Bioresource Technology 121 (2012) 83–92

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bioproduction, statistical optimization and characterization of microbial

plastic (poly 3-hydroxy butyrate) employing various hydrolysates of water
hyacinth (Eichhornia crassipes) as sole carbon source
D. Radhika, A.G. Murugesan ⇑
Manonmaniam Sundaranar University, Sri Paramakalyani Centre of Excellence in Environmental Sciences, Alwarkurichi 627412, Tamil Nadu, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

" The lysates of water hyacinth was

found to be a potential feedstock for
biopolymer production.
" Biopolymer (PHB) is widely
acceptable bioplastic.
" Usage of water hyacinth for PHB
production which will mitigate the
environmental pollution.

a r t i c l e i n f o a b s t r a c t

Article history: Saccharified water hyacinth hydrolysates (acid and enzyme hydrolysate) were used for the efficient pro-
Received 25 April 2012 duction of poly (3-hydroxybutyrate) (PHB) via the Cupriavidus necator bacteria. The bacterium signifi-
Received in revised form 28 June 2012 cantly utilizes the enzymatic hydrolyzate which gave the maximum PHB concentration
Accepted 29 June 2012
(4.3 ± 0.4 g L1), this was greatly exceeded the value of 2 ± 0.1 g L1 obtained from the acid hydrolysate
Available online 7 July 2012
amended media. Moreover, for the optimal PHB production, response surface methodology was used
through central composite rotary design method which gave improved PHB concentration in microbial
Keywords:
cells. After 72 h, 35 g L1 of reducing sugar contained water hyacinth hydrolysate and 1.5 g L1 of
Bioconversion
Water hyacinth
(NH4)2SO4 supplementation in laboratory scale fermentor gave 12 g L1 of dry cell weight and 7 g L1
Glucose of PHB. The produced PHB was characterized under FTIR, GPC and DSC instruments to find out the num-
Fermentation ber average molecular mass, polydispersity index and melting temperature were 1.7  105 kDa, 1.9 and
Hydrolysates 170 °C respectively.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction bioproducts. In addition, one or more fermentable sugars are made

to produce recalcitrant cellulose and the hemicellulosic nature of
Water hyacinth is widely occupied aquatic weed; unable to the water hyacinth plant. Extensive studies have been carried out
control their population growth from the water bodies, which for the production of various bioproducts using water hyacinth
cause severe water pollution. Even though, the easily reproducible hydrolysates such as levulonic acid (Girisuta et al., 2008), ethanol
aptitude and lignocellulose nature of this plant can serve as a (Aswathy et al., 2010), biogases like hydrogen (Ofoefule et al.,
low-cost carbon source for the production of numerous ecofriendly 2009) and methane (Cheng et al., 2010). The issues concerning that
the world-wide energy crisis is also the basic motive for the
invention of bioproducts from the renewable lignocellulosic bio-
⇑ Corresponding author. Tel.: +91 4634 283883; fax: +914634-283270
masses. Cellulosic biomasses such as agricultural residues are the
E-mail address: [email protected] (A.G. Murugesan).

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.06.107
84 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92

potentially inexpensive renewable feedstocks for the biorefineries sterile plastic box until further analysis in room temperature. The
of alternative fuels (Sathesh Prabu and Murugesan, 2011; Bala chemical compositions of the raw material have been analyzed
Amutha and Murugesan, 2011), chemicals and materials have to using standard AOAC manual methods. Water hyacinth powder
be drastically reducing the production cost. One of the bioproduct (5%) was pretreated with 1% wv1 NaOH incubated at room tem-
poly (b-hydroxybutyrate (PHB), a widely acceptable microbial perature for 24 h. Afterward, the insoluble residue was collected
polymer (bioplastic) produced intracellularly by wide variety of by filtration using muslin cloth subsequently washed thoroughly
microbes P ( seudomonas, Azohydromonas, Azotobacter, Bacillus, Cupri- with hot water further, which was neutralized with 0.1 M HCl.
avidus necator, etc.) which provides energy for their growth and After neutralization, the residues were oven dried at 50 °C for over-
development during nutrient starved condition. Moreover, PHB is night then it powdered once more and stored for hydrolysis.
a biodegradable and biocompatible thermoplastic compound, has
similar physical properties to polypropylene. It is used to a wide 2.3. Acid and enzymatic hydrolysis
range of medicinal, veterinary and agricultural practices due to
its biodegradability. The PHB derived from the lignocellulosic Acid hydrolysis was performed to the yield of reducing sugars. The
materials offers environmental advantages over petroleum-de- concentrated acid hydrolysis was performed with 72% of 10 ml (per
rived polymers in terms of non-renewable energy consumption gram pretreated substrate) H2SO4; they were kept at room tempera-
and greenhouse-gas emissions (Kim and Dale, 2008). Nevertheless, ture for 30 min. Insoluble residues were removed through filtration.
their production cost is found to be high compared to the other The filtrates obtained from the hydrolysis was overlimed with 10 M
commercially available thermopolyesters. Over 40% of total oper- NaOH and neutralized with 1 M H3PO4. For the detoxification of acid
ating expense of PHB production is relates to the raw materials, hydrolysate 2% activated charcoal was used. Inhibitor concentration
and more than 70% of this cost is attributed to the carbon source was determined by the total phenol estimation procedure (Folin
(Salehizadeh and Van Loosdrecht, 2004). The thermopolyester res- Ciocalteu assay). For the enzyme hydrolysis experiment pretreated
ins are made from the petroleum wastes. According to Global substrate was further subjected to the action of cellulase enzyme (ex-
Industry Analysts Inc. the global consumption of plastic materials tracted from Aspergillus niger SRL, Mumbai) in the ratio of 20 FPU: 1 g
is increased to 297.5 million tonnes by 2015. Using water hyacinth (Filter Paper Units enzyme: g substrate) with citrate buffer (0.01 M,
like lignocellulosic materials for the synthesis of bioplastic materi- pH 4.8) and incubated at 50 °C for saccharification for 24 h (Mishima
als may help to reduce overall production cost. Recently many re- et al., 2008). Amount of saccharification was estimated in terms of re-
search activities were published about the microbial production of lease of reducing sugars by dinitrosalicylic acid (DNS) method. Unit of
PHAs (polyhydroxyalkanoates) using cheap substrates. In the pres- cellulase was expressed as the amount of enzymes producing 1 lmol
ent study, various hydrolysates were prepared from the water hya- of reducing sugars per minute. All experiments were at least dupli-
cinth through the hydrolysis process, used for PHB production cated, and the mean values were shown as a result. The clear solution
through batch culture experiment via C. necator bacteria. The C. containing sugars were collected by centrifugation (6000g, 10 min)
necator is a gram-negative, facultative chemolithoautotrophic and and used for fermentation.
hydrogen-oxidizing bacterium belonging to the order betaproteo-
bacterium. Unbalanced growth conditions, whenever an available
2.4. Optimization of PHB production and extraction
carbon source is in excess level, which can stores large amounts
of organic carbon in the cytoplasm as PHB while another macroel-
Organic nitrogen [yeast extracts (1.5 g L1) peptone (1.5 g L1),
ement like oxygen is limited (Peplinski et al., 2010). This bacterium
beef extracts (1.5 g L1) and tryptone (1.5 g L1)] and inorganic
is far the most studied microbe when it comes to PHB research. The
nitrogen [(NH4)2 SO4 (1.5 g L1), NH4 Cl (1.5 g L1) and (NH4 H2
reason for that it can produce PHB through the utilization of simple
PO4 (1.5 g L1) sources were optimized. For efficient production
monomeric sugars as its carbon source.
of PHB physical conditions (pH and temperature) and nutrient
compositions were optimized using response surface methodol-
2. Methods ogy. The central composite design was carried out using statistical
software package Design Expert 8.0.7.1 (Stat – Ease Inc., Minneap-
C. necator MTCC-1472 was obtained from the Microbial type olis, USA). The levels of independent factors carbon source (Water-
culture collection, Chandigarh; India used for throughout this hyacinth hydrolysate), nitrogen source ((NH4)2SO4), pH and
study and activated in nutrient agar media by incubating at 27 °C temperature were used by studying each factor in the design at
for 24 h. Glycerol stock was prepared and stored at 80 °C. three different levels (1, 0 and +1) (Table 1). The actual level of
variables for central composite rotary design was selected as a cen-
2.1. Growth condition and media tral point of experiments. For the regression model development,
the variables were transformed to coded variables using the fol-
Culture inoculum was prepared by a loopful of culture inocu- lowing Eq. (1)
lated into 250 ml flask containing 100 ml of a sterile culture media
X i  X i
in which the nutrient concentrations were: peptone – 10 g L1, Xi ¼ ; i ¼ 1; 2; 3::: ð1Þ
DX i
yeast extracts – 5 g L1, NaCl – 10 g L1, glucose – 10 g L1 at
27 °C for 96 h incubation with the agitation speed of 100 rpm. where Xi = coded value, Xi = uncoded value, xi = uncoded value of Xi
The PHB production medium and their compositions were fol- at the centre value of chosen and DXi = step size, for ith test
lowed by the prior literature (Beaulieu et al., 1995).
Table 1
Illustration of coding levels involved in the optimizing the medium components.
2.2. Raw material pretreatment
Independed variables Symbols Coded levels

In this study, raw material of water hyacinth was collected from a 1 0 +1 +a

the Tamirabarani River, Tamil Nadu. Then it was washed with Carbon source (g L1) A 25 30 35 40 45
water by several times intended for the removal of dirts and it Nitrogen source (g L1) B 0.5 1 1.5 2 2.5
pH C 6 6.5 7 7.5 8
dried in the hot air oven at 70 °C for 48 h followed by pulverized
Temperature (°C) D 22.5 25 27.5 30 32.5
into fine powder (5 mm mesh screen) after that packed in the
 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92 85

variable. The optimum value of each factor was taken on a central was defined as the percentage of PHB weight to dry cell weight (Lee
coded value considered as zero. The minimum [coded as (1)] et al., 1994). The biomass and PHB yield coefficients on glucose
and maximum [coded as (+1)] range of the experimental values of were calculated by the DCW produced per unit mass of reducing su-
each factor used and the full experimental plan for central compos- gar consumed.
ite design performed with 31 experiments were listed in Table 2. In
those 31 experiments the central point consists of 4 trials. 3D re- 2.5. Analysis of PHB
sponse graphs were created to understand the interaction of various
factors. The resulting graphs were used to analyze the optimized 2.5.1. FTIR analysis
components of the medium. After performing the experiments, The produced PHB was identified by PHB granules using Sudan
the data were used to develop three-dimensional curves and qua- Black–B staining method. For this Sudan Black-B solution (0.3% in
dratic equation in the form of Eq. (2) 70% ethanol), xylene (decolourizer) and 0.5% of safranine solutions
were used. Produced PHB was confirmed using FTIR spectroscopy
Y ¼ b0 þ b1 A þ b2 B þ b3 C þ b4 D þ b1 b1 A2 þ b2 b2 B2 þ b3 b3 C 2
for that 1 mg of sample and 10 mg of spectral pure anhydrous
þ b4 b4 D2 þ b1 b2 AB þ b3 AC þ b2 b3 BC þ b1 b4 AD þ b2 b4 BD potassium bromide crystals were ground well and was made into
þ b3 b4 CD ð2Þ a pellet. The relative intensity of transmitting light energy was
measured against the wavelength of absorption 4000–400 cm1
where Y is the predicted value, A, B, C, D are the coded independent using FTIR-4100 LE, JASCO, Japan. Growth was monitored spectro-
variables, b1 b1, b2 b2, b3 b3 and b4 b4 are the quadratic coefficients, photometrically by measuring turbidity at 600 nm.
b1 b2, b1 b3, b2 b3, b1 b4, b2 b4 and b3 b4 are the interactive coeffi-
cients, b1, b2, b3 and b4 were linear coefficients, b0 is the constant. 2.5.2. DSC analysis
All assays were conducted in 500-ml Erlenmeyer flasks with Thermal transition of produced PHB was characterized by dif-
250 ml of culture medium on a rotary shaker with the agitation ferential scanning calorimetry (TA Instruments-Q20, USA) with
speed of 120 rpm for 96 h incubation. Experiments were initiated an autocooling accessory calibrated by indium over a temperature
by transferring 5% (vv1) seed culture medium; this was centrifuged range from 30 to 200 °C at a heating rate of 10 °C min1 under
at 2000 rpm for 5 min and the cell pellet was inoculated into the nitrogen atmosphere (50 ml min1). The PHB film was formed by
prepared medium and they were carried out for three days in labo- the powder was dissolved into chloroform and dried at room tem-
ratory conditions. Ten milliters of cultured broth were collected at perature. Sample of PHB film weighed approximately 2 mg were
predefined intervals of time from the flask for the product and bio- packed in an aluminum pan and then heated from 30 to 170 °C
mass analysis. The produced PHB from the bacterial cell pellet was at a scanning rate of 10 °C min1 The DSC curve was used to ana-
recovered by using a dispersion technique. The supernatant and cell lyze the thermal property. The melting temperature (Tm) was
pellets were used for the analysis of residual sugar and PHB extrac- determined from the DSC endothermal peak. In the presence of
tion respectively. The dry cell weight (DCW) was determined by multiple endothermal peaks, the maximum peak temperature
gravimetrically. The washed cell pellet was dried to constant weight was taken as Tm.
at 90 °C for 24 h, cooled in a desiccator, and weighed. PHB content

2.5.3. GPC analysis

Table 2 Molecular mass and poly dispersity index of produced PHB
Experimental design and results of central composite design for response surface were determined by the gel permeation chromatography (GPC)
methodology.
system equipped with Shimadzu LC-6A with RI detector, and
Run No. A B C D DCW (g L1) PHB (g L1) CLC-Polystyrene columns in series. Chloroform was chosen as the
1 1 1 1 1 4.2 3.5 eluent at a flow rate of 1.0 ml min1 Sample concentrations of
2 1 1 1 1 7 4.4 0.5% (w/v) and injection volumes of 100 ml were used. Polystyrene
3 0 2 0 0 9.5 2.6 standards with a low polydispersity were applied to generate a cal-
4 2 0 0 0 6.7 1.8 ibration curve.
5 1 1 1 1 7.1 2.2
6 1 1 1 1 7 2.3
7 2 0 0 0 5.5 4.3 3. Result and discussion
8 1 1 1 1 6.9 3.2
9 0 0 0 0 8 4.6
10 0 0 0 0 8 4.6 3.1. Effect of acid and enzymatic hydrolysate on PHB production
11 1 1 1 1 4 4.4
12 0 0 0 0 8 4.8 Initial screening assays were performed with two hydrolysates
13 0 0 0 0 8 4.8
(Acid hydrolysate and enzymatic hydrolysate) prepared from the
14 0 0 0 2 4.8 3.8
15 0 2 0 0 4.7 3.8
waterhyacinth which comprise a mixture of reducing sugars in-
16 1 1 1 1 5.1 3 cludes pentoses and hexoses. The water hyacinth hydrolysate con-
17 1 1 1 1 6.5 3.6 sisted of xylose, 34 ± 1 g L1; glucose, 20 ± 0.8 g L1; furfural,
18 0 0 2 0 5.5 3.1 0.24 ± 0.1 g L1; hydroxy methyl furfural, 0.145 ± 0.08 g L1; acetic
19 1 1 1 1 4.5 3.3
acid, 0.23 ± 0.2 g L1; total phenol, 2.34 ± 0.2 g L1. Fig. 1 clearly
20 1 1 1 1 4.5 4
21 1 1 1 1 5.5 3 showed that the bacterial culture gave the best results with the
22 0 0 0 2 4.8 3.6 waterhyacinth enzymatic hydrolysate supplementation about the
23 0 0 2 0 5.2 3.7 biopolymer PHB production under nitrogen and phosphorous lim-
24 1 1 1 1 7.2 2.5 ited environment. During this hydrolysate supplementation
25 1 1 1 1 4.3 4.2
26 0 0 0 0 8 4.6
6.2 ± 0.2 g L1 of biomass and 3.7 ± 0.3 g L1 of PHB were obtained
27 0 0 0 0 8 4.6 at after 72 h of incubation time (Fig. 1). Similar results were
28 1 1 1 1 5.4 3.4 observed by Van-Thuoc et al., 2008 who were found that, the
29 1 1 1 1 7.4 2 wheat bran hydrolysate supplemented with mineral media had a
30 0 0 0 0 8 4.5
significant effect on bacterial growth and PHB production. In acid
31 1 1 1 1 7 4
hydrolysate C. necator did not produce considerable amount of
86 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92

Fig. 1. PHB production observed in C. necator during various water hyacinth hydrolsates supplimentation.

PHB as well as biomass; this may due to the inhibitor concentra- production through central composite design methods. The levels
tions in the hydrolysate which were released during acid catalyt- of these factors used in the optimization studies by RSM were
ical hydrolysis. It was produce only 2 ± 0.1 g L1 PHB determined by preliminary experiments. The effect of the four vari-
intracellularly. Fractions of volatile fatty acids were found in the ables, each at five levels, and their interactions in PHB production
hydrolysates could reduce the proton gradient across the mem- have been determined by carrying out 31 experiments given by the
brane, increase osmotic pressure, and reduce acid utilization rates, model. Analysis of variance (ANOVA) for DCW and PHB production
growth rate, and microbial yield (Wang et al., 2010). After that in is presented in Tables 4 and 5. The analysis gave the value of the
this study, for the control of inhibitor concentrations 2% activated model and determines the requirement of a more complex model
charcoal was used as detoxifying agent. It was found to reduce con- with a better fit. The results found that the R2 values were
siderable amount of phenolic compounds (78%) and little amount 0.9996 and 0.9943 indicating that the model was fitted which ex-
reducing sugar (4.2 g L1). After detoxification with charcoal, the plained 99.96% and 99.43% of the variabilities in DCW and PHB
acid hydrolysate was supplemented in the minimal media for the production. F-test for regressions were significant at a level of 5%
production of PHB; as a result the bacterium produce significant (P < 0.05) indicating that the model is fit and can adequately ex-
quantities of PHB (3.2 ± 0.1 g L1) with the cell concentration of plain the variations observed in DCW and PHB productions with
(5.7 ± 0.2 g L1) (Fig. 1). However in this study, all water hyacinth the designed levels of the factors. If the F-test for lack of fit is sig-
hydrolysates used as a sole substrate has the potential to the pro- nificant, then a more complicated model is needed to fit the data.
duction of PHB at optimum environmental conditions (pH value 7 The lack of fit values was not significant at the 5% level (P > 0.05)
and temperature 27 °C). Obtained PHB concentrations from the indicating that the experimental data obtained fitted well with
water hyacinth hydrolysates coincided with the work described the model. The model F-values of 3095.9 and 197.8 implies the
by Koller et al. (2005) who were found that the effect of green grass model is significant. There is only a 0.01% chance that the model
juice and silage juice as carbon substrate for the production of PHB F-values this large could occur due to noise. Thirty-one experi-
using Woutertia eutropha bacteria. The water hyacinth hydroly- ments were carried out from the design and by applying multiple
sates not only provide a carbon source also it can give nitrogen regression analysis on the experimental data; the following second
and phosphate sources at trace amounts for the controlled PHB order polynomial equations were found to explain DCW and PHB
production (Table 3). According to Silva et al., 2007, acid catalyzed production by C. necator bacteria.
saw dust can provide excess carbon source as well as fractions of
nitrogen and phosphate sources for bacteria to the production of 
DCWðg L1 Þ ¼ þ8:00  0:30 A þ 1:17 B  0:083 C  8:333E  003 D þ 0:14 A B þ 0:11 A C


PHB.        2  2
þ 0:12 B C þ 0:075 B D  0:13 C D  0:47 A  0:22 B  0:66 C  0:80 D 2  2
ð3Þ
1   
PHBðgl Þ ¼ þ4:64 þ 0:61 A  0:29 B þ 0:18 C þ 0:025 D þ 0:18 A B þ 0:14 A C  0:025 A D

3.2. Optimization using response surface methodology (RSM) þ 0:075 B C  0:16 B D  0:025 C  D  0:40 A2  0:36 B2  0:31 C 2  0:24 D2 ð4Þ

Regression analysis of the experimental data showed that car-

In this study also stated that the effect of factors such as pH va- bon source, nitrogen source, pH and temperature had positive lin-
lue, substrate concentrations and temperature leading to the PHB ear effects on DCW and PHB production (P < 0.05). Probability (P)
values were used as a tool to check the significance of each of
Table 3 the coefficients. The smaller the level of P value, the more signifi-
Chemical compositions of water hyacinth.
cant was the correlation with the corresponding coefficient.
Chemical parameters Concentration (%) Among the four factors tested, pH value had the highest impact
Cellulose 25 ± 2 on both DCW and PHB production as given by the highest linear
Hemicellulose 35 ± 3.2 coefficients (37.24 and 16.175). Interactions between all parame-
Total carbohydrate 3.35 ± 0.2 ters were also significant. In this case A, B, C, AB, AC, AD, BC, BD,
Protein 9.03 ± 0.6
CD, A2, B2, C2, D2 for DCW and A, B, C, AB, AC, BC, BD, A2, B2, C2, D2
Lignin 10 ± 0.42
Nitrogen 2 ± 0.1 for PHB were interactive significant terms as shown by low P val-
Phosphorous 0.09 ± 0.001 ues (P < 0.05). The 3D response surface plot is a graphical represen-
Moisture 95 ± 2 tation of the regression equation. It is drawned to understand the
Ash 20 ± 1
interaction of the variables and locate the optimal level of each
 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92 87

Table 4
ANOVA for the regression model of DCW production obtained from the experimental results.

Source Sum of squares df Mean squares F value P-value Prob > F

Model 67.7227 14 4.83734 3095.9 <0.0001*
A-Carbon source 2.16 1 2.16 1382.4 <0.0001*
B-Nitrogen source 33.135 1 33.135 21206.4 <0.0001*
C-pH 0.16667 1 0.16667 106.667 <0.0001*
D-Temperature 0.00167 1 0.00167 1.06667 0.3171
AB 0.3025 1 0.3025 193.6 <0.0001*
AC 0.2025 1 0.2025 129.6 <0.0001*
AD 0.0625 1 0.0625 40 <0.0001*
BC 0.25 1 0.25 160 <0.0001*
BD 0.09 1 0.09 57.6 <0.0001*
CD 0.25 1 0.25 160 <0.0001*
A2 6.39544 1 6.39544 4093.08 <0.0001*
B2 1.42098 1 1.42098 909.424 <0.0001*
C2 12.472 1 12.472 7982.1 <0.0001*
D2 18.2061 1 18.2061 11651.9 <0.0001*
Residual 0.025 16 0.00156
Lack of fit 0.025 10 0.0025
Pure error 0 6 0
Cor total 67.7477 30
*
Significant at <0.05% level.

Table 5
ANOVA for the regression model of PHB production obtained from the experimental results.

Source Sum of squares df Mean squares F value P-value Prob > F

Model 23.01541 14 1.643958 197.8048 <0.0001*
A-Carbon source 8.881667 1 8.881667 1068.662 <0.0001*
B-Nitrogen source 2.041667 1 2.041667 245.658 <0.0001*
C-pH 0.806667 1 0.806667 97.05998 <0.0001*
D-Temperature 0.015 1 0.015 1.804834 0.1979
AB 0.49 1 0.49 58.95792 <0.0001*
AC 0.3025 1 0.3025 36.39749 <0.0001*
AD 0.01 1 0.01 1.203223 0.2889
BC 0.09 1 0.09 10.82901 0.0046*
BD 0.4225 1 0.4225 50.83617 <0.0001*
CD 0.01 1 0.01 1.203223 0.2889
A2 4.629949 1 4.629949 557.0861 <0.0001*
B2 3.807183 1 3.807183 458.089 <0.0001*
C2 2.835268 1 2.835268 341.146 <0.0001*
D2 1.645481 1 1.645481 197.9881 <0.0001*
Residual 0.132976 16 0.008311
Lack of fit 0.055833 10 0.005583 0.434259 <0.8832
Pure error 0.077143 6 0.012857
Cor total 23.14839 30
*
Significant at <0.05% level.

variable for maximal response. Each response surface plot for DCW (NH4)2 SO4 supplemented minimal media with the DCW of
and PHB production were explored in Figs. 2 and 3 which repre- 7.5 ± 0.1 g L1. Only 3.5 ± 0.2 g L1 of PHB produce intracellularly
sents the different combinations of two test variables at one time during beef extract addition. The supplementation of other com-
while maintaining the other variable at the zero level. It is also find plex nitrogen sources like tryptone, peptone and yeast extract
the region where the optimal response occurs. The response sur- used in this study respectively produce 4.1 ± 0.2 g L1,
face plots of Figs. 2 and 3 explore the results of optimized environ- 3.8 ± 0.2 g L1 and 4.1 ± 0.3 g L1 (Fig. 4a). Simultaneously, other
mental conditions and nutrient concentrations for biomass (DCW) inorganic nitrogen sources like NH4 Cl and NH4 H2 PO4 enhances
and PHB production. The optimized factors for PHB production are the PHB concentrations in C. necator (Fig. 4b). Usually the PHB
as follows: carbon source, 35 g L1; nitrogen source, 2 g L1; tem- synthesis rate of C. necator depends on the nitrogen source and
perature, 27 °C; pH, 7. their limitation. Under nitrogen depleted conditions, the metabo-
lism of TCA cycle will be induced, resulting in an increase of acet-
3.3. Effect of nitrogen sources yl-CoA (Wang et al., 2009). In the present study 1.5 g L1 of
complex and inorganic nitrogen sources were provided in the
This study proved the best PHB production capability ob- minimal medium. Highest PHB yield obtained when the addition
served in the inorganic nitrogen sources amended minimal med- of inorganic nitrogen sources in the medium. Generally, the lim-
ia. For the optimization studies water hyacinth enzymatic ited nitrogen concentrations reduce the bacterial growth rate; to
hydrolysate (35 g L1 reducing sugar) has been used in the min- compensate that trouble increased concentration of inorganic
imal media. In the present study complex nitrogen sources used nitrogen sources were added in the hydrolysate supplemented
for the PHB production via C. necator produce a lesser amount of medium, than the standard glucose amended medium which ini-
PHB than in inorganic nitrogen sources. Improved concentration tially enhances the biomass growth rates and PHB accumulation
of PHB (4.67 ± 0.2 g L1) has been observed in 1.5 g L1 of capacity of C. necator.
88 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92

Fig. 2. Response surface plots showing the effects of (a) nitrogen source vs. carbon source; (b) pH vs. carbon source; (c) temperature vs. carbon source; (d) pH vs. nitrogen
source; (e) temperature vs. nitrogen source; (f) temperature vs. pH on biomass [DCW(g L1)] production during supplementation of water hyacinth hydrolysate as sole carbon
source.

3.4. Fermentation studies in bioreactor The PHB production capacity of the cultures was strongly influ-
enced by the composition of substrates. The batch experiment with
The results shows that the growth of C. necator on water hya- water hyacinth hydrolysates as a sole carbon substrate, the maxi-
cinth hydrolysate at the initial reducing sugar of 35 g L1 and the mum PHB content about 58% but in glucose addition 62% was ob-
nitrogen source concentration of 1.5 g L1, enter a stationary phase tained. PHB biopolyesters were synthesized and accumulated to
after 54 h of cultivation with the l of 0.36 h1. Other parameters 57 wt.% of cell mass using sugar cane molasses hydrolysated med-
obtained were biomass yield (Yx/s) 0.35 gg1 sugar and product ium (Yu and Stahl, 2008). Recent publications by Mothes et al.
yield (Yp/s) 0.24 gg1 sugar and substrate utilization efficiency (2007) and Cavalheiro et al. (2009) described direct fermentation
reached 97%. Even though, the sugar utilization rate was initially of biodiesel-glycerol to PHB by C. necator (Formerly known as Alca-
much lower in water hyacinth reducing sugar hydrolysate com- ligenes eutrophus, Woutertia eutropha and Ralstonia eutropha) with
pared to the pure glucose addition. The higher Yx/s values and low- polymer production approaching 50% of dry microbial biomass.
er Yp/s values obtained from water hyacinth hydrolysates fed In contrast, Yp/s (product yield) values were also higher when glu-
experiments indicated that fewer substrates was used for product cose was used as the substrate. The product yield was 0.29 gg1,
synthesis, and more substrate was used for biomass production. which is higher than the PHB yield value of a previous report
 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92 89

Fig. 3. Response surface plots showing the effects of (a) nitrogen source vs. carbon source; (b) pH vs. carbon source; (c) pH vs. nitrogen source; (d) temperature vs. carbon
source; (e) temperature vs. nitrogen source; (f) temperature vs. pH on PHB (g L1) production during supplementation of water hyacinth hydrolysate as sole carbon source.

(Sharifzadeh Baei et al., 2011). The high accumulation capacity of C. amended media. Therefore, there is a need for the development
necator using standard carbon source seems strongly related to the of more efficient fermentation strategies for production of these
purity of glucose monomer and there were no inhibitors in its bio- polymers from the cheap carbon sources. Moreover, another
synthesis medium. Therefore, with respect to rates and maximal source to the inhibition of PHB production was the total phenolic
storage capacity, glucose is a more suitable substrate for PHB pro- content of hydrolysate, which also hindered the bacterial growth.
duction by C. necator bacteria. Harsh pretreatment conditions result in the formation of toxic
Earlier studies reported that the PHB production was observed compounds in the hydrolysate that are inhibiting agents to the fer-
in C. necator when the supplementation of glucose, fructose and menting microorganisms. The main inhibitors are the furan deriv-
sucrose in production media as sole carbon sources (Khanna atives, organic acids and phenolic compounds (Nigam, 2002). To
and Srivastava, 2005; Kim et al., 1994; Nurbas and Kutsa, 2004). overcome these inhibitions the hydrolysates require detoxification
In this study, it is proven that this strain had potential to synthesis steps. The hydrolytic byproducts at moderate concentrations are
PHB in their cells when the water hyacinth hydrolysates used as a toxic or inhibitory to many microbial strains and are removed be-
sole carbon source which might be due to the hydrolysates contain fore use in microbial fermentation, such as ethanol fermentation
glucose monomers at maximum concentration. Nevertheless, the (Nigam, 2002).
present study results compared to the glucose based PHB produc- All observed results were focused towards the maximum PHB
tion the lower production rate have been found. Kim et al. (1994) production rates were at the incubation period of 72 h. After the
reported the higher PHB production rate of 2.42 g L1 h1 was ob- increased incubation period above 72 h and up to 96 h the PHB
tained by the Ralstonia eutropha bacteria grown under glucose production ability of bacteria was declined. PHB loss because of
90 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92

Fig. 4. Effect of complex organic (a) and inorganic (b) nitrogen sources with water hyacinth hydrolysate amended minimal media for PHB production.

hydrolysis was observed upon reaching the stationary phase, sug-

gesting critical importance at the time of harvest. Because PHB is
quite stable in aqueous suspensions (Tamer et al., 1998), the ob-
served hydrolysis was associated to enzyme mediated metabolic
action in a low-sugar environment. In most microorganisms, PHB
is a food reserve which means it is degraded to provide carbon
and energy when an external carbon source is exhausted (Grothe
et al., 1999). Moreover, in this study concludes that the biomass
growth has been enhanced with the addition of enzymatic hydro-
lysate while the PHB production was increased at 72 h incubation
period.

3.5. Characterization of produced PHB

3.5.1. FTIR results

FTIR spectrum of PHB was recorded in the range of 4000–
400 cm1. Obtained spectral peak values and the presence of broad
Fig. 5. DSC thermogram of produced PHA from the C. necator using water hyacinth
band’s responding to the groups OH, C–O and C@O indicating the enzymatic hydrolysate as carbon source.
structure similar to PHB. Mainly two intense absorption bands at
1720.19 cm1 and 1276.65 cm1 corresponding to C@O and C–O
stretching groups respectively. Stretched absorption band at (Ram Kumar Pandian et al., 2010; Sathesh Prabu and Murugesan,
3640–3610 cm1 found to shows as O–H group of polyester. Smal- 2010).
ler bands from 3010 to 2960 cm1 have been proven that as CH3
group whereas the band in the field of 2984 cm1 belongs to a 3.5.2. Molecular mass
group depending on the amorphous state. The C–H bond (where Gel permeation chromatography (GPC) was used to estimate
the hydrogen is attached to a carbon which is singly-bonded to the molecular mass of PHB sample, since this is an important factor
everything else) absorbs the range from 3000 to 2850 cm1. The to determine physical properties of polymers. However, high
peak values obtained in this study coincides with previous results molecular mass is a high quality of PHB, and it has a less limited
 D. Radhika, A.G. Murugesan / Bioresource Technology 121 (2012) 83–92 91

industrial application. The weight average molecular mass of PHB Beaulieu, M., Beaulieu, Y., MeLinard, J., Pandian, S., Goulet, J., 1995. Influence of
ammonium salts and cane molasses on growth of Alcaligenes eutrophus and
produced by wild-type strain of C. necator ranges from 100 to
production of polyhydroxybutyrate. Appl. Environ. Microbiol. 61, 165–169.
1000 kDa with a polydispersity (Mw/Mn) of around 2. In the pres- Cavalheiro, J.M.B.T., Dealmeida, M.C.M.D., Grandfils, C., Dafonseca, M.M.R., 2009.
ent study relatively low molecular mass PHB (1. 77  105 kDa) was Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste
produced in the cells with the polydispersity index of 1.9 for the glycerol. Proc. Biochem. 44, 509–515.
Chen, G.Q., Page, W.J., 1994. The effect of substrate on the molecular weight of
water hyacinth hydrolysate. These results indicated that the PHB polyhydroxybutyrate produced by Azotobacter vinelandii UWD. Biotechnol. Lett.
produced by the water hyacinth hydrolysate supplemented cul- 16, 155–160.
tures had much lower Mn and Mw than the cultures grown in glu- Cheng, J., Xie, B., Zhou, J., Song, W., Cen, K., 2010. Cogeneration of H2 and CH4 from
water hyacinth by two-step anaerobic fermentation. Int. J. Hydrogen Energy 35,
cose. The PHB produced from waste activated sludge alkaline 3029–3035.
fermentation liquid had a molecular mass of 8.5  105 Da (Jiang Doi, Y., Segawa, A., Kawaguchi, Y., Kunioka, M., 1990. Cyclic nature of poly(3-
et al., 2009). The molecular mass of the final polymers strongly de- hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol. Lett.
55, 165–169.
pended on the composition of the substrate. Chen and Page (1994) Girisuta, B., Danon, B., Manurung, R., Janssen, L.P.B.M., Heeres, H.J., 2008.
reported substrates and culture conditions, which affect the molec- Experimental and kinetic modelling studies on the acid- catalysed hydrolysis
ular mass of PHB, including the method of PHB isolation, which of the water hyacinth plant to levulinic acid. Bioresour. Technol. 99, 8367–8375.
Grothe, E., Moo-Young, M., Chisti, Y., 1999. Fermentation optimization for the
causes severe damage to the granules and led to loss of molecular production of poly(b-hydroxybutyric acid) microbial thermoplastic. Enzyme
mass of polymer (Marchessault, 1996). In future studies, the Microb. Technol. 25, 132–141.
copolymer induction and the growth conditions used should be Jiang, Y., Chen, Y., Zheng, X., 2009. Efficient polyhydroxyalkanoates production from
a waste-activated sludge alkaline fermentation liquid by activated sludge
modified in order to obtain the high molecular mass PHB for prac-
submitted to the aerobic feeding and discharge process. Environ. Sci. Technol.
tical applications. 43, 7734–7741.
Khanna, S., Srivastava, A.K., 2005. Statistical media optimization studies for growth
and PHB production by Ralstonia eutropha. Proc. Biochem. 40 (6), 2173–2218.
3.5.3. Thermal characteristics of the PHB produced by the C. necator
Kim, B.S., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K., Woo, S.I., 1994. Production of
The thermal properties of PHB sample obtained from the C. poly (3-Hydroxybutyric acid by fed – batch culture of Alcaligenes eutrophus with
necator using cheap substrate water hyacinth was investigated by glucose concentration control. Biotech. Bioeng. 43, 892–898.
Kim, S., Dale, B.E., 2008. Energy and greenhouse gas profiles of
differential scanning calorimetry (DSC) and the results were inter-
polyhydroxybutyrates derived from corn grain: a life cycle perspective.
preted with the results of Doi et al. (1990). PHB recovered from the Environ. Sci. Technol. 42, 7690–7695.
C. necator presented melting temperatures and percentages of Koller, M., Bona, R., Hermann, C., Horvat, P., Martinz, J., Neto, J., Pereira, L., Varila, P.,
crystallinity slight vary to the standard PHB. Fig. 5 shows the ther- Braunegg, G., 2005. Biotechnological production of poly (3-hydroxybutyrate)
with Wautersia eutropha by application of green grass juice and silage juice as
mogram of produced PHB, the melting temperature and crystalline additional complex substrates. Biocat. Biotrans. 23, 329–337.
temperature are an important characteristic of a polymer in order Lee, S.Y., Yim, K.S., Chang, H.N., Chang, Y.K., 1994. Construction of plasmids,
to regulate the mechanical properties of the material. Since the estimation of plasmid stability, and use of stable plasmids for the production of
poly(3-hydroxybutyric acid) in Escherichia coli. J. Biotechnol. 32, 203–211.
melting temperature of PHB is around 170–180 °C, while that of Marchessault, R.H., 1996. Tender morsels for bacteria: recent developments in
the PHB sample is in the range around 170 °C, which is matching microbial polyesters. Trends Poly Sci. 4, 163–168.
to that of polypropylene. Highly crystalline polymers are usually Mishima, D., Kuniki, M., Sei, K., Soda, S., Ike, M., Fujita, M., 2008. Ethanol production
from candidate energy crops: Water hyacinth (Eichhornia crassipies) and Water
stiff and brittle resulting in very poor mechanical properties with lettuce (Pistia stratiotes L.). Bioresour. Technol. 99, 2495–2500.
low extension at break (Savenkova et al., 2000), but they have very Mothes, G., Schnorpfeil, C., Ackermann, J.U., 2007. Production of PHB from crude
low resistance to thermal degradation. glycerol. Eng. Life Sci. 7, 475–479.
Nigam, J.N., 2002. Bioconversion of water-hyacinth (Eichhornia crassipes)
hemicellulose acid hydrolysate to motor fuel ethanol by xylose–fermenting
4. Conclusions yeast. J. Biotechnol. 97, 107–116.
Nurbas, M., Kutsa, T., 2004. Production of PHB and P(HB-co-HV) biopolymers by
using Alcaligenes eutrophus. Iran Poly J. 13 (1), 45–51.
The development and implementation of renewable feedstocks Ofoefule, U., Uzodinma, E.O., Onukwuli, O.D., 2009. Comparative study of the effect
for the production of multifunctional chemicals have received of different pretreatment methods on biogas yield from water Hyacinth
much attention from the food and Pharmaceutical industries. In (Eichhornia crassipes). Int. J. Phys. Sci. 4, 535–539.
Peplinski, K., Ehrenreich, A., Doring, C., Bomeke, M., Reinecke, F., Hutmacher, C.,
this study, it is concluded that the addition of water hyacinth enzy- Steinbuchel, A., 2010. Genome-wide transcriptome analyses of the ‘Knallgas’
matic hydrolysate in the minimal mineral media gave higher PHB bacterium Ralstonia eutropha PHB H16 with regard to polyhydroxyalkanoate
production. During acid hydrolysate supplementation lesser metabolism. Microbiology 156, 2136–2152.
Ram Kumar Pandian, S., Deepak, V., Kalishwaralal, K., Rameshkumar, N., Muniyandi,
amount of PHB has been observed owing to phenolic load which J., Gurunathan, S., 2010. Optimization and fed-batch production of PHB utilizing
hindered partial utilization of sugars, microbial growth rate and dairy waste and sea water as nutrient sources by Bacillus megaterium SRKP-3.
PHB storage capacity. The production of PHB was 58% on the dry Bioresour. Technol. 101, 705–711.
Salehizadeh, H., Van Loosdrecht, M.C.M., 2004. Production of
cell weight basis when the organism was grown in a water hya- polyhydroxyalkanoates by mixed culture: recent trends and biotechnological
cinth hydrolysate amended medium at optimum environmental importance. Biotechnol. Adv. 22, 261–279.
conditions. Sathesh Prabu, C., Murugesan, A.G., 2010. Effective utilization and management of
Coir Industrial waste for the Production of poly- b- hydroxybutyrate (PHB)
using the bacterium Azotobacter Beijerinickii. Int. J. Environ. Res. 4, 519–524.
Acknowledgement Sathesh Prabu, C., Murugesan, A.G., 2011. Potential utilization of sorghum field
waste for fuel ethanol production employing Pachysolen
tannophilus and Saccharomyces cerevisiae. Bioresour. Technol. 102, 2788–2792.
Financial assistance of the Tamil Nadu State Council for Science Savenkova, L., Gercberga, Z., Nikolaeva, V., Dzene, A., Bibers, I., Kalnin, M., 2000.
and Technology, Government of Tamil Nadu, Chennai in the form Mechanical properties and biodegradation characteristics of PHB-based films.
of a major research project to Prof. A.G. Murugesan is gratefully Proc. Biochem. 35, 573–579.
Sharifzadeh Baei, M., Najafpour, G.D., Younesi, H., Tabandeh, F., Issazadeh, H.,
acknowledged.
Khodabandeh, M., 2011. Growth kinetic parameters and biosynthesis of
polyhydroxybutyrate in cupriavidus necator DSMZ 545, on selected substrates.
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