with other heavy metals.
Other techniques like the dialysis sac bioreactor,anion-
exchange membrane reactor,continuous stirred tank
bioreactor,artificial biofilms and rotating biological
contactor could be applied complementarily with cells of
B.megaterium
TKW3 to enhance the overall efficiency
in Cr
6+
reduction (Ganguli & Tripathi 2002).Current
investigations are being conducted to study the frac-
tion(s) of protein (periplasmic,cytoplasmic or both) of
B.megaterium
TKW3 responsible for Cr
6+
reduction to
delineate the pathway and mechanism involved,as well
as the interaction with other heavy metals on Cr
6+
reduction by this strain.
Acknowledgements
This project was supported by an ITF grant (276/00)
from the Innovative Technology Commission of Hong
Kong SAR,and industrial partnerships with Kou Hing
Hong Scientific Supplies,Peako Engineering Co.We
thank Jessie Lai for laboratory assistance and Paula
Wang and Li Pan for the DNA sequence alignment.
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Chromate reduction by
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219
Formation of
Yersinia pseudotuberculosis
biofilms on multiple surfaces on
Caenorhabditis elegans
Marc A.Nascarella* and Steven M.Presley
The Institute of Environmental & Human Health,Department of Environmental Toxicology,Texas Tech University,
MS 41163,Lubbock,TX 79409-1163,USA
*Author for correspondence:Tel.:+1-806-885-4567,Fax:+1-806-885-4577,E-mail:marc.nascarella@ttu.edu
Received 1 October 2004;accepted 25 October 2004
Keywords:
Biofilm,
Caenorhabditis elegans
,
endotokia matricide
,
Yersinia pseudotuberculosis
Summary
A liquid-based assay was used to evaluate the ability of
Yersinia pseudotuberculosis
to form a bacterial biofilm on
the nematode
Caenorhabditis elegans
.After 3 days of incubation in the liquid assay a biofilm was clearly visible by
light microscopy on both the head and vulva region of the worms.At times,the biofilm formation on the vulva
appeared to prevent the laying of eggs by the adult hermaphrodite;the eggs would later hatch inside of the worm.
One possible explanation for the biofilmformation observed on the vulva may be the increased motion of the cuticle
surrounding the vulva when the wormis immersed in a liquid culture.This is the first report of biofilmformation on
the vulva of
C.elegans
.
Introduction
Bubonic plague is a zoonotic disease transmitted by
fleas infected with the bacterium
Yersinia pestis
.The
disease is spread rapidly due to a
Y.pestis
-induced
extracellular mass that obstructs the flea’s alimentary
canal,and results in repeated attempts to feed by the
flea (Bacot & Martin 1914).Recently (Hinnebusch
2003),this extracellular matrix has been termed a
biofilm (Costerton
et al.
1978).Similarly,
Yersinia
pseudotuberculosis
will form an extracellular biofilm
on the anterior cuticle of the nematode
Caenorhabditis
elegans
and inhibit feeding (Darby
et al.
2002).Thus,
the
Y.pseudotuberculosis

C.elegans
model of biofilm
formation provides an experimental system in which to
study the obstruction of the fleas digestive tract by
Y.pestis
(Darby
et al.
2002;Joshua
et al.
2003;Tan &
Darby 2004).
Biofilm accumulation on
C.elegans
was previously
determined to be dependent upon movement through a
bacterial lawn (Tan & Darby 2004).The formation of
this movement-dependent biofilmwas also limited to the
anterior portion of the worms.Here we show that
worms infected in a liquid-based assay produce biofilms
on both the anterior (head) and vulva of the worm.To
our knowledge,this is the first report of a biofilm on the
vulva of
C.elegans
.
Materials and methods
Bacterial strains and growth media
Yersinia pseudotuberculosis
YPIII (a gift of Dr.Creg
Darby,University of Alabama,Birmingham,AB,USA)
was cultured in 5 ml of Luria-Bertani broth (LB).The
culture was incubated overnight (17 h) at 28

C.
The
C.elegans
wild-type N2 (var.Bristol) and
E.coli
OP50 strains that were used in this study were obtained
from the
Caenorhabditis
Genetics Center (CGC,Bio-
logical Sciences Center,University of Minnesota,1445
Gortner Ave.,St.Paul,MN,USA).Nematodes were
cultured at 20

C on nematode growth medium (NGM)
that had been previously seeded with
E.coli
OP50 and
cultured overnight at 37

C (Sulston & Hodgkin 1988).
To maintain the nematode colony,cultures were trans-
ferred to freshly seeded plates every 7 days.
Synchronizing
C.elegans
cultures
Age-synchronized cultures of
C.elegans
were obtained
following exposure to a sodium hypochlorite (bleach)/
sodium hydroxide solution that has been previously
described (Sulston & Hodgkin 1988).Four days prior to
the assay,overgrown plates of
C.elegans
were washed
three times with 5 ml of K-medium (3.075 g NaCl,
World Journal of Microbiology & Biotechnology (2005)
21
:229–231

Springer 2005
DOI 10.1007/s11274-004-5299-x
2.42 g KCl,and 1 l of distilled water).The rinsing liquid
following each wash was combined in a 15 ml centrifuge
tube.The worms were then pelleted by centrifugation at
3000 g for 7 min and resuspended in 10 ml of the
sodium hypochlorite/sodium hydroxide solution.The
suspension was allowed to sit for 7 min and received
careful shaking every few minutes,killing all the
nematodes except for the eggs.The suspension was then
pelleted again (2000 g for 7 min),and washed three
times in K-medium.The final pellet was resuspended in
approximately 1 ml of K-medium and incubated at
20

C for 1 day on an NGMplate.On day 2 the worms
were washed off the plate with K-medium and trans-
ferred to a seeded NGM plate until the worms had
reached adulthood.
Infection assay
Two 5 ml borosilicate tubes containing an overnight
(17 h) culture of
Yersinia pseudotuberculosis
in LB Broth
was centrifuged at 2000 g for 15 min and the superna-
tant was removed.The pellet was resuspended in 10 ml
of K-medium and 1 ml of the resulting solution was
transferred into a standard 24-well tissue culture plate
(Falcon 3047).Approximately 10 adult worms were
pipetted into each well.The plate was then placed at
20

C,in the dark.
Microscopy
Worms were observed daily for biofilm formation using
a stereomicroscope with epi-illumination and/or trans-
mitted light.Photomicrographs (
Fig
ure 1) were taken
using a digital camera after viewing
C.elegans
on 1%
agarose pads (Sulston & Horvitz 1977) with a 20
·
objective,on a compound light microscope (Olympus
BX-51) using transmitted light.In some instances
(Figure 1b & c),worms were first stained with 0.1%
crystal violet.
Results
After approximately 3 days of incubation in the test
solution a biofilm was observed on both the head and
vulva of the worms (Figure 1).Worms that had formed
anterior biofilms were easily identified as the worms
would violently thrash in an apparent attempt to free
themselves from the obstruction.Often times,multiple
worms would become joined at the head or vulva and
form an asterisk (*) appearing structure (Figure 1a).At
times,the biofilm formation on the vulva appeared to
prevent the laying of eggs by the adult hermaphrodite;
the eggs would later hatch inside of the worm(Figure 1b
& c).After 7 days the biofilm on the vulva became
increasing thick with material accumulating on top of
the initially formed layer (Figure 1d).The accumulation
of biofilm appeared similar to what has been observed
with
C.elegans
infected with agar lawn-based
Yersinia
infections (Darby
et al.
2002;Joshua
et al.
2003;Tan &
Darby 2004).
Discussion
The biofilm formation on the vulva may not have led to
the hermaphrodite progeny hatching inside of the adult
worm.Internal hatching,or
endotokia matricida
as it has
been called in the general nematode literature (Johnigk
& Ehlers 1999),has previously been observed in
C.elegans.
This phenomenon,generally referred to as
producing ‘bags of worms’ (Trent
et al.
1983) or more
simply as ‘bagging’,will usually occur as a response to a
defect in vulval cell development,inadequate motility of
the vulval muscles,or will occasionally occur in older
animals (Kornfeld 1997).However,bagging has also
been observed in response to stressors such as antimi-
crobials,high salt,and antagonistic bacteria (Chen &
Caswell-Chen 2003,2004).The cited benefits of bagging
under stressful or food-limited situations have been to
provide adequate nutrition (by way of the adult’s
internal organs) as to allow larvae to become dauers.
It has therefore been suggested (Chen & Caswell-Chen
2004) that bagging may actually be a type of facultative
vivipary and a normal important life-history trait of
C.elegans
where parental resources are offered to their
progeny.Therefore,it is difficult to say with complete
certainty that the bagging observed in these experiments
was not a secondary response to biofilm formation that
followed the food-limiting situation created by the
anterior biofilm blocking food intake.We feel confident
Figure 1
.Biofilm growth on adult
Caenorhabditis elegans
hermaphro-
dites following infection by
Yersinia pseudotuberculosis
in a liquid
medium.(a) A group of larvae that have become joined at the head by
a biofilm mass.(b,c) The vulva of this single worm has become
obstructed (see arrow on inset b) and the eggs have hatched into larvae
that are trapped within the hermaphrodite.(d) After 5 days of
infection the vulva has become infected with a large biofilm mass and
additional worms have become trapped.
230
M.A.Nascarella and S.M.Presley
that this is not a secondary response,as bagging has not
been reported in similar lawn-based assays (Darby
et al.
2002;Joshua
et al.
2003;Tan & Darby 2004).It seems
likely that the bagging is a primary response to biofilm
formation,whereby biofilm formation on the vulva
prevented the laying of eggs,either by physically
obstructing the vulval opening or by preventing the
motility of vulval muscles.It is unlikely that the bagging
observed here was due to the age of the worms,as
similar experiments have been conducted in this lab with
E.coli
OP50 and no bagging was observed.
Why a worm’s vulva would become preferentially
infected in a liquid-based assay and not on an agar-
based assay is not known.Tan & Darby (2004) have
shown that
C.elegans
movement is necessary in order
for a
Yersinia
spp.biofilm to form.They also speculate
that the necessary movement in the biofilm-mediated
transfer of
Y.pestis
in an infected flea is provided by the
peristaltic action of the flea’s digestive and feeding
muscles.One possible explanation for the dichotomy
observed in the two infection phenotypes (liquid versus
agar-based infection assays) may be the increased
motion of the cuticle surrounding the vulva when the
worm is immersed in a liquid assay.The increased
thrashing of the worm may provide the necessary
movement around the centre of the worm’s cuticle for
the biofilm to form.In fact,the cuticle is located almost
exactly at the apex of the thrashing ‘V’ that is formed
when a worm is added to a liquid solution (see
Figure 1d).However,the physiology of the vulva has
some apparent interaction as the infection is specifically
located on the vulva and is not observed on the worms
cuticle located 180

from the vulva.
Future work involves the use of lectins as
in situ
probes for earlier detection of biofilm binding (Tan &
Darby 2004).Future work will also evaluate host
resistance factors by evaluating the response of
C.elegans
mutants;mutations leading to aberrations
in feeding,surface recognition,and movement (Joshua
et al.
2003).
Acknowledgements
The authors gratefully acknowledge Dr.Creg Darby,
University of Alabama,Birmingham,for suggestions on
laboratory methods,for providing a copy of a manu-
script prior to publication,and for providing the
Yersinia pseudotuberculosis
III strain used in this study;
and Dr.Phil Williams,University of Georgia,Athens,
for his assistance with the cultivatoin of
C.elegans
.
Partial support for this work was provided by a Colgate-
Palmolive/Society of Toxicology Award for Research
Training in Alternative Methods.The nematode strain
used in this work was provided by the
Caenorhabditis
Genetics Center,which is funded by the National
Institutes of Health (NIH) National Center for Research
Resources (NCRR).
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Biofilm growth on
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Use of RAPD to investigate the epidemiology of
Staphylococcus aureus
infection in
Malaysian hospitals
V.Neela
1,
*,N.S.Mariana
1
,S.Radu
3
,S.Zamberi
1
,A.R.Raha
3
and R.Rosli
2
1
Department of Clinical Laboratory Sciences
2
Department of Human Growth and Development,Faculty of Medicine and Heath Sciences
3
Department of Biotechnology,Faculty of Food Science and Biotechnology,University Putra Malaysia,43400 UPM
Serdang,Selangor,Malaysia
*Author for correspondence:Tel.:+603-89468497,Fax:+603-89412787,E-mails:neela2000@hotmail.com,
neela2010@yahoo.com
Received 5 April 2004;accepted 12 July 2004
Keywords:
Dendrogram,MRSA,non-MRSA,randomly amplified polymorphic DNA,
Staphylococcus aureus
Summary
Randomly amplified polymorphic DNA (RAPD) with four different decamer oligonucleotide primers was
performed on 50 clinical
Staphylococcus aureus
isolates obtained from different hospitals in Malaysia.All the four
primers generated polymorphisms in all 50 isolates of
S.aureus
studied
,
revealing DNA markers with sizes ranging
from100 to 7000 bp.The dendrogramgenerated fromthe RAPDanalysis revealed two major groups (Groups I–II)
with three clusters each in one group.
S.aureus
strains isolated from the same hospital were found to be genetically
closely related and most of them were placed in the same cluster.In addition RAPD differentiated between MRSA
and non-MRSA based on the clustering,where all MRSA and non-MRSA were placed in their respective clusters.