Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Utilization of agricultural residues for

poly(3-hydroxybutyrate) production by Halomonas
boliviensis LC1
D. Van-Thuoc1,2, J. Quillaguamán1, G. Mamo1 and B. Mattiasson1
1 Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Lund, Sweden
2 Department of Micro-biotechnology, Faculty of Agro-Biology, Hanoi University of Education, Hanoi, Viet Nam

Keywords Abstract
anaerobic digestion, Halomonas boliviensis,
moderate halophile, poly(3-hydroxybutyrate), Aims: Utilization of cheap and readily available agricultural residues as cheap
potato waste, wheat bran hydrolysate. carbon sources for poly(3-hydroxybutyrate) (PHB) production by Halomonas
boliviensis.
Correspondence Methods and Results: Wheat bran was hydrolysed by a crude enzyme prepara-
Jorge Quillaguamán, Department of
tion from Aspergillus oryzae NM1 to provide a mixture of reducing sugars
Biotechnology, Centre for Chemistry and
Chemical Engineering, Lund University, PO
composed mainly of glucose, mannose, xylose and arabinose. Growth of
Box 124, Lund SE-221 00, Sweden. H. boliviensis using a mixture of glucose (0Æ75% w ⁄ v) and xylose (0Æ25% w ⁄ v)
E-mail: [email protected] in the medium led to a PHB content and concentration of 45 wt% and 1 g l)1,
respectively, after 30 h. A similar PHB concentration was attained when
2007 ⁄ 0161: received 19 July 2006, revised 28 H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB
June 2007 and accepted 3 July 2007 content, 34 wt%. In a batch cultivation mode in a fermentor, using 1Æ8%
(w ⁄ v) reducing sugars, the maximum PHB accumulation by H. boliviensis was
doi:10.1111/j.1365-2672.2007.03553.x
attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid
(0Æ8% v ⁄ v), sodium acetate (0Æ8% w ⁄ v) and decreasing the reducing sugars
concentration to 1Æ0% w ⁄ v in the medium, PHB accumulation and concentra-
tion were increased to 50 wt% and 4 g l)1, respectively, after 20 h. Butyric acid
and sodium acetate for PHB production could also be provided by anaerobic
digestion of solid potato waste.
Conclusions: Cheap and readily available agricultural residues can be used as
substrates to produce PHB. The production of PHB by H. boliviensis using
wheat bran hydrolysate as source of carbon is expected to reduce the produc-
tion cost and motivates further studies.
Significance and Impact of the Study: Large-scale commercial utilization of
PHB is mainly hampered by its high production cost. Carbon source for PHB
production accounts up to 50% of the total production costs. Thus, the use of
waste agricultural residues can substantially reduce the substrate cost (and in
turn even provide value to the waste), and can downsize the production costs.
This improves the market competitiveness. Studies on PHB production by
moderate halophiles were recently initiated with H. boliviensis and findings
show that it has potential for commercial exploitation. PHB production by
H. boliviensis using wheat bran and potato waste is hence interesting.

(c. 1$ per kg) (Panda et al. 2006). However, it is well

Introduction
known that these materials are not biologically degrad-
Petrochemical-based plastics such as polyethylene and able, causing an increasing solid waste stream with
polypropylene are amongst the most used materials negative environmental effects. Biodegradability of plas-
because of their physicochemical properties and low cost tics has been proposed as a solution to overcome this

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420 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428
D. Van-Thuoc et al. Utilization of agricultural residues by Halomonas boliviensis LC1

problem. Interest in biodegradable plastics for packaging, production when utilized under optimum fermentation
medical, agricultural and fisheries applications has conditions.
increased in recent years (Palmisano and Pettigrew 1992;
Rehm 2006).
Materials and methods
Polyhydroxyalkanoates (PHAs), a group of biodegrad-
able polymers of biological origin, have attracted consid-
Microbial strains and maintenance
erable industrial interest. PHAs are accumulated
intracellularly as reserves of carbon and energy by a Halomonas boliviensis LC1T (=DSM 15516T) was main-
wide variety of bacteria (Anderson and Dawes 1990; tained at 4C on solid HM medium (Quillaguamán et al.
Bertrand et al. 1990; Sudesh et al. 2000), usually when 2004), containing (%, w ⁄ v): NaCl, 4Æ5; MgSO4Æ7H2O,
grown under limitation of a nutrient such as O, N, P, 0Æ025; CaCl2Æ2H2O, 0Æ009; KCl, 0Æ05; NaBr, 0Æ006; pep-
S, or trace elements like Mg, Ca, Fe and in the presence tone, 0Æ5; yeast extract, 1Æ0; glucose, 0Æ1; and granulated
of excess carbon (Lee 1996b; Sudesh et al. 2000; Kessler agar, 2Æ0. The pH of the medium was adjusted to 7Æ5
and Witholt 2001). Of the large family of PHAs, the using 5 mol l)1 NaOH solution.
homopolymer poly(3-hydroxybutyrate) (PHB) is the Aspergillus oryzae NM1 was maintained by monthly
most extensively studied. It possesses mechanical proper- subculture on solid medium containing (%, w ⁄ v): KNO3,
ties similar to the common petrochemical-based syn- 0Æ3; MgSO4Æ7H2O, 0Æ05; KH2PO4, 0Æ1; KCl, 0Æ05; NaCl,
thetic thermoplastics, and has been used to make 0Æ05; starch, 2Æ0 and agar, 2Æ0, pH 5Æ5.
various products, including films, coated paper, compost
bags, disposable food service-ware, and moulded prod-
Production and recovery of polysaccharide hydrolysing
ucts such as bottles and razors. After use, it can be
enzymes
degraded to carbon dioxide and water (or methane
under anaerobic conditions) by micro-organisms in the Aspergillus oryzae NM1 was grown in a medium using
environment (Mas-Castella et al. 1995; Lee 1996b; Du wheat bran (Ceralia, Järna, Sweden) as the main carbon
et al. 2001; Nonato et al. 2001; Tokiwa and Calabia source. Wheat bran composition is shown in Table 1. The
2004). strain was grown at 30C in 1 l flasks with shaking at 200
Nevertheless, the production cost of PHB (c. 4–6$ per rev min)1 in 200 ml liquid medium containing (%, w ⁄ v):
kg) is high compared with that of chemical synthetic KNO3, 0Æ3; KH2PO4, 0Æ1; MgSO4Æ7H2O, 0Æ05; KCl, 0Æ05;
nonbiodegradable plastics (Choi and Lee 1999). The effi- NaCl, 0Æ05; rice bran, 0Æ5 for 72 h. The fungus was sepa-
ciency and economics of the manufacturing process of rated by centrifugation at 8000 g for 20 min at 4C; then
PHB is determined by the price of the carbon source by the clarified supernatant was concentrated 10 times by
the fermentation process and the downstream processing ammonium sulfate precipitation, sterilized by filtering
of the polymer. About 40–50% of the total production through a 0Æ2 lm membrane, and stored at )20C until
cost is attributed to the carbon source – often refined
substrates, e.g. glucose and sucrose are supplemented in
the medium to produce the polymer (Bertrand et al. Table 1 Wheat bran composition per 100 g as described by the
1990; Lee 1996a; Choi and Lee 1999). Hence, the develop- supplier Cerealia (Järna, Sweden)
ment of fermentation strategies that allow high PHB con-
tent and productivity from cheap carbon sources is Component Amount

important. Fibre 50 g
Recently, PHB production by Halomonas boliviensis Carbohydrates 15 g
LC1, a moderately halophilic bacterium, has been free sugars 1Æ5 g
reported (Quillaguamán et al. 2005, 2006, 2007). This Protein 14 g
Fats 6g
bacterium is able to produce high PHB content (50–80
Sodium 0Æ003 g
wt%) from various carbon sources including volatile
Thiamine 0Æ66 mg
fatty acids (VFAs), mono- and disaccharides and starch Riboflavin 0Æ32 mg
hydrolysate. In the present work, we studied the produc- Niacin 26 mg
tion of PHB by H. boliviensis from wheat bran hydroly- Vitamin B6 1Æ4 mg
sate (obtained by treatment with hydrolytic enzymes Folacin 0Æ26 mg
from Aspergillus oryzae NM1) and potato residuals Potassium 1100 mg
Iron 11 mg
(obtained after anaerobic digestion). Such hydrolysates
Magnesium 480 mg
are inexpensive carbon and nitrogen sources (Choi and
Zinc 7Æ3 mg
Lee 1999), and may be readily available for polyester

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428 421
Utilization of agricultural residues by Halomonas boliviensis LC1 D. Van-Thuoc et al.

required. The concentrate was used as a source of poly- initially set to 1Æ0 l min)1 and 700 rev min)1 for all fer-
saccharide hydrolysing enzymes. mentations, and were increased up to 4Æ0 l min)1 and
900 rev min)1 after 2 h when a decrease from the initial
dissolved oxygen concentration was detected. The effect
Wheat bran hydrolysis by Aspergillus oryzae NM1
of various nutrients such as yeast extract, wheat bran
enzyme concentrate
hydrolysate and organic acids was studied under these
Different amounts of the enzyme concentrate, as obtained conditions.
above, were mixed with 30 g of wheat bran and 150 ml
of a phosphate buffer solution at pH 6 in 1 l flasks. The
Poly(3-hydroxybutyrate) isolation for nuclear magnetic
mixture was incubated at 50C and the stirrer speed was
resonance-spectroscopic analysis
set at 160 rev min)1. The mixture was centrifuged and fil-
tered through a Munktell analytical filter paper (Gry- PHB chemical structure was determined as reported pre-
cksbo, Sweden), and the filtrate containing the reducing viously (Quillaguamán et al. 2006). Halomonas boliviensis
sugars was used as carbon source for polyester production cells containing the polymer, were harvested from 500 ml
by H. boliviensis. of culture broth by centrifugation at 10 000 g for 10 min,
washed once with water and finally resuspended in water
and lyophilized. PHB was recovered from lyophilized cells
Hydrolysis of potato waste
by extraction for 30 h with chloroform in a Soxhlet appa-
The hydrolysis was achieved in cylindro-conical anaerobic ratus (Duran, Germany), and concentrated by evaporating
reactors with 2 dm3 working capacity as previously the solvent under vacuum. The polymer was precipitated
described (Parawira et al. 2004). Subsequently, the hydro- from the concentrated solution with 10 volumes of etha-
lysate was adjusted to pH 9, heated for 5 min in a boiling nol and the resulting PHB granulates were filtered twice.
water bath and filtered through a Munktell analytical The 1H nuclear magnetic resonance (NMR) spectrum was
filter paper to eliminate suspended solids. recorded at 500 MHz with a Bruker ARX500 Spectro-
meter (Bruker, Sikerstrifen, Germany) at room temperature
using deuterated chloroform as internal reference solvent.
Poly(3-hydroxybutyrate) production in flasks
The spectrum was evaluated using standard Bruker uxnmr
Seed culture of H. boliviensis was grown in a modified software.
HM medium, HM-I (Quillaguamán et al. 2005) contain-
ing (% w ⁄ v): glucose, 1Æ0; yeast extract, 0Æ2; NaCl, 4Æ5;
Quantitative analysis
MgSO4Æ7H2O, 0Æ038; CaCl2Æ2H2O, 0Æ013; KCl, 0Æ075;
NaBr, 0Æ02. Halomonas boliviensis was grown in 30 ml of Samples were withdrawn at defined time intervals during
HM-I medium in 100 ml flasks with shaking at the cultivation of H. boliviensis and were analysed for cell
200 rev min)1 at 30C until the culture broth reached an dry weight (CDW), PHB content and residual cell mass
optical density (OD600) of 0Æ50–0Æ55. Subsequently, 2 ml (RCM).
of this culture was inoculated in 1 l flasks containing CDW was determined by centrifuging 3 ml of the culture
100 ml HM-II medium (having the same composition as samples at 2000 g for 15 min in a preweighed centrifuge
HM-I medium but without glucose) with different carbon tube, the pellet washed twice with 3 ml distilled water, and
sources. The cultures were incubated at 35C with shak- dried at 75C until constant weight was obtained.
ing at 200 rev min)1. PHB quantification was performed according to the
method of Law and Slepecky (1961). The dried pellets
containing intracellular PHB were hydrolysed using 10 ml
Poly(3-hydroxybutyrate) production in a fermentor
of concentrated sulfuric acid for 1 h to obtain crotonic
Halomonas boliviensis was first grown at 30C in 150 ml acid, and the mixture was then cooled to room tempera-
HM-I medium in 1 l flasks, with shaking at ture and quantified by measuring absorbance at 235 nm.
200 rev min)1, for about 13 h until the cell density of Analysis was performed in triplicates for all samples.
1 g l)1 (OD600 about 0Æ48–0Æ50) was reached. This culture RCM was defined as the CDW minus PHB concentra-
medium was used to inoculate a 2-l fermentor vessel tion, while PHB content (wt%) was obtained as the
(Voyager, Luton, UK) containing 1Æ35 l of cultivation percentage of the ratio of PHB concentration to CDW
medium. Temperature was kept at 35C during the pro- (Lee et al. 2000).
cess, antifoam was added when needed and the pH of the Wheat bran hydrolysate composition was analysed by
medium was maintained at 7Æ7 by using 5 mol l)1 HPLC using an Aminex HPX-87P column (Bio-Rad,
NaOH ⁄ HCl. The air inflow rate and agitation speed were Hercules, CA, USA) and refractive index detector. The

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422 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428
D. Van-Thuoc et al. Utilization of agricultural residues by Halomonas boliviensis LC1

chromatography was performed at 85C using deionized In all subsequent experiments, the wheat bran was trea-
water as mobile phase at a flow rate of 0Æ6 ml min)1. ted with the enzyme solution containing 5Æ1 U ml)1 amy-
Reducing sugars concentration was determined by the lase and 6Æ8 U ml)1 xylanase for 5 h, which was enough
dinitrosalicylic acid (DNS) method (Miller 1959). to yield the maximal amount of reducing sugar. The
Total protein concentration in the hydrolysate was wheat bran hydrolysate had a final concentration of
quantified using the BCA (Bicinchoninic Acid)-method 32 g l)1 reducing sugars, which was mainly composed of
(Stoscheck 1990). 52Æ8% glucose, 17Æ2% xylose, 17Æ4% mannose and 12Æ6%
VFAs in the potato hydrolysate were determined using arabinose. The total protein concentration in the hydroly-
HPLC with a Bio-Rad column 125-0115. The column sate was 15Æ6 g l)1.
temperature was 65C. Sulfuric acid (1 mmol l)1) was
used as mobile phase and the liquid flow rate was
Effect of carbon source on PHB accumulation in shake
0Æ8 ml min)1. The fatty acids were monitored by UV
flasks
absorption at 208 nm (Parawira et al. 2004).
Wheat bran hydrolysate, composed of a complex mixture
of hexoses and pentoses, was used as C-source for PHB
Enzyme assay
production. PHB chemical structure was determined by
Enzyme activities were determined from the amount of NMR-spectroscopic analysis (Fig. 1). Substrate uptake
reducing sugar formed using the DNS method (Miller and metabolic assimilation of both kinds of sugars are
1959). different, and hence should have different effects on cell
concentration and PHB accumulation by H. boliviensis.
Amylase activity PHB production was first studied using mixtures of glu-
The crude enzyme preparation (50 ll) was incubated with cose and xylose at varying ratios as carbon source, and
450 ll of 3 g l)1 starch solution in 50 mmol l)1 phos- was compared with the production obtained with wheat
phate buffer (pH 6Æ0) at 50C for 5 min. bran hydrolysate (Table 2). At two different concentra-
tions, glucose showed to be a better substrate for PHB
Xylanase activity accumulation with respect to xylose when supplemented
The crude enzyme preparation (50 ll) was incubated with in the medium. Increasing glucose : xylose ratio was
450 ll of 10 g l)1 xylane solution in 50 mmol l)1 phos-
phate buffer (pH 6Æ0) at 50C for 5 min.
The reactions were stopped by the addition of DNS
reagent, after which the samples were placed in a boiling
water bath for 10 min. The samples were cooled in the
water for 10 min and then absorbance read at 540 nm.
Glucose was used as the calibration standard for amylase
activity and xylose was used as the calibration standard
for xylanase activity. One unit of enzyme was defined as
the amount of enzyme releasing 1 lmol reducing sugar
per minute under the standard assay conditions.

Results

Effect of enzyme concentration on total reducing sugar

Aspergillus oryzae produced 15 U ml)1 xylanase and
11 U ml)1 amylase activities under the culture conditions
provided and using wheat bran as the main carbon
source. The enzymes were concentrated by ammonium
sulfate precipitation to reach activity levels of 110 U ml)1
xylanase and 80 U ml)1 amylase activities. Wheat bran
hydrolysis was studied using enzymes concentration up to Figure 1 1H-nuclear magnetic resonance spectrum showing the car-
7Æ4 U ml)1 amylase and 9Æ8 U ml)1 xylanase at 50C. In bon composition of the monomers belonging to the polymer
all cases, the reducing sugars concentration reached con- extracted and purified from Halomonas boliviensis cells. Deuterated
stant levels after 5 h of treatment (data not shown). chloroform was used as internal reference solvent.

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428 423
Utilization of agricultural residues by Halomonas boliviensis LC1 D. Van-Thuoc et al.

Table 2 Influence of carbon source on

Carbon source poly(3-hydroxybutyrate) (PHB) accumulation
Residual
Glucose Xylose % PHB content PHB conc. cell mass and cell mass concentration by Halomonas
% (w ⁄ v) (w ⁄ v) CDW (g l)1) (wt%) (g l)1) (g l)1) boliviensis

1 – 2Æ1 (±0Æ04) 39Æ3 (±1Æ11) 0Æ83 1Æ29

0Æ5 – 1Æ7 (±0Æ13) 32Æ4 (±1Æ07) 0Æ60 1Æ24
0Æ75 0Æ25 2Æ2 (±0Æ07) 44Æ9 (±0Æ41) 1Æ00 1Æ23
0Æ5 0Æ5 2Æ2 (±0Æ02) 43Æ3 (±0Æ83) 0Æ96 1Æ26
0Æ25 0Æ75 2Æ0 (±0Æ09) 29Æ3 (±0Æ52) 0Æ6 1Æ43
– 0Æ5 1Æ4 (±0Æ02) 13Æ9 (±0Æ50) 0Æ19 1Æ17
– 1 1Æ5 (±0Æ10) 23Æ1 (±1Æ83) 0Æ35 1Æ17
Wheat bran hydrolysate 3Æ19 (±0Æ09) 33Æ8 (±2Æ17) 1Æ08 2Æ11
(1% w ⁄ v reducing sug-
ars)

Experiments were performed in 1 l flasks containing 200 ml of HM-I medium at 35C and 200
rev min)1 for 30 h. Between 5 and 7 mg of dried cells were used to determine the PHB con-
tent. The numbers in brackets represent the SD of the average values.

found to be favourable for PHB accumulation (Table 2). to partial anaerobic digestion. The hydrolysate contained
Highest PHB concentration of 1Æ0 g l)1 was obtained mainly acetic and butyric acids in a solution. Halomonas
when cells were grown with 0Æ75% (w ⁄ v) glucose and boliviensis was grown in HM-II medium with 1% (w ⁄ v)
0Æ25% (w ⁄ v) xylose in the HM-II medium (Table 2). A wheat bran hydrolysate and 1% (w ⁄ v) digested potato
similar PHB concentration (1Æ08 g l)1) was reached when extract as C-source. The digested potato extract added to
wheat bran hydrolysate was supplied as C-source; though the medium led to a final concentration of 0Æ56% (w ⁄ v)
PHB content was lower and the RCM generated was acetic acid and 0Æ4% (w ⁄ v) butyric acid. Under these con-
higher (Table 2). ditions, cell mass concentration obtained was 6Æ6 g l)1
and maximum PHB content was 43 wt% after 20 h of
growth (Fig. 3).
Effect of N- and C-sources on PHB production by
Halomonas boliviensis in a fermentor
Discussion
We have previously determined that yeast extract can
enhance both cell mass and PHB content even at concen- The carbon source contributes most significantly to the
trations as high as 1Æ5% (w ⁄ v) when sucrose is used as substrate costs in PHA production by bacteria (Bertrand
sole carbon source and under controlled conditions in a et al. 1990; Lee 1996a; Choi and Lee 1999). The use of
fermentor (Quillaguamán et al. 2007). For this work, the cheap and readily available agricultural residues is
influence of yeast extract concentration on PHB produc- expected to substantially reduce the cost of the carbon
tion was also studied. HM-II medium was supplemented source that can be used in fermentation (Choi and Lee
with the wheat bran hydrolysate to reach a reducing sugar 1999). The use of agricultural residues as fermentation
concentration of 1Æ8% (w ⁄ v) (found to be optimum in substrates involves a hydrolysis step that releases easily
shake flask experiments). As depicted in Fig. 2, increase metabolizable sugars. Nevertheless, the hydrolysis step
in yeast extract concentration from 0Æ8 to 1Æ5% (w ⁄ v) led should be straightforward and inexpensive to not affect
to an increase in cell dry weight but the PHB content negatively the overall economics. Moreover, the hydrolysis
decreased from 30 to 18 wt%. process should not result in compounds that inhibit the
While maintaining the yeast extract concentration at fermentation process. In this regard, the biocompatibility
0Æ5% (w ⁄ v) in HM-II medium, the effect of mixing wheat of enzymatically hydrolysed agricultural residues to fer-
bran hydrolysate with other carbon sources on PHB pro- mentation process makes it interesting. However, in
duction was studied. PHB accumulation was improved, large-scale applications the enzyme cost can be a bottle-
reaching a maximum value of 50 wt% and a PHB con- neck. Alternative ways of reducing the enzyme cost
centration of 4 g l)1 using 1% (w ⁄ v) hydrolysate, 0Æ8% includes the use of cheap production substrate and using
(w ⁄ v) sodium acetate and 0Æ8% (w ⁄ v) butyric acid in the crude enzyme preparation for the target application. In
medium (Fig. 3). Furthermore, the cell mass concentra- this study, the hydrolytic enzymes were produced cheaply
tion was 8Æ0 g l)1 after 20 h (Fig. 3). In order to obtain using wheat bran as carbon source and used as a crude
VFAs from a cheap source, potato residue was subjected concentrate.

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424 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428
D. Van-Thuoc et al. Utilization of agricultural residues by Halomonas boliviensis LC1

Figure 2 Influence of yeast extract concentration on poly(3-hydrox- Figure 3 Influence of the carbon sources on cell dry weight (a) and
ybutyrate) (PHB) production: (a) CDW and (b) PHB accumulation by poly(3-hydroxybutyrate) accumulation (b) by Halomonas boliviensis.
Halomonas boliviensis. HM-II medium containing 1Æ8% (w ⁄ v) reducing Symbols used refer to different combinations of C-sources: (r), 1%
sugars of wheat bran hydrolysate was supplemented with different (w ⁄ v) wheat bran hydrolysate + 0Æ5% (w ⁄ v) sodium acetate + 0Æ5%
concentrations of yeast extract % (w ⁄ v): , 0Æ8; , 1Æ2; r, 1Æ5. All (w ⁄ v) butyric acid; ( ), 1% (w ⁄ v) wheat bran hydrolysate + 0Æ8%
experiments were performed in a fermentor maintaining constant pH (w ⁄ v) sodium acetate + 0Æ8% (w ⁄ v) butyric acid; ( ), 1% (w ⁄ v)
(7Æ5), temperature (35C) and avoiding oxygen limitation by the cells. wheat bran hydrolysate + 1% (w ⁄ v) waste potato hydrolysate. In all
The error bars refer to the SD of the average values. cases, 0Æ5% (w ⁄ v) yeast extract was added to H-II medium. The error
bars refer to the SD of the average values.

The hydrolysis of wheat bran by A. oryzae NM1

resulted in a reducing sugar mixture suitable for PHB (Shamanna and Sanderson 1979). As shown in Table 2,
production by H. boliviensis. Reducing sugars compo- the mixture of xylose and glucose provided the best
sition in the hydrolysate was composed mainly of hexo- conditions for cell growth and PHB accumulation by
ses (glucose and mannose) and pentoses (xylose and H. boliviensis in shake flasks.
arabinose). Glucose was the preferred substrate for PHB In the fermentor, maximum PHB content in H. bolivi-
synthesis (Table 2). Pentose catabolism affects the ensis cells was attained in 20 h, a reduction of 10 h in the
growth kinetics of various Gram-negative bacteria production process with respect to shake flasks, although
(Wood 1966; Shamanna and Sanderson 1979), e.g. with low intracellular amounts of PHB (up to 30 wt%)
Escherichia coli and Salmonella typhimorium generation (Fig. 2). Previous studies, using sucrose as C-source,
times increase twofold when glucose is replaced by showed that a high yeast extract concentration (up to
xylose under otherwise similar culture conditions 1Æ5% w ⁄ v) in the medium provides favourable conditions

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428 425
Utilization of agricultural residues by Halomonas boliviensis LC1 D. Van-Thuoc et al.

Figure 4 Pathways for the biosynthesis of poly(3-hydroxybutyrate) (PHB) from different carbon sources: (I) Glucose Embden–Meyerhof pathway;
(II) Xylose via pentose-P pathway; (III) Butyric acid via acetoacetyl CoA and (IV) acetic acid. GAP refers to glyceraldehide-3-phosphate. Pathway for
the biosynthesis of PHB from acetyl CoA involves three enzymes: (i) 3-keto-thiolase, (ii) acetoacetyl-CoA reductase and (iii) PHB synthase. The
scheme was adapted from previous reports (Braunegg et al. 1998; Steinbüchel and Füchtenbush 1998).

for both growth and PHB production by H. boliviensis NAD(P)H that inhibits citrate synthase within the TCA,
but excess of a complex N-source hinders PHB synthesis and allows PHB synthesis. Furthermore, this process
(Quillaguamán et al. 2007). PHB accumulation by this results in a decrease in concentration of acetyl-CoA,
organism using wheat bran hydrolysate was improved by hence increasing the concentration of free CoASH. High
reducing the amount of yeast extract in the medium concentration of CoASH is known to inhibit the 3-keto-
(Fig. 2), implying that there was an excess of N-source in thiolase condensation reaction in the PHB synthesis
HM-II medium. This is most likely related to the high (Jackson and Dawes 1976; Anderson and Dawes 1990;
protein concentration found in the hydrolysate, which Koyama and Doi 1993; Du et al. 2001; Kessler and
provided, besides carbon, also nitrogen for the cells. Witholt 2001). In general, PHB synthesis depends upon
The accelerated cell growth observed in H. boliviensis intracellular NADPH concentration and ⁄ or NAD(P)H ⁄
(Fig. 2) is to be accompanied by a high concentration of NADP ratio (Dawes and Senior 1973; Shi et al. 1997).

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426 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 420–428
D. Van-Thuoc et al. Utilization of agricultural residues by Halomonas boliviensis LC1

Different substrates lead to characteristic amounts of the rhodesianum as an expression for an internal bottleneck.
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This study was supported by the Swedish International Lee, S.Y., Wong, H.H., Choi, J., Lee, S.H., Lee, S.C. and Han,
Development Cooperation Agency (SIDA). C.S. (2000) Production of medium-chain-length polyhy-
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