World Journal of Microbiology and Biotechnology

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World Journal of Microbiology and Biotechnology
Volume 21, Number 1
A comparison of proline, thiol levels and GAPDH activity in
cyanobacteria of different origins facing temperature-stress
(1 - 9)
A. P. Singh, R. K. Asthana, Aravind M. Kayastha, S. P. Singh
DOI: 10.1007/s11274-004-0872-x
Growth kinetics of EDTA biodegradation by Burkholderia
cepacia
(11 - 16)
Shih-Chin Chen, Kuo-Hsu Li, Hung-Yuan Fang
DOI: 10.1007/s11274-004-0876-6
Biosynthesis of short-chain-length-polyhydroxyalkanoates
during the dual-nutrient-limited zone by Ralstonia eutropha
(17 - 21)
Qun Yan, Ying Sun, Lifang Ruan, Jian Chen, Peter Hoi Fu Yu
DOI: 10.1007/s11274-004-0877-5
Purification of lipase from Cunninghamella verticillata by
stepwise precipitation and optimized conditions for
crystallization
(23 - 26)
T. S. Kumarevel, S. C. B. Gopinath, A. Hilda, N. Gautham, M. N. Ponnusamy
DOI: 10.1007/s11274-004-1005-2
Secondary metabolites from endophytic fungi isolated from the
Chilean gymnosperm Prumnopitys andina (Lleuque)
(27 - 32)
G. Schmeda-Hirschmann, E. Hormazabal, L. Astudillo, J. Rodriguez, C. Theoduloz
DOI: 10.1007/s11274-004-1552-6
Preparation and testing of Sardinella protein hydrolysates as
nitrogen source for extracellular lipase production by Rhizopus
oryzae
(33 - 38)
Sofiane Ghorbel, Nabil Souissi, Yosra Triki-Ellouz, Laurent Dufossé, Fabienne Guérard,
Moncef Nasri
DOI: 10.1007/s11274-004-1556-2
Comparative assessment of fermentation techniques useful in
the processing of ogi
(39 - 43)
O. D. Teniola, W. H. Holzapfel, S. A. Odunfa
DOI: 10.1007/s11274-004-1549-1
Production of antibacterials from the freshwater alga Euglena
viridis (Ehren)
(45 - 50)
B. K. Das, J. Pradhan, P. Pattnaik, B. R. Samantaray, S. K. Samal
DOI: 10.1007/s11274-004-1555-3
Partial purification properties of human epidermal growth factor
from recombinant Escherichia coli by expanded bed adsorption
(51 - 57)
Wang-Yu Tong, Shan-Jing Yao, Zi-Qiang Zhu
DOI: 10.1007/s11274-004-1554-4
Antimicrobial activity of chicken egg white cystatin (59 - 64)
Ewelina Wesierska, Yousif Saleh, Tadeusz Trziszka, Wieslaw Kopec, Maciej Siewinski,
Kamila Korzekwa
DOI: 10.1007/s11274-004-1932-y




Production and characterization of xylanases of a Bacillus strain
isolated from soil
(65 - 68)
Banu Avcioglu, Banu Eyupoglu, Ufuk Bakir
DOI: 10.1007/s11274-004-1934-9
Metabolism of betaine as a carbon source by an osmotolerant
bacterium isolated from the weed rhizosphere
(69 - 73)
Triwibowo Yuwono
DOI: 10.1007/s11274-004-1935-8
Kinetic sedimentation of Rhizobium-aggregates produced by
leguminous lectins
(75 - 82)
Cosme R. Martínez, André M. Netto, Márcia V.B. Figueiredo, Benildo S. Cavada, José L.
Lima-Filho
DOI: 10.1007/s11274-004-2777-0
Ethanol production in multistage continuous, single stage
continuous, Lactobacillus-contaminated continuous, and batch
fermentations
(83 - 88)
Dennis P. Bayrock and W. M. Ingledew
DOI: 10.1007/s11274-004-2781-4
Short communication

Tolerance to copper and zinc of Acidithiobacillus thiooxidans
isolated from sewage sludge
(89 - 91)
Renata P. R. Barreira, Luciene D. Villar, Oswaldo Garcia
DOI: 10.1007/s11274-004-1551-7
Short Communication

Chitinase production by Beauveria felinaRD 101: optimization of
parameters under solid substrate fermentation conditions
(93 - 95)
Pankaj Patidar, Deepti Agrawal, Tushar Banerjee, Shridhar Patil
DOI: 10.1007/s11274-004-1553-5
Short Communications

Acetate-enhanced polymerized triacylglycerol utilization by Mucor
circinelloides
(97 - 99)
M. Joseph, J. L. F. Kock, C. H. Pohl, P. J. Botes, E. van Heerden, A. Hugo
DOI: 10.1007/s11274-004-1557-1

A comparison of proline,thiol levels and GAPDH activity in cyanobacteria of
different origins facing temperature-stress
A.P.Singh
1
,R.K.Asthana
1,
*,Arvind M.Kayastha
2
and S.P.Singh
1
1
Centre of Advanced Study in Botany,Banaras Hindu University,Varanasi 221 005,India
2
School of Biotechnology,Banaras Hindu University,Varanasi 221 005,India
*Author for correspondence:Tel.:+91-0542-2307146/2307147,Fax:+91-0542-2368174,E-mail:rasthana@bhu.ac.in
Received 20 November 2003;accepted 6 May 2004
Keywords:Cyanobacteria,GAPDH activity,mesophile,proline,psychrophile,thermophile,thiol
Summary
Three cyanobacterial strains originating from different habitats were subjected to temperature shift exposures and
monitored for levels of proline,thiol and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Thermophile Mastigocladus laminosus (growth optimum,40 C),raised the proline level 4.2-fold at low temperature
(20 C),for the psychrophile Nostoc 593 (growth optimum,20 C),it was raised 8-fold at 40 C while in the
mesophile Nostoc muscorum (growth optimum,30 C),the imino acid level increased 2.3-fold during temperature
‘shiftdown’ to 20 C or 3.5-fold in sets facing ‘shiftup’ (40 C).Alterations in thiol levels in the above strains were in
line with proline.It is suggested that such fluctuations reflect metabolic shifts as a response to stress.Interestingly,
GAPDHactivity was maximumat the respective growth temperature optimumof M.laminosus (122 nmol NADPH
oxidized min
)1
mg
)1
protein) and Nostoc 593 (141 nmol NADPH oxidized min
)1
mg
)1
protein) while in N.
muscorum,it increased at 40 C (101 nmol NADPH oxidized min
)1
mg
)1
protein) and to 93.3 nmol NADPH
oxidized min
)1
mg
)1
protein (20 C) relative to 86 nmol NADPH oxidized min
)1
mg
)1
protein at 30 C.It seems
that extremophiles maintain the GAPDHactivity/level during growth at their respective temperatures optimal while
the mesophile increases it in order to cope up with temperature-stress.
Introduction
Cyanobacteria colonize varied habitats like thermal
springs,Antarctica,saline conditions,rice fields as well
as tree bark.Their biotechnological importance as
sources of single cell protein and biofertilizer is well
documented (Vonshak 1997).Their wide range of adap-
tability,therefore,needs acclimation studies to elucidate
the possible metabolic shifts.Temperature shift exposures
of cyanobacteria may provide information on how such
prokaryotes could have acquired thermotolerance during
their long evolutionary history (Fork et al.1987).The
limited evidence available suggests that acclimation
begins within a few hours of fluctuations in temperature
(Lynch &Thompson 1984;Michel et al.1989).
The role of proline is well documented in stress man-
agement by metal-tolerant Silene vulgaris (Schat et al.
1997) as well as Rhizobium (Chien et al.1992;Singh
et al.2001) but little is known in cyanobacteria except
the reports of Wu et al.(1995) in heavy metal-stressed
Anacystis nidulans or Nostoc muscorum facing salinity
stress (Singh et al.1996).Thiols participate in multifar-
ious reactions in the cell ranging from electron transfer
(Barron 1951) to signal transduction during environ-
mental stress (Foyer et al.1997).In this context,
glutathione has been studied in Nostoc muscorum
(Fahey et al.1978),Synechococcus 6311 and Nostoc
muscorum 7119 (Tel-Or et al.1985).
GAPDH (glyceraldehyde-3-phosphate dehydroge-
nase) has been reviewed extensively (Wrba et al.1990)
and is a key enzyme involved in glycolysis,gluconeogen-
esis and the carbon reduction cycle in microbes (Leusch-
ner &Antranikian 1995) including cyanobacteria (Papen
et al.1986).Studies on homologous enzymes/proteins
from organisms with different temperature optima are
imperative in deciphering the acclimation process (van
der Oost et al.1996).This tempted us to monitor
alterations in selected parameters such as proline,thiol
and GAPDH activity that can possibly provide insight
into cyanobacterial adaptations using thermophilic Mas-
tigocladus laminosus,mesophilic Nostoc muscorum and
psychrophilic Nostoc 593 as model organisms.
Materials and methods
Organisms and growth conditions
The diazotrophic Mastigocladus laminosus was isolated
from a hot spring at Gangnani,Uttarkashi,India,
World Journal of Microbiology & Biotechnology 2005 21:1–9
DOI:10.1007/s11274-004-0872-x

Springer 2005
Nostoc muscorum ISU (Anabaena ATCC 27893),was
the kind gift from R.Haselkorn,USA and Nostoc 593,
was an Antarctic strain from Dr.Paul A.Broady,New
Zealand.Cyanobacteria were grown in modified Chu-10
medium (Gerloff et al.1950),free from any combined
nitrogen sources under continuous tungsten plus fluo-
rescent illumination (14.4 W/m
2
) at 40 ± 1 C,
30 ± 1 C or 20 ± 1 C.
Measurement of k
The specific growth rate constant (k) was calculated as
prescribed by Kratz & Myers (1955).
Dry weight determination
Cells were concentrated by centrifugation,washed and
dried (60 C) to constant weight (expressed as g l
)1
).
Protein estimation
Protein was estimated by the Lowry method as modified
by Herbert et al.(1971) using lysozyme (Sigma,St.
Louis,USA) as standard.
Proline and thiol levels were determined (upto 12 h) in
M.laminosus (growing at 40 C) was shifted to 30 or
20 C,N.muscorum cells (growing at 30 C) shifted to
20 or 40 C while Nostoc 593 (growing at 20 C) to 30 or
40 C.
Proline determination
Proline was measured according to Bates et al.(1973).
Cyanobacterial cells (400 lg ml
)1
) were harvested sub-
sequent to a common incubation of 12 h,resuspended in
10 ml of 3% (v/v) sulphosalicylic acid (SRL,Mumbai,
India) and subjected to ultrasonic disruption (Labsonic
L.Malayasia) for 5 min.The resulting cell extract was
centrifuged (9000 · g,15 min) to remove cell debris and
the supernatant with free proline was treated with
freshly prepared acidic ninhydrin at 80 C (1 h) and the
reaction terminated in an ice bath.The coloured
complex was extracted in toluene and absorbance read
at 520 nm.A proline standard was prepared by dissolv-
ing proline (SRL,Mumbai,India) in 3%(v/v) sulphos-
alycylic acid.The proline level is expressed as nmol mg
)1
protein.
Thiol determination
Thiol in concentrated cells (400 lg ml
)1
) was deter-
mined by the method of Fahey et al.(1978).Thiol was
extracted in boiling ethanol (80%,v/v;15 min),and
determined using DTNB [5,5¢-dithio-bis(2-nitrobenzoic
acid)],Sigma (Ellman 1959).DTNB (6 mM in 0.1 M
phosphate buffer,pH 7.5) was allowed to react (5 min)
with thiol in the cell extract followed by cold centrifu-
gation (9000 · g,15 min).The intensity of yellowcolour
of 2-nitro-5-mercaptobenzoate was measured at
412 nm.Thiol level is expressed as lmol g
)1
cyanobac-
terial dry wt.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
extraction and activity assay
The three target organisms growing at their respective
optimal temperatures were exposed to temperature-
shifts for a common duration of only 4 h as compared to
proline or thiol.Cyanobacterial biomass (400 lg pro-
tein ml
)1
) was broken in liquid nitrogen,suspended in
sodium phosphate buffer (0.1 M,pH 7.0) and centri-
fuged (9000 · g,1 h) at 4 C to procure the supernatant
for enzyme assay as described by Ferdinand (1964):
GAPDH activity assay buffer
The buffer consisted of triethanolamine hydrochloride
(TEA-Cl,40 mM),orthophosphoric acid (50 mM),
EDTA (25 lM) and pH was adjusted to 8.6 with dilute
ammonia.
GAPDH activity assay
The enzyme activity was assayed spectrophotometrically
using glyceraldehyde-3-phosphate (G-3-P) and nicotin-
amide adenine dinucleotide phosphate (NADP
+
,
Sigma,USA) as substrates and by monitoring the
time-dependent formation of NADPH at 340 nm.The
reaction mixture contained 0.69 ml assay buffer,0.05 ml
G-3-P (10 mM) and 0.05 ml NADP
+
(10 mM) at 30 C
and reaction was started by adding a 0.01 ml of
appropriately diluted enzyme solution (test volume,
0.8 ml).The increase in absorbance at 340 nm was
noted at 10 s interval for 2 min.The enzyme unit is
defined as ‘that amount of enzyme which reduces
1 lmol of NADP
+
to NADPH in one min’ under
specified test conditions.Enzyme activity is expressed as
nmol NADPH oxidized min
)1
mg
)1
protein.
Statistical analysis
All experiments were carried out in triplicate with
standard errors represented as bars wherever necessary.
Results
Proline biosynthesis
The sequence of photoautotrophic growth in terms of
generation time at the respective temperature optimum
was:Nostoc 593 (70.6 h) >M.laminosus (33 0h) >N.
muscorum (26 h) (data not shown).For control M.
laminosus cells,the basal proline level (8.64 nmol mg
)1
protein) was raised by almost 1.5-fold within 4 h
followed by a slow decline until 8 h (12.1 nmol mg
)1
protein) or even upto 12 h (10 nmol mg
)1
protein)
(Figure 1).Cells facing temperature downshift (30 C)
seemed to have triggered proline biosynthesis by more
than 2.3-fold (19.7 nmol mg
)1
protein) during 4 h,but
2 A.P.Singh et al.
the level went down to 15 nmol mg
)1
protein (8 h) or
ultimately to 14 nmol mg
)1
protein (12 h).Also,proline
level was raised by more than 4.2-fold (36 nmol mg
)1
protein) for temperature downshift sets (20 C) during
4 h,but the extent of decline was maximum during 8 h
to reach 18.6 nmol mg
)1
protein.The declining trend
ended with 16.9 nmol mg
)1
protein (12 h).
The basal proline level in N.muscorum(10 nmol mg
)1
protein) as control,could be raised by almost 1.7-fold
just within 4 h followed by a slow decline until 8 h
(13 nmol mg
)1
protein) and stabilized thereafter (Fig-
ure 2).Cells facing temperature downshift (20 C),
raised proline by more than 2.3-fold (22.6 nmol mg
)1
protein).However,the subsequent decline in proline
level did not follow the control,as cells still retained a
sufficient amount until 8 h that stabilized thereafter
(20.1 nmol mg
)1
protein).Significantly,the imino acid
amount for the 40 C sets was on top of the rest
(34.8 nmol mg
)1
protein) during 4 h,thus correspond-
ing to 3.5-fold over the basal level or even 1.5-fold over
the 20 C sets.A drastic decline in proline
(15.3 nmol mg
)1
protein) accompanied subsequent
incubation upto 8 or 12 h.
For control Nostoc 593,the basal proline level
(9 nmol mg
)1
protein) registered a 2-fold rise within
4 h,followed by a slow decline until 8 h
(13.5 nmol mg
)1
protein) or stabilization thereafter
(Figure 3).Atemperature shift-up (30 C) raised proline
>3.8-fold (34 nmol mg
)1
protein) even during 4 h,but
it invariably declined to 20.7 nmol mg
)1
protein with
incubation and stabilized thereafter.For shift-up
(40 C) sets,proline registered about 8-fold rise
(72 nmol mg
)1
protein) only during 4 h,as it declined
finally to 17 nmol mg
)1
protein (>8 h).Proline accu-
mulation by target organisms during temperature-shifts
was in the sequence:Nostoc 593 >M.laminosus >
N.muscorum,and data indicate that thermotolerance
may possibly be caused by accumulation of this imino
acid although transiently,and in a stress-specific man-
ner.
Thiol biosynthesis
Thiol in the three target strains was analysed keeping
protocol as applicable to proline.For control
Figure 1.Time-course of proline biosynthesis in M.laminosus during
temperature shift:40 C (.),30 C (s) and 20 C (d).
Figure 2.Time-course of proline biosynthesis in N.muscorum during
temperature shift:30 C (s),20 C (d) and 40 C (.).
Figure 3.Time-course of proline biosynthesis in Nostoc 593 during
temperature shift:20 C (d),30 C (s) and 40 C (.).
A comparison of proline,thiol levels and GAPDH activity 3
M.laminosus,the basal thiol level (20 lmol g
)1
dry wt.)
could be raised marginally to 22 lmol g
)1
dry wt.
during 4 h;a level that remained unaltered until 12 h
(Figure 4).Cells facing temperature downshift (30 C),
raised thiol by more than 1.5-fold (28.9 lmol g
)1
dry
wt.) during 4 h,but it went subsequently down to
24 lmol g
)1
dry wt.(8 h) to remain constant until end
(12 h).Also,the level of sulphydryl was raised by more
than 2-fold (40.3 lmol g
)1
dry wt.) for temperature
downshift (20 C) sets during 4 h,but cells failed to
sustain it with passage of time as it reached 28 lmol g
)1
dry wt.during 8 h or declined further to 21.1 lmol g
)1
dry wt.during 12 h;a value slightly lower to control.
For control N.muscorum,the basal thiol level
(17.8 lmol g
)1
dry wt.) was raised to 24 lmol g
)1
dry
wt.following 4 h but remained constant until 12 h
(Figure 5).Cells facing temperature downshift (20 C),
raised thiol by almost 1.7-fold (29.9 lmol g
)1
dry wt.)
even during 4 h but it reached 26.5 lmol g
)1
dry wt.
during 8 h and remained unaltered until end (12 h).
Cyanobacterial response to temperature shift-up (40 C)
was quite significant as evident from an almost 4-fold
rise in thiol (68.8 lmol g
)1
dry wt.) but the subsequent
incubation recorded a sharp decline to 30.1 lmol g
)1
dry wt.during 8 h or 21 lmol g
)1
dry wt.during 12 h to
approximate the basal level.
For control Nostoc 593,the basal thiol level
(23.2 lmol g
)1
dry wt.) registered an almost 1.2-fold rise
(28 lmol g
)1
dry wt.) during 4 h but remained constant
thereafter (Figure 6).Temperature shift-up(30 C) raised
thiol 1.7-fold(40.2 lmol g
)1
dry wt.) evenduring 4 h,but
it declined with incubation to reach 36 lmol g
)1
dry wt.
during 8 h or 31.3 lmol g
)1
dry wt.during 12 h;a value
very close to the control (20 C).The sulphydryl level was
significantly raised exceeding 4-fold (94.8 lmol g
)1
dry
wt.) for cells in temperature shift-up (40 C) trials even
during 4 h.However,this unusual rise was instantly
followed by a sharp decline to 31.3 lmol g
)1
dry wt.
during 8 h or to just 24 lmol g
)1
dry wt.(12 h),and this
was the least of all sets including control.Overall data
indicate that thiol was synthesizedinall the strains evenas
a response to fresh growth mediumbut more significantly
to temperature-induced stress and organisms responded
positively by switching over to elevated thiol biosynthesis
as quickly as within 4 h.
Figure 4.Time-course of thiol biosynthesis in M.laminosus during
temperature shift:40 C (.),30 C (s) and 20 C (d).
Figure 5.Time-course of thiol biosynthesis in N.muscorum during
temperature shift:30 C (s),20 C (d) and 40 C (.).
Figure 6.Time-course of thiol biosynthesis in Nostoc 593 during
temperature shift:20 C (d),30 C (s) and 40 C (.).
4 A.P.Singh et al.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
activity
Stress causes the shortage of reducing power in general
and GAPDH,the key enzyme in glycolysis and gluco-
neogenesis aids in the generation of reducing power.The
data presented herein refer to target cyanobacteria as
before,exposed to temperature fluctuations as applica-
ble to proline or thiol.As a 4 h exposure to temperature
shifts corresponded to optimal expression of proline or
thiol,the same duration was adopted for monitoring
GAPDH activity in cyanobacterial cell-free extracts.
Figure 7 shows the rate of NADPH oxidation by
GAPDH in extracts from M.laminosus exposed to 20,
30 or 40 C and its possible correlation with thiol
biosynthesis.The highest enzyme activity (120.6 nmol
NADPH oxidized min
)1
mg
)1
protein) was typical of
only 40 C sets as it went down by 20 units for 30 C set.
Also,enzyme activity remained unaltered even for 20 C
set (100.4 nmol NADPH oxidized min
)1
mg
)1
protein).
Therefore,unlike thiol,temperature-stress did not
trigger GAPDH activity.
The enzyme activity in N.muscorum increased for
both the temperature regimes but more significantly for
cells exposed to 40 C (101 nmol NADPH oxidized
min
)1
mg
)1
protein) thus amounting to 15 units (Fig-
ure 8).However,the extent of stimulation was limited to
just 7.5 units (93.3 nmol NADPH oxidized min
)1
mg
)1
protein) for 20 C sets.
Data in Figure 9 incorporate the spectrum of GAP-
DHactivity in Nostoc 593 facing the temperatures 20,30
or 40 C.The declining slope indicates maximum
expression of enzyme at the growth temperature opti-
mum of the organism as it approached as high as
141 nmol NADPH oxidized min
)1
mg
)1
protein.By
contrast,for a 10 rise in temperature (30 C),the
enzyme activity declined by 11 units (130 nmol NADPH
oxidized min
)1
mg
)1
protein).A further rise in temper-
ature (40 C) seemed detrimental to the enzyme as the
activity declined by almost 19 units from the level
achieved at 20 C.The overall data with regard to
GAPDH in three targets indicated following salient
features:(1) M.laminosus and Nostoc 593 had the
highest enzyme activity at their respective growth
temperature optimum in contrast to N.muscorum,and
(2) the stimulation of enzyme activity as the stress
response was the characteristic of N.muscorum only.
Figure 8.Thiol (j) and GAPDH activity (d) vs.temperature in N.
muscorum.
Figure 9.Thiol (j) and GAPDH activity (d) vs.temperature in
Nostoc 593.
Figure 7.Thiol (j) and GAPDH activity (d) vs.temperature in M.
laminosis.
A comparison of proline,thiol levels and GAPDH activity 5
Discussion
Proline biosynthesis
Studies on proline accumulation/metabolism have con-
centrated mainly on rice (Anbazhagan et al.1988;Iyer &
Caplan 1998;Lutts et al.1999),lettuce (Costa & Morel
1994),metal-tolerant and non-tolerant ecotypes of
Silene vulgaris (Schat et al.1997),wheat (Bassi &
Sharma 1993),Lotus corniculatus (Borsani et al.1999)
and soybean (van Heerden & Kru
¨
ger 2002).There are
reports of proline accumulation/biosynthesis in Sticho-
coccus bacillaris facing salinity (Brown & Hellebust
1978),in Chlorella vulgaris challenged with heavy metals
(Mehta & Gaur 1999) and in Salmonella typhimurium
facing osmotic stress (Csonka,1981,1988).The basal
proline level in M.laminosus (8.64 nmol mg
)1
protein),
N.muscorum (10 nmol mg
)1
protein) and Nostoc 593
(9 nmol mg
)1
protein) (ref.Figures 1–3) and such a
proximity indicates that cyanobacteria irrespective of
the habitat,do maintain the same range of proline.Also,
the values are in the close range of 11.5 lmol g
)1
protein
in N.muscorum (Singh et al.1996) but higher than in
Anacystis nidulans (5.6 nmol mg
)1
protein,Wu et al.
1995).Other prokaryotes S.typhimurium
(<1.2 nmol mg
)1
protein) or R.leguminosarum
(2.4 nmol mg
)1
protein) show proline still in the lower
range (Csonka 1981;Singh et al.2001).
For all the targets as control,proline level attained its
maximum within 4 h but declined subsequently to
match the starter culture thus reflecting its consump-
tion/degradation (ref.Figures 1–3).Such a short time
span (4 h) for proline climax is also reported in
A.nidulans (Wu et al.1995) or the green alga C.vulgaris
(Mehta & Gaur 1999),in contrast to higher plants (Alia
& Saradhi 1991).The overall trend of proline biosyn-
thesis in M.laminosus was indicative of stress caused by
lower temperatures (30 or 20 C) relative to growth
temperature optimum (40 C).Likewise,N.muscorum
and Nostoc 593 subjected to temperature shifts,could
take it as stress,and triggered proline biosynthesis.It is
also a fact that stimulation in proline biosynthesis or the
attainment of peaks was also limited to 4 h irrespective
of the stress.
The data clearly demonstrate that proline is synthe-
sized/accumulated by cyanobacteria as a response to
new environment irrespective of origin and that too as
quickly as within 4 h.Synthesis,accumulation and
catabolism of proline in plants proceed via two routes,
i.e.either glutamate or ornithine (Adams & Frank 1980;
Delauney & Verma 1993).It has been suggested that
proline accumulation in plants under stress involves the
loss of feedback regulation owing to a conformational
change in the P5C5 protein (D
1
-pyrroline-5-carboxylate
synthetase) (Boggess et al.1976a,b).In bacteria,proline
biosynthesis is regulated by the end product inhibition
of c-GK (c-glutamyl kinase) activity (Smith
et al.1984).Proline is also accumulated as a response
to low and high temperatures in barley,radish or wheat
seedlings (Chu et al.1974;Naidu et al.1991).A
reciprocal increase in P5C5 and PDH (proline dehydro-
genease) during stress and recovery from stress controls
the proline level in plants (Peng et al.1996).
In general,homeostasis of the ‘proline cycle’ depends
on the physiological state of the target tissue (Verma
1999) and the imino acid scavenges free radicals rather
than acting as the simple osmolyte (Hong et al.2000).
This aspect virtually opens the new research avenues on
metabolic engineering and stress-tolerance.Singh et al.
(1996) suggested that N.muscorumharbours two proline
transport systems:one under normal growth conditions
for the imino acid transport and also to be metabolized
like a N source,and the other,an osmoinducible that
functions under salt-stress for uptake and even overac-
cumulation of cellular proline as compatible osmolyte to
counteract the osmotic/salt stress.Each proline mole-
cule after oxidation produces 30 ATP equivalents
(Atkinson 1977) therefore,catabolism donates electrons
to the respiratory electron transport chain (Hare &
Cress 1997),and transfer of amino nitrogen and
reducing power to cells for recovery from stress (Peng
et al.1996;Verbruggen et al.1996;Xin & Browse 1998)
in addition to support of N
2
-fixation (Kohl et al.1988).
The data at present demonstrate that proline accumu-
lation in cyanobacteria is strain-specific.The superiority
of Nostoc 593 over M.laminosus or N.muscorum opens
curiosity for details of the enzymes of anabolism and/
catabolism.However,it is certain that such a metabo-
lism shift is related to acclimation of cyanobacteria.
Thiol biosynthesis
The hyperactive soluble thiols such as glutathione and
cysteine regulate cell respiration,act as binding posts
between proteins and prosthetic groups,and support the
electron transfer system(Barron 1951).In higher plants,
thiol/disulphide exchange involving the glutathione pool
regulates signal transduction during environmental
stress (Foyer et al.1997).In almost all stress cases,thiol
level is raised slightly over the basal,and the dynamics
possibly reflect metabolism adjustment to overcome
stress imposed by temperature shifts in the present case.
The idea in monitoring total thiol content in the present
work,was to ascertain if thiols could be taken as the
stress parameter.The three cyanobacterial targets while
growing at their respective temperature optima,had the
lowest basal thiol level for N.muscorum (17.8 lmol g
)1
dry wt.) followed by M.laminosus (20 lmol g
)1
dry wt.)
and the highest in Nostoc 593 (23.1 lmol g
)1
dry wt.)
(ref.Figures 4–6).Such values are in close proximity
with those of N.spongiaeforme (22.5 lmol g
)1
dry wt.)
as reported by Singh et al.(1999).As such,there is no
other report on the amount of total thiol in cyanobac-
teria to offer a comparison with the exception of
glutathione in N.muscorum (2.5 lmol g
)1
,Fahey et al.
1978),however,endogenous level of GSSG (oxidized
glutathione) + GSH (reduced glutathione) was in the
range of 0.6–0.7 mM(Karni et al.1984).In a subsequent
6 A.P.Singh et al.
report,Synechococcus 6311 and N.muscorum 7119 had
glutathione plus glutathione disulphide in the range of
2.3–4.1 mM in the former and 3.2 mM for the latter
(Tel-Or et al.1985).These investigators also observed
that 89%of the amount was glutathione while the rest as
glutathione disulphide.Glutathione level in Mycobacte-
rium smegmatis MC
2
6 was 19 lmol g
)1
residual dry wt.
and 12 lmol g
)1
residual dry wt.for M.tuberculosis
ATCC 25618 (Newton et al.1996).The level of c-
glutamyl cysteine (as free thiol) was 2.9 lmol g
)1
pellet
dry wt.in Halobacterium halobium (Sundquist & Fahey
1989).
In M.laminosus,stimulation of thiol biosynthesis was
indicative of stress imposed during exposure to lower
temperatures (30 or 20 C) relative to optimum (40 C)
(ref.Figure 4).Stimulation of thiol biosynthesis or the
attainment of peaks was also limited to 4 h in line with
the control.Thiol biosynthesis in M.laminosus (at
40 C) with the average slow rise rate of 0.5 lmol g
)1
dry wt.h
)1
,attained 2.25 lmol g
)1
dry wt.h
)1
for cells
facing a temperature ‘shift down’ (30 C).Noticeably,
cells facing stress of 20 C below the optimum,triggered
thiol biosynthesis to 5.07 lmol g
)1
dry wt.h
)1
(ref.
Figure 4,topmost curve).Similarly,N.muscorum and
Nostoc 593 cells subjected to temperature shifts,
responded as if the cells were facing stress (ref.Figures
5 and 6).The overall comparison clearly demonstrates
that thiol is synthesized/accumulated by cyanobacterial
cells as a response to temperature stress irrespective of
their origin and also,as quickly as within 4 h and
stabilized thereafter.In other words,thiol synthesizing
enzyme(s) might be active optimally until 4 h and thiol
available,was consumed in various metabolic events.
The observations indicate possible involvement of thiols
in acquisition of thermotolerance at least in the target
strains;nevertheless more details are needed to ascertain
whether these alone or in conjunction with other
metabolites,do the job.
Glyceraldehyde-3-phosphate dehydrogenase activity
GAPDH has been characterized from mesophiles to
moderately thermophiles and hyperthermophiles (Wrba
et al.1990).Therefore,the present work on GAPDH
activity in cyanobacteria facing temperature-stress
seems justified.The vital metabolic role of the enzyme
is in the glycolytic transformation of glucose to pyruvic
acid,the event highly crucial to carbohydrate metabo-
lism (Harris & Waters 1976).Analogous report in
cyanobacteria is limited to one by Papen et al.(1986),
who reported GAPDHin heterocyst and vegetative cells
of Anabaena variabilis,A.cylindrica and Anabaena 7119
that utilized both NAD and NADP
+
for its activity.
The NADP
+
-dependent enzyme activity in extract of
whole filaments was 74 nmol min
)1
mg
)1
protein in A.
variabilis,61 nmol min
)1
mg
)1
protein in A.cylindrica
and 85 nmol min
)1
mg
)1
protein in Anabaena 7119.A
comparison in this regard,puts the basal enzyme
activity level in the range of 120.6 nmol min
)1
mg
)1
protein for M.laminosus,85.8 nmol min
)1
mg
)1
protein
for N.muscorum and 141 nmol min
)1
mg
)1
protein for
Nostoc 593 (ref.Figures 7–9).The elevated basal level of
enzyme activity only in M.laminosus or Nostoc 593
possibly indicates the survival strategies adopted by the
two extremophiles to sustain effective metabolism under
conditions of temperature extremes although not
excluding other stresses in the respective habitat.
The pattern of temperature vs.GAPDH activity
collectively suggests that:(a) the thermophile and
psychrophile possessed maximum enzyme activity re-
stricted only to their respective growth temperature
optima and the temperature shift-down (in M.lami-
nosus) or temperature shift-up (in Nostoc 593) was
inhibitory to enzyme expression,and (b) N.muscorum
was at variance in this regard as temperature shifts from
the optimum,promoted enzyme activity (ref.Figure 8).
Wrba et al.(1990) observed that in mesophilic yeasts,
rise in temperature from 35 to 50 C inhibited GAPDH
activity and similar was the case in Thermotoga maritima
facing temperature shift down from the optimum of
100 C.It is therefore,substantiated by the present
observations that extremophiles manage to sustain
optimum GAPDH activity at their respective growth
temperature optimum.The possible physiological link-
age between thiol and GAPDH has been the target of
investigation in the past (Harris & Waters 1976;Papen
et al.1986).Attempts made in the present study,offer
some variations from the expected positive correlation
between thiol and GAPDH activity.Temperature stress
in all the target strains promoted thiol biosynthesis
invariably,nevertheless the opposite response in terms
of GAPDH makes it difficult to link thiol and enzymes
activity at least in the case of M.laminosus and Nostoc
593 (ref.Figures 7 and 9) and not in N.muscorum as
thiol biosynthesis and GAPDH activity were both
stimulated during temperature shift-up or shift-down
exposures (ref.Figure 8).
Acknowledgements
We are grateful to the Head and Programme Coordi-
nator,Centre of Advanced Study in Botany and
Coordinator,School of Biotechnology,Banaras Hindu
University,Varanasi,India for laboratory facilities.
Financial support of CSIR(Ref.No.38(0964)/99/EMR-
II),New Delhi is gratefully acknowledged by RKA.
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A comparison of proline,thiol levels and GAPDH activity 9
Growth kinetics of EDTA biodegradation by Burkholderia cepacia
Shih-Chin Chen
1
,Kuo-Hsu Li
2
and Hung-Yuan Fang
2,
*
1
Graduate School of Engineering Science and Technology (Doctoral Program),National Yunlin University of Science
and Technology,Touliu,Yunlin 640,Taiwan
2
Graduate Institute of Safety,Health and Environmental Engineering,National Yunlin University of Science and
Technology,Touliu,Yunlin 640,Taiwan
*Author for correspondence:E-mail:csc@ylepb.gov.tw
Received 24 December 2003;accepted 7 May 2004
Keywords:Biodegradation,Burkholderia cepacia,EDTA,kinetic parameters
Summary
A pure culture of an EDTA-degrading strain was isolated from the Taiwan environment.It was identified as
Burkholderia cepacia,an aerobic bacterium,elliptically shaped with a length of 5–15 lm.The degradation assay
showed that the degradation efficiency of Fe-EDTA by B.cepacia was approximately 91%.Evaluation of kinetic
parameters showed that Fe-EDTA degradation followed substrate inhibition kinetics.This is evident from the
decrease in specific growth rate with an increase in the initial substrate concentration greater than 500 mg/l.To
estimate the kinetic parameters – l
max
,K
S
and K
I
,five substrate–inhibition models were used.From the results of
non-linear regression,the value of l
max
ranged from 0.150 to 0.206 d
)1
,K
S
from 74 to 87 mg/l,and K
I
from 890 to
2289 mg/l.The five models were found to underestimate the maximumspecific growth rate by 1.5–3.7%.Therefore,
predictions based on these models would result in lower predicted value than those from the experimental kinetic
data.
Introduction
Ethylenediaminetetracetate (EDTA) widely used in
industries and households,is an aminopolycarboxylic
acid (Witschel et al.1997).It can chelate with metal ions
to form a stable and soluble chelate compound.Che-
lating agents form soluble complexes with heavy metals,
increasing their mobility in subsurface environments
(Cleveland & Ress 1981).A report (van Ginkel et al.
1997) showed that there are only three EDTA-degrading
bacterial strains that have isolated such as:Agrobacte-
rium sp.ATCC 55002 (Lauff et al.1990),gram-negative
BNC1 (No
¨
rtemann 1992) and a gram-negative bacte-
rium DSM 9103 (Witschel et al.1997).These EDTA-
degrading bacterial strains utilize Fe-EDTA or EDTA
as the sole sources of energy and carbon (Lauff et al.
1990;No
¨
rtemann 1992;Witschel et al.1997).Hence,
EDTA can be used as a substrate for bacterial growth.
In the present study,Fe-EDTA was used as major
substrate of carbon source in the growth medium to
isolate and screen for an EDTA-degrading strain.
The other objective of the present study was to estimate
the growth kinetic parameters from the relationship
between the substrate concentration (Fe-EDTA) and the
specific growth rate.Much previous kinetic research has
been done using non-inhibitory substrates for which
substrate-limited growth usually is represented by the
well-known Monod equation (Monod 1949).The
Monod equation has been applied with some success in
stable,continuous culture systems for the steady-state
biodegradation of inhibitory compounds (Neufeld &
Valiknac 1979;Kim et al.1981a,b;Colvin & Rozich
1986;Gaudy et al.1986).In such systems,the contam-
inant concentration is below the inhibition threshold.
However,the Monod equation cannot be used success-
fully to predict washout (Kumaran &Paruchuri 1997).A
number of substrate–inhibition models have been pro-
posed for use with inhibitory substrates (Webb 1963;
Yano et al.1966;Aiba et al.1968;Edwards 1970;Yang &
Humphrey 1975;Wayman & Tseng 1976;Sokol &
Howell 1981;Han & Levenspiel 1988).The simplest
and most commonly used model,proposed by Haldane
(1930),is given as Equation (1) and four other substrate–
inhibition models,Equations (2)–(5),are listed in
Table 1.The five substrate–inhibition models will be
tested with the experimental data of this study.
Materials and methods
Chemicals and culture media
The cultures of the bacteria grew in a sterile basal
medium prepared with deionized water and contained
World Journal of Microbiology & Biotechnology 2005 21:11–16
DOI:10.1007/s11274-004-0876-6

Springer 2005
substrates and mineral salts in the following concentra-
tion (mg/l):Fe-EDTA,500;CH
3
COOK,125;Ur-
ea+(NH
4
)SO
4
(ratio of 1:1,w/w ),500;KH
2
PO
4
,
1000;NaCl,100;CaCl
2
,25;FeSO
4
Æ 7H
2
O,20;
MgSO
4
Æ 7H
2
O,100;ZnSO
4
Æ 6H
2
O,10;CuSO
4
Æ 6H
2
O,
10;MnSO
4
Æ 4H
2
O,5;CoCl
2
Æ 6H
2
O,0.5;H
3
BO
3
,0.5;
Na
2
MoO
4
Æ 2H
2
O,0.5.All of these chemicals were
reagent grade.Fe-EDTA was used as major carbon
source,while CH
3
COOK as cometabolic carbon source.
Urea and ammonium sulphate were used as the major
nitrogen source.
Isolation and screening of microbial strain
Isolation samples of sludge were obtained from a
wastewater stream that had contained Fe-EDTA for a
long period of time.The enrichment culture method was
used to isolate and screen for an EDTA-degrading
strain.An enrichment mediumof 200 ml was inoculated
with 1 g of the sample and incubated at 30 C in a shake
flask running at 150 rev/min for 10–15 days.Cell
material from this enrichment culture was streaked on
an agar plate and incubated at 30 C until colonies
appeared.Then a single colony was picked up and
incubated on an agar slant.This colony was spread,
picked and restreaked repeatedly more than three times.
The pure strain was used as inoculumand transferred to
liquid medium contained in a flask.The liquid culture
medium containing Fe-EDTA as carbon source and
phosphate buffer in basal medium at pH 7.0 was
incubated at 30 C in a flask shaken at 150 rev/min.
Cell growth was measured by the optical density (OD)
via a spectrophotometer (Model UVIKON930,Kon-
torn,Italy) at 660 nm for 12 h.
Strain identification
After growth had taken place in Fe-EDTA medium,
cells were harvested by centrifuging at 17,300 · g for
5 min,(Model CR22E,Hitachi,Japan) and the super-
natant was withdrawn for analysis.Biochemical prop-
erties of the strain were examined by API 20 NE test kit
(BioMerieux,Marcy l’Etoile,France).The morphology
of the strain was observed by a scanning electron
microscope (JEOL JSM-5410 LV,Japan).
Measurement of EDTA,and COD
High-performance liquid chromatography analysis was
performed to determine EDTA concentration (Janne
Virtapohja & Raimo Alen 1999).The column was
250 · 4.6 mm (Spherisorb C18).Sample peaks were
separated by isocratic elution at 1.0 ml/min where
0.05 M sodium acetate was used as the mobile phase
and buffer solution.The pH was adjusted to 4.5 by
adding acetic acid.EDTA concentration was detected
by ultraviolet spectrophotometer at 254 nm.Chemical
oxygen demand (COD) was analysed according to
Standard Method for Examination of Water and
Wastewater (APHA et al.1992).
Growth kinetic experiment
After serial culture procedures,the EDTA-degrading
bacterium Burkholderia cepacia was transferred to a
flask with 200 ml culture medium and cultivated for 7–
10 days on a shaker running at 150 rev/min,pH7.0 and
30 C.As the growth of bacterium reached the expo-
nential phase at OD
660 nm
greater than 1.0,the broth
was harvested as seed.Then 200 ml of these cultures
were transferred to a 5-l fermentor with 3-l working
volume (as shown in Figure 1) for further growth
experiment.Batch growth experiments with initial Fe-
EDTA concentration in a range of 10–1500 mg/l were
performed in the fermentor.The composition of culture
medium in the fermentor was the same as mentioned in
the previous section except that the concentration of
CH
3
COOKwas 1/4 of that of Fe-EDTA.The fermentor
was stirred constantly at 500 rev/min.The cultivation
temperature was maintained at 30 C and pH 7.0.The
fermentor was supplied with filter-sterilized air (Advan-
tec JP050,0.2 lmproe size,Japan).Sampling took place
every 12 h,and cell concentration was measured by
protein content.Protein content was measured by the
Folin-Ciocalteu phenol reagent method and calculated
from a standard curve constructed in this experiment.
The cell concentration (X) was calculated from protein
content (P) measurement using the linear relationship
X ¼ P/0.6 we had constructed in this experiment.
The growth kinetics of Fe-EDTA degradation of B.
cepacia were determined by the data from batch
fermentation experiments.In batch fermentation exper-
iments,the specific growth rate was determined as the
slope of ln X (where X is the cell concentration) vs.time
measured immediately after the lag phase.The initial
Fe-EDTA concentration (S) was used to represent the
characteristic Fe-EDTA concentration for each specific
growth rate.
The kinetic parameters l
max
(maximum specific
growth rate),K
S
(saturation constant) and K
I
(inhibition
constant) were determined by non-linear regression on S
and l from the full dataset using the statistical software
package,SPSS (SPSS 10.0 for Windows).Initial esti-
mates for the kinetic parameters l
max
and K
S
were
obtained using a linearized form of the Haldane model
Table 1.Substrate–inhibition models.
Source Model
Haldane (1930) l ¼
l
max
S
S þK
S
þ
S
2
K
I
Webb (1963) l ¼
l
max
S 1 þ
S
K
 
S þK
S
þ
S
2
K
I
Yano et al.(1966) l ¼
l
max
S
S þK
S
þ
S
2
K
I
1 þ
S
K
 
Aiba et al.(1968) l ¼
l
max
Se
S=K
I
S þK
S
Edwards (1970) l ¼ l
max
e
S=K
I
e
S=K
S
 
12 S.-C.Chen et al.
at low Fe-EDTA concentration (SK
I
).By taking the
derivative of Haldane model equation,S ¼
ffiffiffiffiffiffiffiffiffiffiffi
K
S
K
I
p
can
be found when
dl
dS
¼ 0 (Humphrey 1978).Thus,the
initial estimate of K
I
value can be calculated by using the
experimental data S when l is the maximum value.The
95% confidence level was used for all tests of signifi-
cance.
Results and discussion
Isolation,screening,and identification of the EDTA
degradation strain
A bacterial strain was selected from the Taiwan envi-
ronment.It was isolated by Fe-EDTA enrichment
cultivation on a shaker,with 150 rev/min at pH 7.0,
30 C,for 30 days,and the strain YL-6,which possesses
the greatest degradation capability,was isolated.
The growth is shown in Figure 2.Since the colour of
Gramstaining was red,strain YL-6 is to Gram-negative.
The oxidase test showed a dark-coloured colony,which
means oxidase is present.The catalase test revealed O
2
generation,which proves catalase is present.Through
these tests,YL-6 was found to be an aerobic species.The
biochemical properties of the isolated strain were
examined by using the API 20 NE test kit (99%,
probability).The photograph of YL-6 shown in Fig-
ure 3,taken by a scanning electron microscope at ·1500,
shows that the cells are elliptical and 5–15 lm in length.
Finally,the isolated strain YL-6 was determined as
Burkholderia cepacia.
Figure 1.Schematic illustration of the batch fermentor.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
0 3 6 9 12 15 18 21 24 27 30
YL-1
YL-2
YL-3
YL-4
YL-5
YL-6
Isolated Sources
Culure Time (days)
Growth (OD
660nm)
Figure 2.Accumulation of different isolated source in the Fe-EDTA medium.
EDTA biodegradation 13
Degradation assay
To evaluate the degradation efficiency of the strain,the
strain was incubated in a shake flask running at 150 rev/
min,pH 7.0 and 30 C with 1000 mg/l Fe-EDTA.The
time course of cell growth and Fe-EDTA utilization by
B.cepacia is shown as Figure 4.Obviously on the first
day,the cell growth was still at lag phase.The second
day,the growth entered the exponential growth phase
slowly and reached the stationary phase after day 15.
The Fe-EDTA concentration was degraded by B.
cepacia from1000 to 90 mg/l at day 17.The degradation
efficiency of Fe-ETDA was approximately 91%and the
removal of COD was 78%.This shows that B.cepacia
YL-6 has the ability to degrade Fe-EDTA.
Growth of batch culture
Figure 5 shows the specific growth rate (l) of strain YL-
6 in batch experiments with initial Fe-EDTA concen-
tration in a range from10 to 1500 mg/l in the fermentor.
From the corresponding value of the specific growth
rate of each substrate concentration,the value of l
increased with increasing substrate at the beginning.
When the substrate concentration reached to 500 mg/l,
the specific growth rate reached its maximum value
(=0.131 h
)1
).At substrate concentrations greater than
500 mg/l,the specific growth rate began to decline.This
shows that cell growth follows substrate inhibition
kinetics,which is evident from the decrease in specific
growth rate with increase in the initial substrate con-
centration.The generation time calculated from exper-
imental values of the maximum specific growth rate for
B.cepacia growing on Fe-EDTA was 5.3 days.
Estimation of kinetic parameters
Five substrate–inhibition models were used to estimate
the values of kinetic parameters – l
max
,K
S
and K
I
.
Multiple parameter sets were obtained by varying the
initial estimates.
For the fit of Haldnne model,the method of double-
reciprocal plot (l/l vs.l/S) was used.From the result of
data fitting,the estimated value of kinetic parameters
l
max
,K
S
and K
I
were 0.206 d
)1
,80 mg/l and 890 mg/l,
respectively.The r
2
value of Haldane model was about
0.997 and the predicted value of maximum specific
growth rate (l*) was 0.129 d
)1
.By comparing with the
Figure 3.Photograph of Burkholderia cepacia YL-6 by the scanning
electron microscope.
0
100
200
300
400
500
600
700
800
900
1000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0.0
0.5
1.0
1.5
2.0
2.5
COD
Cultivation time (days)
Optical Density (OD660nm)
Concentration (mg/L)
EDTA
OD
660nm
Figure 4.The time course of cell growth and Fe-EDTA utilization by Burkholderia cepacia.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0 200 400 600 800 1000 1200 1400 1600
Substrate concentration (mg/L)
(d-1)
experimental data
fitted curve
Figure 5.Data (¤) was from the batch experiments with initial Fe-
EDTA concentration from 10 to 1500 mg/l.The fitted curve [—] was
plotted using the parameters:l
max
¼ 0.206 h
)1
,K
S
¼ 80 mg/l,
K
I
¼ 890 mg/l.The Haldane model equation was l ¼ 0.206/(1+80/
S+S/890).
14 S.-C.Chen et al.
corresponding experimental data (=0.131 d
)1
),the
Haldane model was found to underestimate l* value
by 1.5%.Figure 5 is the fitted curve of Haldane model
for l vs.S using the kinetic parameters described above.
The predicted value and the experimental value of
maximum specific growth rate were slightly different by
1.5%.Therefore,prediction based on the Haldane
model would result in lower predicted value than those
from the experimental kinetic data.
Compared with Haldane model,the Webb and Yano
models have an additional factor (S/K).When the value
of K approaches infinity,the Webb and Yano models
simplify to the Haldane model.For the Webb model,
after different initial estimate of regression,when the
value of K is larger than 10
4
,the r
2
values are larger than
0.95 and the difference of kinetic parameters is very
small.Figure 6 is the fit of the Webb model using the
kinetic parameters listed in Table 2.Similarly,after
regression,the fit of the Yano model is shown in
Figure 7.The r
2
value is 0.95.For the Webb and Yano
models,the predicted values of l* were 0.127 and
0.129 d
)1
,respectively.Compared to the corresponding
experimental value of 0.131 d
)1
,the Webb and Yano
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0 200 400 600 800 1000 1200 1400 1600
Substrate concentration (mg/L)
experimental data
fitted curve
(d-1)
Figure 6.The fitted curve [—] was plotted using the parameters:
l
max
¼ 0.203 h
)1
,K
S
¼ 87 mg/l,K
I
¼ 891 mg/l,K ¼ 18969 mg/l.The
Webb model equation was l ¼ 0.203(1+S/18969)/(1+87/S+S/891).
Table 2.Parameter estimates and regression for different substrate–inhibition models.
Temperature (C) Model Estimates
l
max
(d
)1
) K
S
(mg/l) K
I
(mg/l) K (mg/l) r
2
value
30
Haldane (1930) 0.206 80 890 – 0.997
Webb (1963) 0.203 87 891 18969 0.958
Yano et al.(1966) 0.206 81 904 22737 0.950
Aiba et al.(1968) 0.189 75 1703 – 0.975
Edwards (1970) 0.15 74 2289 – 0.952
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0 200 400 600 800 1000 1200 1400 1600
Substrate concentration (mg/L)
(d-1)
experimental data
fitted curve
Figure 7.The fitted curve [—] was plotted using the parameters:l
max
¼ 0.203 h
)1
,K
S
¼ 87 mg/l,K
I
¼ 891 mg/l,K ¼ 18969 mg/l.The Webb
model equation was l ¼ 0.203(1+S/18969)/(1+87/S+S/891).
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0 200 400 600 800 1000 1200 1400 1600
Substrate concentration (mg/L)
(d-1)
experimental data
fitted curve
Figure 8.The fitted curve [—] was plotted using the parameters:l
max
¼ 0.189 h
)1
,K
S
¼ 75 mg/l,K
I
¼ 1703 mg/l.The Aiba model equation was
l ¼ 0.189Se
)S/1703
/(S+75).
EDTA biodegradation 15
models were found to underestimate l* by 1.5–3.7%as
well.Compared with the Haldane model,the difference
of kinetic parameters l
max
,K
S
and K
I
are small.
The other two models’ Aiba and Edwards,after
different initial estimates of regression,gave the results
shown in Table 2.Figures 8 and 9 are the fit of the Aiba
and Edwards models.The r
2
values of the Aiba and
Edwards models are 0.975 and 0.952 and the predicted
values of l* are 0.127 and 0.129 d
)1
,respectively.
Compared to the corresponding experimental value,
the Aiba and Edwards model still underestimate l* by
3.7–1.5%.Compared with the Haldane,Webb and Yano
models,the kinetic parameters l
max
and K
S
of Aiba and
Edwards models are smaller,but the value of K
I
is larger.
References
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Edwards,V.H.1970 The influence of high substrate concentrations on
microbial kinetics.Biotechnology and Bioengineering 12,679–712.
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Haldane,J.B.S.1930 Enzymes.London:Longmans,Green.
Han,K.&Levenspiel,O.1988 Extended monod kinetics for substrate,
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Humphrey,A.E.1978 Biochemical reaction engineering.American
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Janne Virtapohja & Raimo Alen 1999 Behaviour of EDTA in marine
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Kim,J.W.,Humenick,M.J.& Armstrong,N.E.1981a A comprehen-
sive study on the biological treatabilities of phenol and methanol.
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Kim,J.W.,Humenick,M.J.& Armstrong,N.E.1981b A comprehen-
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Research 15,1233–1247.
Kumaran,P.& Paruchuri,Y.L.1997 Kinetics of phenol biotransfor-
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Lauff,J.J.,Steele,D.B.,Coogan,L.A.& Breitfeller,J.M.1990
Degradation of the ferric chelate of EDTA by a pure culture of an
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3346–3353.
Monod,J.1949 The growth of bacterial cultures.Annual Review of
Microbiology 3,371–394.
Neufeld,R.D.& Valiknac,T.1979 Inhibition of phenol biodegrada-
tion by thiocyanate.Journal of the Water Pollution Control
Federation 51,2283–2291.
No
¨
rtemann,B.1992 Total degradation of EDTA by mixed cultures
and a bacterial isolate.Applied and Environmental Microbiology 58,
671–676.
Sokol,W.&Howell,J.A.1981 Kinetics of phenol oxidation by washed
cells.Biotechnology and Bioengineering 23,2039–2049.
Van Ginkel,C.G.,Vandenbroucke,K.L.& Stroo,C.A.1997 Biolog-
ical removal of EDTA in conventional activated-sludge plants
operated under alkaline conditions.Bioresource Technology 59,
151–155.
Wayman,M.& Tseng,M.C.1976 Inhibition threshold substrate
concentrations.Biotechnology and Bioengineering 18,383–387.
Webb,J.L.1963 Enzyme and Metabolic Inhibitors.Boston:Academic
Press.
Witschel,M.,Nagel,S.& Egli,T.1997 Identification and character-
ization of the two-enzyme systemcatalyzing oxidation of EDTAin
the EDTA-degrading bacterial strain DSM 9103.Journal of
Bacteriology 179,6937–6943.
Yang,R.D.&Humphrey,A.E.1975 Dynamic and steady-state studies
of phenol biodegradation in pure and mixed cultures.Biotechnol-
ogy and Bioengineering 17,1211–1235.
Yano,T.,Nakahara,T.,Kamiyama,S.& Yamada,K.1966 Kinetic
studies on microbial activities in concentrated solutions.I.Effect
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system.Agricultural and Biological Chemistry 30,42–48.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0 200 400 600 800 1000 1200 1400 1600
Substrate concentration (mg/L)
(d-1)
experimental data
fitted curve
Figure 9.The fitted curve [—] was plotted using the parameters:l
max
¼ 0.150 h
)1
,K
S
¼ 74 mg/l,K
I
¼ 2289 mg/l.The Edwards model equation
was l ¼ 0.15 (e
)S/2289
)e
)S/74
).
16 S.-C.Chen et al.
Biosynthesis of short-chain-length-polyhydroxyalkanoates during the
dual-nutrient-limited zone by Ralstonia eutropha
Qun Yan
1
,Ying Sun
1
,Lifang Ruan
1
,Jian Chen
2
and Peter Hoi Fu Yu
1,
*
1
Open Laboratory of Chirotechnology of the Institute of Molecular for Drug Discovery and Synthesis,Department of
Applied Biology and Chemical Technology,The Hong Kong Polytechnic University,Hong Hum,Kowloon,Hong Kong
2
Ministry of Education,Key Laboratory of Industrial Biotechnology,Southern Yangtze University,Wuxi 214036,
China
*Author for correspondence:Tel.:+852-27666558,Fax:+852-23649932,E-mail:bcpyu@inet.polyu.edu.hk
Received 3 February 2004;accepted 7 May 2004
Keywords:Ralstonia eutropha,scl-PHAs,dual-nutrient-limitation zone,biosynthesis
Summary
Biosynthesis of PHAs by Raltonia eutropha during the dual nutrient-limitation-zone was investigated with mixed
organic acids as carbon sources and (NH
4
)
2
SO
4
as nitrogen source.Two different methods of maintaining the dual-
nutrient-limitation zone were adopted by feeding mixed acids and (NH
4
)
2
SO
4
at determined rates into the
fermentation cultures which were initially free of carbon sources (method A) or nitrogen sources (method B).The
results indicate that,firstly,with the increase of the width of the dual-nutrient-limitation zone,the yield of short-
chain-length-polyhydroxyalkanoates also increases and it suggests that most of the short-chain-length-polyhy-
droxyalkanoates were biosynthesized during the dual-nutrient-limitation zone.Secondly,in contrast with the
dual-nutrient-limitation method of limiting the nitrogen source first (method B),the dual-nutrient-limitation
method of limiting the carbon source first (method A) was more favourable for the production of short-chain-
length-polyhydroxyalkanoates,and the maximum production of short-chain-length-polyhydroxyalkanoates of
these two methods are 3.72 and 2.55 g/l,respectively.
Introduction
Polyhydroxyalkanoates (PHAs) are a class of polyesters
biosynthesized by a variety of microorganisms under
unbalanced growth conditions,such as the limitation of
carbon,nitrogen,oxygen,magnesium,phosphorus and
even trace elements (Asenjo et al.1995).Short-chain-
length-PHAs (scl-PHAs) are those polymers with the
chain length of the monomers varying from 3 to 5,in
contrast with medium-chain-length-PHAs (mcl-PHAs),
with the monomer chain length varying from6 to 14 and
long-chain-length-PHAs (lcl-PHAs) with the monomer
chain length longer than 14 (Luengo et al.2003).PHAs
are supposed to partly take the place of traditional
plastics made from petroleum in the near future since
they are harmless to the environment and biodegradable.
However,the growth conditions,the cost of the raw
materials and their recovery have limited the bulk
production of the PHAs.Thus,exploiting the biosyn-
thesis of PHAs using cheap carbon sources may be the
better strategy (Anderson & Dawes 1990).Hereby
organic acids (mainly butyrate,lactate,propionate and
acetate,etc.) obtained fromagricultural waste materials,
industrial wastes,and food wastes after anaerobic
digestion may be the first choice to be used as carbon
sources to produce scl-PHAs (Wong et al.2000;Yu
2001).At the same time,since acids are inhibitory to cell
growth of microorganisms,a relative low initial con-
centration of acids in the media or slow feeding at a
controlled speed is required (Douglas & James 1995).
Dual-nutrient-limitation means to limit two nutrient
factors (usually are carbon and nitrogen sources)
simultaneously by controlling the feeding rates of these
two kinds of nutrients.Durner et al.tried to investigate
the biosynthesis of mcl-PHAs during the dual-nutrient-
limitation zone and even the triple-nutrient-limitation
zone by Pseudomonas oleovorans,they found that the
dry cell mass (DCW) of the microorganism and the
composition of the PHAs were both affected by the dual
limitation of carbon and nitrogen sources.Besides,the
yield of mcl-PHAs could be determined by the width of
the dual-nutrient-limitation zone (Durner et al.2000,
2001).However,studies on the biosynthesis of scl-PHAs
during the dual-nutrient-limitation zone have not yet
been reported.
This paper investigates the formation of scl-PHAs
during the dual-nutrient-limitation (carbon and nitrogen
sources) zone by Ralstonia eutropha with mixed organic
acids and ammonium sulphate used as carbon and
nitrogen source,respectively.
World Journal of Microbiology & Biotechnology 2005 21:17–21
DOI:10.1007/s11274-004-0877-5

Springer 2005
Material and methods
Microorganism and media
Ralstonia eutropha ATCC 17699 was used throughout
this study.
The seed culture medium contained (per l):20 g glu-
cose,3 g (NH
4
)
2
SO
4
,1.2 g MgSO
4
,1.7 g citric acid,
13.3 g KH
2
PO
4
,10 ml mineral solution (per 1 HCl
(1 mol/l):10 g FeSO
4
Æ 7H
2
O,2.25 g ZnSO
4
Æ 7H
2
O,
2.25 g CuSO
4
Æ 5H
2
O,0.5 g MnSO
4
Æ 5H
2
O,2 g Ca-
Cl
2
Æ 2H
2
O,0.3 g H
3
BO
4
,0.1 g (NH
4
)
6
Mo
7
O
24
) and the
pH was adjusted to 7.0.
Concentrations of the feeding stock of carbon (the
mass ratio of the four component acids was butyric acid:
propionic acid:acetic acid:lactic acid ¼ 3:3:1:1,and
which was simulated as once of the result of the
anaerobic digestion of food wastes) and nitrogen
sources of the fermentation media were 100 and 20 g/l,
respectively.Other constituents of the fermentation
culture were the same as those of the seed culture.
Culture methods
Inocula were cultivated in 250 ml flasks containing
75 ml of seed culture at 30 C and 200 rev/min for 24 h.
Fermentation was conducted in a 5 l fermentor (KBT,
Korea) with the initial volume of 3 l and the inoculation
volume of was 10%.Carbon and nitrogen source were
fed by two different peristaltic pumps (Figure 1).The
culture temperature was maintained at 30 C and the
pH was controlled at 7.0 ± 0.1 by adding 3 M NaOH
and 3 M HCl.The aeration was 1.5 v/v/min and
agitation speed 400 rev/min.The maintaining condition
of the dual-nutrient-limitation zone was shown as
Table 1.
Analytic methods
Acids,DCWand PHAs were detected using the method
of Yu et al.2002.Organic acids were detected by HPLC
(Agilent 1100 equipped with a RID detector,USA) with
an Aminex HPX-87 column (300 · 7.8 mm,Bio-rad,
USA) at a column temperature of 60 C,and 0.0055 M
H
2
SO
4
as mobile phase.The eluted times for lactate,
acetate,propionate and butyrate were at about 10.7,
12.7,14.9 and 18.1 min,respectively.(NH
4
)
2
SO
4
was
detected using the Nessler’s reagent colorimetric method
(National Environment Protect Bureau 1989).
Results
Biosynthesis of scl-PHAs in the dual-nutrient-limitation
method A
The dual-nutrient-limitation method A was designed to
feed mixed acids to the fermentation culture,which
initially only contained nitrogen sources and no carbon
sources,at a constant rate in order to limit the supply of
carbon from the beginning of the fermentation.Nitro-
gen source was fed from hour 6 of the fermentation
process since the concentration of the residual nitrogen
in the media was rather low at that time.With the
forthcoming of logarithmic phase of the cell growth and
the starting of scl-PHA biosynthesis,most of the fed
mixed acids and ammonium sulphate was utilized,thus
the so-called dual-nutrient-limitation zone appeared
(Figure 2-A2,A3).Finally,whencell growthandpolymer
biosynthesis ceased,the residual concentration of carbon
and nitrogen sources in the media began to increase
rapidly as the dual-nutrient-limitation zone terminated.
Figure 2-A1,A2 and A3 show the effect of three dif-
ferent feeding rates on the length of the dual-nutrient-
limitation zone.As shown by Figure 2-A2,the length of
the dual-nutrient-limitation zone was the longest of the
three whenfeeding rate of carbonsource is about 18 ml/h,
dual-nutrient-limitation zone started from hour 8 of the
fermentation process and ended at hour 25,the whole
lengthwas about 17.Onthe other hand,nodual-nutrient-
limitation zone appeared when the feeding rate was
12 ml/h.Furthermore,the residual concentrationof acids
was under the detect limit throughout the process.While
the whole length of the dual-nutrient-limitation zone was
only about 10 h when the feeding rate was 25 ml/h,the
residual concentration of the mixed acids was higher than
2 g/l fromhour 20.
Figure 2-B,C show the time courses of the cell growth
and the production of scl-PHAs of the above three
Figure 1.Schematic illustration of dual-nutrient-limitation fermenta-
tion.
Table 1.Maintaining method of the dual-nutrient-limitation zone.
a
Batches A1,A2,A3 B1,B2,B3
C
0
(g/l) 0 2
N
0
(g/l) 1.5 0
F
carbon
(ml/h) 0–6 h 12,18,25 0
6–48 h 12,18,25 12,18,25
F
nitrogen
(ml/h) 0–6 h 0 16
6–48 h 11 11
a
(1) C
0
,N
0
refer to the initial media concentration of carbon and
nitrogen sources.F
carbon
and F
nitrogen
refer to the feeding rates of
carbon and nitrogen sources.(2) A1,A2,A3 and B1,B2,B3 stand
for the three batches of PHAs fermentation with different feeding
strategies of carbon and nitrogen sources so as to maintain the
corresponding dual-nutrient limitation methods of A and B,
respectively.
18 Qun Yan et al.
batches of dual-nutrient-limitation method of A.Cor-
respondingly,the yields of DCW and scl-PHAs were
also the highest of the three when feeding rate of carbon
sources was 18 ml/h,which were 8 and 3.72 g/l,respec-
tively.In addition,there was more than 50%of the net
increase of the scl-PHAs produced for A2 during the
dual-nutrient-limitation zone in this case,which was
about 1.5–1.7 g,while the yields of DCWand scl-PHAs
of the other two batches were 6.45,2.4 g/l (when feeding
rate of carbon sources was 12 m/l) and 6.58,2.15 g/l
(when feeding rate of carbon sources was 25 m/l)
respectively.
Biosynthesis of scl-PHAs in the dual-nutrient-limitation
method B
The dual-nutrient-limitation method B was designed to
feed ammonium sulphate to the fermentation culture,
which initially contained only carbon sources and no
nitrogen source,at a constant rate in order to limit the
supply of nitrogen from the beginning of the fermenta-
tion.Carbon sources started to be fed at hour 6.Others
were the same as method A.
Figure 3-B1,B2 and B3 shows the effect of three
feeding rates on the length of the dual-nutrient-limitation
0
0.02
0.04
0.06
0.08
0.1
0 10 20 30 40 50
Time (h) Time (h) Time (h)
Acids(g/L)
0
0.5
1.5
(NH4)2SO4(g/L)
0
2
4
6
10
0 10 20 30 40 50
Acids(g/L)
0
0.5
1
1.5
(NH4)2SO4(g/L)
0
1
2
3
5
0 10 20 30 40 50
Acids(g/L)
0
0.5
1.5
(NH4)2SO4(g/L)
1
8
4
1
A1 A2 A3
0
3
6
9
0 10 20 30 40 50
Time (h)
DCW(g/L)
DCW-A1
DCW-A2
DCW-A3
0
1
2
4
0 10 20 30 40 50
Time (h)
scl-PHAs (g/L)
PHAs-A1
PHAs-A2
PHAs-A3
3
B C
Figure 2.Time course of scl-PHAs fermentation by R.eutropha in the dual-nutrient-limitation method of A.Figure A1–A3 indicate the
corresponding time course of the utilization of carbon and nitrogen sources of the three batches of PHAs fermentation described as Table 1,
respectively.Symbols:

:Acids
¤
:(NH
4
)
2
SO
4
Shadow:dual-nutrient-limitation zone.
0
1
2
3
4
5
6
8
0 10 20 30 40 50
Acids(g/L)
0
0.2
0.4
0.6
(NH4)2SO4(g/L)
0
1
2
3
0 10 20 30 40 50
Time (h) Time (h) Time (h)
Acids(g/L)
0
0.2
0.4
0.6
(NH4)2SO4(g/L)
0
1
2
3
0 10 20 30 40 50
Acids(g/L)
0
0.2
0.4
0.6
(NH4)2SO4(g/L)
7
B1 B2 B3
0
3
6
9
0 10 20 30 40 50
Time (h)
DCW(g/L)
DCW-B1
DCW-B2
DCW-B3
A C
0
1
2
3
0 10 20 30 40 50
Time (h)
s/L)cl-PHAs (g/L)
PHAs-B1
PHAs-B2
PHAs-B3
Figure 3.Time course of scl-PHAs fermentation by R.eutropha in the dual-nutrient-limitation method of B.Figure B1–B3 indicate the
corresponding time course of the utilization of carbon and nitrogen sources of the three batches of PHAs fermentation described as Table 1,
respectively.Symbols:

:Acids
¤
:(NH
4
)
2
SO
4
Shadow:dual-nutrient-limitation zone.
Biosynthesis of PHA 19
zone.Started from hour 10 and ending at hour 40,the
whole length of the dual-nutrient-limitation zone was
about 30 h,and which was also the largest of the three
when feeding rate of the carbon sources was 18 ml/h.
Similarly,no dual-nutrient-limitation zone appeared
when the feeding rate was 12 ml/h (Figure 3-B1) and
the length of the dual-nutrient-limitation zone was only
10 h (Figure 3-B2) just as seen in Figure 2-A3.
In the case of the cell growth and scl-PHA produc-
tion,the highest yields could be obtained when the
feeding rate of the carbon sources was 18 ml/h,which
were 8.28 and 2.55 g/l,respectively.Of the net increase
of scl-PHAs,about 40% was biosynthesized during the
nutrient-limitation zone,which was about 0.8–1.0 g.
Moreover,yields of DCW and scl-PHAs were 5.25 and
1.98 g/l,respectively,when the feeding rate of the
carbon sources was 12 ml/h,and the yields of DCW
and scl-PHAs were 5.24 and 2.13 g/l,respectively,when
feeding rate of the carbon sources was 25 ml/h.
Discussion
To maximize the yield of DCW and the scl-PHAs,it is
essential to feed more carbon and nitrogen sources to
the fermentation medium,hence the limitation of mixed
acids or (NH
4
)
2
SO
4
did not mean to limit the supply of
them,but to feed as much of carbon and nitrogen
sources as possible in order to achieve maximum cell
growth and PHA formation by R.eutropha.On the
other hand,it is indispensable to make the residual
concentration of carbon nitrogen sources as low as
possible since organic acids are inhibitory to cell growth
(Suzuki et al.1986),and most importantly,only under
the condition of low concentration of nitrogen could R.
eutropha produce more PHAs than any other unbal-
anced growth condition.
In both of the dual-nutrient-limitation methods when
the feeding rate of carbon sources was 12 ml/h,carbon
mixed acid feeding was not sufficient for cell growth,
therefore,the dual-nutrient-limitation zone did not
appear,since inadequate acid supply cannot lead to
the fast utilization of ammonium sulphate.At the same
time,if excess carbon sources were fed,the dual-
nutrient-limitation zone may be terminated early be-
cause the higher concentration of residual acids would
prevent bacterial growth,and result in the presence of a
higher concentration of residual nitrogen.Only when
the carbon and nitrogen sources were fed at appropriate
rates could the length of dual-nutrient-limitation zone
be as long as possible since most of the nutrients fed
were almost all consumed.Therefore,yields of DCW
and scl-PHAs would be higher when the nutrients were
fed at appropriate values (18 ml/h) than those at lower
(12 ml/h) and higher (25 ml/h) rates.Moreover,yield of
scl-PHAs would increase with the increase of the length
of the dual-nutrient-limitation zone since the limitation
of one or two nutrients would lead to the scl-PHAs
production.
Dual-nutrient-limitation method Aseemed to be more
favourable for the biosynthesis of scl-PHAs than
method B,since yields of DCW and scl-PHAs of the
dual-nutrient-limitation method A were larger than
those of method B.This is particularly evident when
both the feeding rates were 18 ml/h,the yield of scl-
PHAs yield of method A (3.72 g/l) being about 46%
higher than that of method B (2.55 g/l).That is because
biosynthesis of scl-PHAs under unbalanced growth
condition of either carbon source limitation or nitrogen
source limitation both depend on the excess of acetyl-
CoA,which could enter the scl-PHAs biosynthesis
pathway as the precursor of the HB or HV component
of the polymer (Kessler & Witholt 2001).However,due
to the inadequate nitrogen supply during the first few
hours of the fermentation when nitrogen sources was
limited first (method B),although biosynthesis of scl-
PHAs could also be initiated after the GS-GOGAT
pathway was induced (Gostomski et al.1996),the
cytoplasmic enzymes,especially those scl-PHAs syn-
thetic enzymes may not developed so well as when
carbon source was limited first,correspondingly,not so
much of the scl-PHAs was produced.
From above all,dual-nutrient-limitation zone was
proved to be efficient to produce the highest possible
concentration of scl-PHAs since it could utilize organic
acids,which maybe come from wastes,at the largest
extent.However,a more calculated feeding method
should be studied further in order to keep the dual-
nutrient-limitation zone as long as possible.
Acknowledgement
The authors would like to show their sincere gratitude to