Accepted Manuscript

Microbial Production, Ultrasound–Assisted Extraction and Characterization of

Biopolymer Polyhydroxybutyrate (PHB) from Terrestrial (P. hysterophorus)
and Aquatic (E. crassipes) Invasive Weeds

Sushobhan Pradhan, Arup Jyoti Borah, Maneesh Kumar Poddar, Pritam Kumar
Dikshit, Lilendar Rohidas, Vijayanand S. Moholkar

PII: S0960-8524(17)30398-X
DOI: http://dx.doi.org/10.1016/j.biortech.2017.03.117
Reference: BITE 17821

To appear in: Bioresource Technology

Received Date: 23 January 2017

Revised Date: 18 March 2017
Accepted Date: 20 March 2017

Please cite this article as: Pradhan, S., Borah, A.J., Poddar, M.K., Dikshit, P.K., Rohidas, L., Moholkar, V.S.,
Microbial Production, Ultrasound–Assisted Extraction and Characterization of Biopolymer Polyhydroxybutyrate
(PHB) from Terrestrial (P. hysterophorus) and Aquatic (E. crassipes) Invasive Weeds, Bioresource Technology
(2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.117

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Microbial Production, Ultrasound–Assisted Extraction and

Characterization of Biopolymer Polyhydroxybutyrate (PHB) from

Terrestrial (P. hysterophorus) and Aquatic (E. crassipes) Invasive Weeds

Sushobhan Pradhan,1,# Arup Jyoti Borah,2,§ Maneesh Kumar Poddar,1,§ Pritam Kumar
Dikshit,1,§ Lilendar Rohidas3 and Vijayanand S. Moholkar1,2,*

1
Department of Chemical Engineering, 2 Center for Energy, Indian Institute of Technology
Guwahati, Guwahati – 781 039, Assam, India

3
Department of Chemical Engineering, National Institute of Technology (NIT),
Tiruchirapalli – 620 015, Tamil Nadu, India

* Author for correspondence. Fax: +91 361 258 2291. E–mail: [email protected]

# Present address: School of Chemical Engineering, Oklahoma State University, Stillwater,

OK 74078, USA.

§ Equal contribution by these authors.

Abstract

This study reports synthesis of biodegradable poly(3–hydroxybutyrate) (PHB) polymer from

two invasive weeds, viz. P. hysterophorus and E. crassipes. The pentose and hexose–rich

hydrolyzates obtained from acid pretreatment and enzymatic hydrolysis of two biomasses

were separately fermented using Ralstonia eutropha MTCC 8320 sp. PHB was extracted

using sonication and was characterized using FTIR, 1H and 13C NMR and XRD. PHB content

of dry cell mass was 8.1–21.6% w/w, and the PHB yield was 6.85×10–3–36.41×10–3 % w/w

raw biomass. Thermal properties of PHB were determined by TGA, DTG and DSC analysis.

PHB obtained from pentose–hydrolyzate had glass transition temperatures of 6o–9oC, while

PHB from hexose–rich hydrolyzate had maximum thermal degradation temperatures of 370o–

389oC. These thermal properties were comparable to the properties of commercial PHB.

Probable causes leading to differences in thermal properties of pentose and hexose–derived

PHB are: extent of crystallinity and presence of impurity in the polymer matrix.

Keywords: PHB, Ralstonia eutropha, Parthenium hysterophorus, Eichhornia crassipes,

Fermentation
1. Introduction

Use of synthetic polymers and plastics derived from petrochemicals as substitutes of

conventional materials of fabrication (such as wood, glass and metals) in domestic, industrial

and transport sectors has immensely increased in past few decades. Polymer and plastic have

comparable or even better thermal, mechanical, electrical and optical properties than

conventional materials, in addition to low cost. However, biorecalcitrant nature of these

plastics leads to their persistent presence in the environment and accumulation in terrestrial

and aquatic habitats (Khanna and Srivastava, 2005). Efficient disposal of the synthetic

polymers and plastics has been a global environmental challenge. Use of biodegradable

polymers has thus become an urgent need of the hour. Polyhydroxyalkanoates (PHAs) are

one of the potential bioplastics that are completely degraded by microorganisms (Mergaert et

al., 1992). PHAs also possess similar physical properties as synthetic plastics which assist in

their use as substitutes of synthetic plastics (Mozumder et al., 2015). Among the PHA family

of biopolymers, polyhydroxybutyrate (PHB) has physical properties that closely resemble

common synthetic polymers like polyethylene and polypropylene (Yousuf and Winterburn,

2016). By virtue of these properties, PHB can processed using common techniques of

extrusion, moulding, spinning into fibers or casting into thin films (Batcha et al., 2014).

Moreover, it can also be blended with other synthetic polymers and plastics. Major hurdle in

large–scale production of PHB is the high cost of production (Du et al., 2012; Lenz and

Marchessault, 2005). PHB can be produced through microbial fermentation of reducing

(primarily hexose) sugars. PHB in the form of intracellular granules is synthesized by several

microbial species as energy reserve materials under environmental stresses such as limited

concentrations of nitrogen, oxygen and phosphate nutrients, but an excess carbon source

(Steinbuchel, 1991; Lee, 1996). Depending on the microbial species, the intracellular PHB

accumulation could be as high as 80% of the dry cell weight. Among the PHB synthesizing
microbial cultures, Ralstonia eutropha (also known as Cupriavidus necator) has been widely

studied due to its potential of producing significant quantities of PHB from simple carbon

substrates such as hexose and pentose sugars or fatty acids/acetic acid (Chee et al., 2010;

Khanna and Srivastava, 2005).

In most fermentation based processes, a major fraction of the total operating cost is

contributed by the cost of substrate (Wang et al., 2013). Conventional carbon source for PHB

fermentation is glucose. However, as per previous studies, approx. 3 tons of glucose is

required for synthesis of 1 ton of PHB (Collins, 1987). The production cost of PHB can be

significantly reduced by use of renewable and inexpensive carbon sources for fermentation

(Obruca et al., 2015). Lignocellulosic biomasses in the form of agro– and forest residues and

waste biomasses such as grasses or weeds can be employed as substrate for PHB production

(Keenan et al., 2006; Kumar et al., 2008; Cesario and de Almeida, 2015). The cellulose

content of these biomasses (after dilute acid treatment and delignification) can be converted

into fermentable hexose sugar through enzymatic hydrolysis (Borah et al., 2016a; Singh et

al., 2014a). A summary of recent studies on PHB production by fermentation of various

inexpensive substrates is given in Table S.1 in the Supplementary Material provided with this

paper.

The present study deals with production of PHB from invasive weeds such as

Parthenium hysterophorus and Eichhornia crassipes (or water hyacinth) using R. eutropha as

the microbial culture. This study also includes ultrasound–assisted extraction of PHB from

microbial cells and the characterization of PHB using standard techniques. P. hysterophorus

is regarded as world’s seventh most devastating and hazardous terrestrial invasive weed that

has infested thousands of hectares of arable land. Exposure to this weed also causes severe

health problems in both humans and animals. E. crassipes is an aquatic invasive weed that

covers surfaces of rivers and lakes, and poses major threat to the aquatic life. Utilization of
these waste biomasses for biopolymer production offers simultaneous solution for control of

these weeds and economically viable route for PHB production.

2. Materials and Methods

2.1 Materials and microbial culture

Microbial strain R. eutropha (MTCC No. 8320) was a kind gift from Prof. Kannan

Pakshirajan, Department of Biosciences and Bioengineering, Indian Institute of Technology,

Guwahati, India. Poly–3–hydroxybutyrate, corn steep liquor, deuterated chloroform (CDCl3),

and the enzymes cellulase (6 U/mg, produced by Trichoderma reesei) and cellobiase (or β–

glucosidase, 250 U/g, produced by Aspergillus niger) were procured from Sigma–Aldrich.

D–fructose, chloroform and methanol were procured from Sisco Research Laboratory Pvt.

Ltd., India. Nutrient for the growth and fermentation media, viz. beef extract, yeast extract,

peptone, sodium chloride, KH2PO4, nickel chloride (NiCl2·6H2O), calcium hydroxide

(Ca(OH)2), sulphuric acid (H2SO4), sodium molybdate (NaMoO4·2H2O) and magnesium

sulphate (MgSO4·7H2O) were obtained from HiMedia Pvt. Ltd, India. Calcium chloride

(CaCl2), urea (N2H4CO), potassium bromide (KBr), boric acid (H3BO4), cobalt chloride

(CoCl2·7H2O), cuprous chloride (CuCl2·2H2O) and zinc sulphate (ZnSO4·7H2O) were

procured from Merck India Ltd. Manganese chloride (MnCl2·4H2O) and disodium hydrogen

phosphate (Na2HPO4) were obtained from Loba Chemie Pvt Ltd., India.

2.2 Pretreatment of biomass

Collection / preliminary processing of biomass: Samples of P. hysterophorous and E.

crassipes biomass were collected from the campus of I.I.T. Guwahati. Entire plant body

except root was used as substrates. The biomass samples were dried in ambient air followed

by chopping into small pieces. The chopped biomass was washed with water and again dried

in hot air oven at 60oC for 24 h. The particle size of dried biomass was further reduced to < 1
mm using a domestic mixer grinder. The grinded biomass samples were stored in air–tight

containers at room temperature for further experiments.

Acid hydrolysis: Conditions for dilute acid pretreatment (or acid hydrolysis) of both

biomasses were adopted from results of Singh et al. (2014b). The optimum conditions for

acid hydrolysis of P. hysterophorus biomass, as reported by Singh et al. (2014b) were: 1%

(v/v) H2SO4 (equivalent to 0.36 N), 10% w/v biomass, autoclaving at 121oC and 15 psi for 30

min followed by rapid steam release. The dried biomasses of both P. hysterophorus and E.

crassipes were pretreated under these conditions. Dilute acid pretreatment hydrolyzed the

hemicellulose fraction of biomass resulting in release of pentose sugars. The filtrate resulting

from acid pretreatment (or the acid hydrolysate) was analysed for the concentration of

reducing or fermentable sugars. The hydrolysate was then detoxified to remove the inhibitory

compounds. The biomass from reaction mixture was filtered using double–layered muslin

cloth. The residual chemicals left on the biomass surface after acid pre–treatment were

removed by successive water wash till the neutral pH of the wash water was achieved. This

was followed by drying of biomass residue in hot air oven for 24 h at 60oC. The resultant

cellulose rich biomass (with traces of lignin) was used for enzymatic hydrolysis.

Detoxification of acid hydrolysate: Detoxification of hydrolysate resulting from dilute acid

pretreatment was done in two steps: Initially, the pH of the hydrolysate was increased to 10

with addition of Ca(OH)2 followed by neutralization using conc. H2SO4. It was then

centrifuged at 10,000 × g for 15 min to remove suspended solids. Next, 1.5% w/v activated

charcoal was added to hydrolysate with continuous stirring for 30 min. The inhibitory

compounds formed during acid hydrolysis were adsorbed on activated charcoal. Activated

charcoal particles were removed by vacuum filtration of the hydrolysate.

Delignification of biomass: Delignification of the P. hysterophorus and E. crassipes biomass

obtained after acid hydrolysis was carried out using the procedure outlined by Bharadwaja et
al. (2015) and Borah et al. (2016b), respectively. This procedure applies sonication (or

ultrasound irradiation) during delignification at optimized conditions of temperature, biomass

concentration and NaOH concentration. The physical and chemical effects of ultrasound and

cavitation enhance the kinetics of the delignification process. A probe type programmable

and microprocessor controlled ultrasonic processor (Sonics & Materials, Model VCX 500)

with maximum power 500 W and frequency 20 kHz was used for sonication of reaction

mixture. The reaction was carried out in 100 mL beaker. The total volume of reaction mixture

was 80 mL with alkali concentration of 1.5% w/v NaOH and biomass loading of 2% w/v. The

ultrasound probe was set at 30% amplitude with theoretical power consumption of 150 W at

duty cycle of 83% (i.e. pulse mode sonication with 50 s ON and 10 s OFF cycle in 1 min).

The total sonication period was 10 min. The temperature of the reaction medium was

maintained at 30oC during sonication treatment. After completion of sonication, the reaction

mixture was filtered through a double layered muslin cloth to remove the solid biomass

residue after the delignification. This cellulose rich biomass residue (after removal of lignin

and hemicellulose) was washed with hot water until the pH of wash water was neutral,

indicating complete removal of traces of residual chemicals from biomass surface. The

biomass residue was dried for ~ 12 h in hot air oven at 60°C, and was used for enzymatic

hydrolysis.

Biomass saccharification: The enzymatic hydrolysis (or saccharification) of delignified

biomasses of P. hysterophorus was carried out using commercial cellulase and cellobiase

enzymes at the optimum conditions reported by Bharadwaja et al. (2015). The hydrolysis was

carried out in an incubator shaker (Make: Scigenics Biotech, India; Model: Orbitek LE) in 50

mM citrate phosphate buffer solution (pH 4.8) at 50oC and 150 rpm. The reaction mixture

was taken in 1000 mL Erlenmeyer flask with total reaction volume of 500 mL. The

concentration of pre–treated biomass in reaction mixture was 4.2% w/v, with cellulase and
cellobiase concentrations of 135 and 75 FPU/g biomass, respectively. The hydrolysis was

carried out for 120 h. 0.005% w/v sodium azide solution was added to the mixture to avoid

external microbial contamination. 0.1 mL samples of reaction mixture were withdrawn

periodically during enzymatic hydrolysis and were analysed for release of sugar.

Determination of reducing sugar in hydrolysate: Both pentose–rich hydrolyzate (resulting

from dilute acid pretreatment) and hexose–rich hydrolyzate (resulting from enzymatic

hydrolysis) were subjected to centrifugation for 10 min at 10,000 × g at 4oC for removal fine

suspended biomass particles. Concentration of total reducing sugar in the hydrolyzate was

estimated using method of Nelson and Somogyi (Nelson, 1944; Somogyi, 1945).

2.3 Microorganism growth and maintenance

The cells of R. eutropha were revived in Nutrient Broth (NB) medium and placed in

rotary incubator shaker at 30oC, 150 rpm for 24 h and the composition of NB medium was as

follows: beef extract 1.0 g, yeast extract 2.0 g, peptone 5.0 g, NaCl 5.0 g, fructose 10.0 g in 1

L of distilled water. The pH of the medium was adjusted to 7.0 prior to autoclaving. The

autoclaving was done at 121oC, 15 psi for 15 min. The cultures were stored at 4°C and sub–

cultured in every month.

2.4 Preparation of inoculum and fermentation media

The cells of R. eutropha were precultured in NB medium at 30oC for 24 h. The

fermentation experiments were carried out in mineral salt medium with composition as

follows: KH2PO4 – 2.0 g, Na2HPO4 – 4.0 g, MgSO4·7H2O – 0.51 g, CaCl2 – 0.02 g, urea –

1.0 g, corn steep liquor – 0.5 g, and (NH4)2SO4 – 2.0 g in 1 L distilled water. 10 mL/L trace

element solution containing ZnSO4·7H2O – 0.01 g, MnCl2·4H2O – 0.003 g, H3BO4 – 0.003 g,

CoCl2·7H2O – 0.02 g, CuCl2·2H2O – 0.001 g, NiCl2·6H2O – 0.002 g, and NaMoO4·2H2O –

0.003 g in 1 L distilled water was added to the fermentation medium. Concentrations of

pentose and hexose sugar hydrolyzates in fermentation media was kept at 10 g/L for both
biomasses. All components of fermentation media such as carbon source (or hydrolyzates),

phosphates, sulphates, corn steep liquor (CSL), urea chlorides and trace element solution

were sterilized separately to avoid precipitation of these compounds at high temperature. All

components were autoclaved at 121oC, 15 psi pressure for 15 min. The pH of the medium

was adjusted to 7.0 using 1 N NaOH / HCl. 5% (v/v) inoculum was transferred to the

fermentation medium. Batch fermentation was carried out in an Erlenmeyer flask (capacity:

500 mL) with working volume of 200 mL. The fermentation mixture was incubated at 30oC,

150 rpm for 50 h in an incubator shaker.

2.5 Extraction and purification of PHB

Bacterial cells after fermentation were harvested using centrifugation (SIGMA D–

37520 Osterode am Harz, Germany) at 10000 × g for 10 min at 4oC. The cell pellets obtained

after centrifugation were washed twice with distilled water to remove impurities, followed by

freeze drying in lyophilizer (SIGMA: Alpha 2–4 LD plus, 0.1 millibar, –80oC) to prevent

further degradation. Cell dry weight obtained after lyophilisation was noted. For PHB

extraction, 1 g dry cell mass was blended with 100 mL of hot chloroform in 200 mL round–

bottom flask, and was sonicated in an ultrasound bath (Make: JeioTech, South Korea;

Capacity: 10 L; Frequency: 40 kHz; Power: 200 W) for 2 h. In order to prevent the cells from

degradation the temperature of sonication bath was maintained at 20oC. After complete

dispersion of cells in chloroform, the entire solution was taken in 200 mL beaker and was

subjected to high intensity sonication by ultrasonic probe at 20 kHz and 40% amplitude

(Make: Sonics; Model: VCX500; Power: 500 W) for 30 min. High intensity sonication

generates strong transient cavitation in the medium. The transient cavitation bubbles generate

intense shock waves that disrupt the microbial cells with release of intracellular PHB

granules in the medium. Due to strong microturbulence created in the liquid medium, the

PHB granules released from microbial cells get completely dissolved in chloroform. The
polymer solution was then filtered to remove any suspended solids. The PHB dissolved in the

polymer solution was then precipitated by dropwise addition of polymer solution into 1 L

chilled methanol (99.8%). White precipitate of the PHB polymer was collected and dried at

40oC in vacuum oven for evaporation of any residual solvent. The dried PHB polymer in

powder form was used for further characterization.

2.6 Analytical method and characterization of PHB

The optical density of R. eutropha cells in fermentation broth was monitored with a UV–Vis

spectrophotometer by measuring absorbance at 600 nm (Make: Perkin Elmer, Model:

LAMBDA 35). The powdered PHB samples were characterized as follows:

Nuclear Magnetic Resonance (NMR) spectroscopy: 1H NMR and 13C NMR analysis of the

PHB (20 mg of purified PHB was dissolved in 1 mL of CDCl3) was performed using a

Bruker NMR spectrometer (Make: Bruker, 600 MHz).

Fourier Transform Infrared (FTIR) spectroscopy: Major functional groups and moieties in

the PHB polymer were identified using FTIR spectroscopy (Make: Shimadzu; Model: IR

Affinity–1). The FTIR spectra were recorded in the range of 4000 to 500 cm–1.

X–ray diffraction (XRD) analysis: The crystalline nature of the PHB powder was analyzed

using X–ray diffraction (Make: Bruker; Model: D8–Advance) spectroscopy with Cu –Kα

radiation (λ = 1.54056 A˚) in the range of 2θ = 1–50o.

Thermo Gravimetric analysis (TGA): Thermal stability of the PHB was determined by

thermogravimetric analysis in the temperature range of 30oC to 600oC at a heating rate of

10oC/min in nitrogen atmosphere (N2 flow rate = 40 mL/min). T5%, T10% and T50%

temperatures obtained from TGA and DTG analysis of PHB synthesized by microbial

fermentation were compared with standard PHB obtained from Sigma Aldrich.

Differential Scanning Calorimetry (DSC): The glass transition temperatures (Tg) of the

extracted PHB were determined with DSC analysis (Make: Mettler Toledo–1 series) with
heating and cooling rate of 10oC/min in N2 environment with a gas flow of 20 mL min–1. For

analysis, 3.5 mg sample was loaded in aluminium pan and heated in the temperature range of

–10oC to 200oC. The first heating cycle was operated from –10oC to 200oC at a heating rate

of 10oC min–1 followed by cooling cycle from 200oC to –10oC. The second heating cycle was

operated from –10oC to 200oC at a heating rate of 10oC min–1. The point of inflection in the

DSC curve between onset and endset temperatures corresponds to glass transition

temperature.

3. Results and Discussion

3.1 Biomass pretreatment, enzymatic hydrolysis and fermentation

The holocellulose contents of dried and ground raw biomasses were as follows: P.

hysterophorus = 72.1  2.9% (with lignin content of 23.6  0.87% w/w); E. crassipes = 82.2

 2.1% w/w (with lignin content of 4.1  0.4% w/w). The net cellulose content of the two

biomasses increased after pretreatment as follows: P. hysterophorus: past acid pretreatment =

64.1  1% w/w, past delignification = 96.1  0.94% w/w; E. crassipes: past acid pretreatment

= 72.7  2.1% w/w, past delignification = 96.7  3.9% w/w (Bharadwaja et al., 2015; Borah

et al. 2016b). The reducing sugar content of the acid and enzymatic hydrolyzates was

determined as follows: (1) P. hysterophorus: acid hydrolysate (or pentose rich hydrolysate) =

285.3 mg/g raw biomass, enzyme hydrolysate (or hexose rich hydrolysate) = 293.2 mg/g raw

biomass; (2) E. crassipes: pentose rich hydrolysate = 238.6 mg/ g raw biomass, hexose rich

hydrolysate = 379.3 mg/g raw biomass.

The results of the fermentation of pentose and hexose rich hydrolysate by R. eutropha

are depicted in Table 1. For both biomasses, hexose rich enzymatic hydrolysate gave greater

cell yield (dry cell weight) with higher PHB content. The total PHB yield per unit raw

biomass is higher for E. crassipes (43.57 × 10–3 w/w raw biomass) than P. hysterophorus
(24.44 × 10–3 w/w raw biomass). The probable factors that could have contributed to higher

PHB yield from fermentation of hydrolyzates of E. crassipes are: (1) relative higher

concentration of glucose and xylose (which are preferentially consumed by microbial

species) in the hydrolysate due to large (> 90%) holocellulose content of raw biomass, and

(2) smaller concentrations of inhibitors in hydrolysate lower lignin content of raw biomass

that could retard the kinetics and yield of fermentation. The yields of dry cell mass and PHB

in the present study is relatively lower than recent study of Annamalai and Sivakumar (2016),

in which the hydrolysate of wheat bran was used for fermentation. However, the microbial

strain employed in this work was R. eutropha NCIMB 11599, which essentially is a glucose

utilizing mutant strain.

3.2 Characterization of PHB

As noted earlier, PHB obtained from the microbial fermentation was characterized

using standard techniques. The results of these characterizations are given below:

NMR: The PHB was characterized using 1H NMR spectroscopy and 13C spectroscopy. Fig.

S.1 in the Supplementary Material depicts the 1H and 13C NMR spectra of PHB obtained

from fermentation of hexose–rich hydrolysate of P. hysterophorus, while Fig. S.2 shows the

same spectra for PHB obtained from E. crassipes. The summary of the peaks and assignment

is depicted in Table 2. 1H and 13C NMR spectra of PHB produced from pentose–rich

hydrolyzates of both P. hysterophorus and E. crassipes are shown in Fig. S.3 and Fig. S.4,

respectively in the Supplementary Material provided with this paper. It could be inferred

from Table 2 that both 1H and 13C spectrum of PHB identical irrespective of the source, viz.

type of hydrolyzate (viz. pentose–rich or hexose–rich) or the biomass itself (P. hysterophorus

or E. crassipes). The doublet at 1.30 ppm in 1H NMR spectrum is attributed to methyl group,

while multiplet peaks between 2.45–2.62 are assigned to methylene group adjacent to

asymmetric carbon atom. The multiplet at 5.25 ppm is attributed to –CH group or proton
splitting. The 13C NMR spectrum revealed following peaks confirming the structure of PHB

synthesized by microbial fermentation: methyl (–CH3) – 19.96 ppm, methylene (CH2) –

40.97 ppm, methane (CH) – 67.81 ppm and carbonyl (C=O) – 169.36 ppm. Both 1H and 13C

NMR spectra of PHB synthesized by microbial fermentation match with the spectra reported

in previous studies as well as spectra of commercial grade PHB.

FTIR: Fig. S.5A in the Supplementary Material depicts the FTIR spectra of PHB synthesized

from pentose and hexose rich hydrolyzates of both biomasses. Similar to NMR spectra, the

FTIR spectrum of PHB obtained from fermentation of pentose–rich and hexose–rich

hydrolyzate of both biomasses match closely. The assignments of various peaks in the FTIR

spectra are given in Table 3. The strong peaks in the range 1852–2976 cm–1 correspond to C–

H stretch of alkanes. Band at 1726–1745 cm–1 represents C=O (carbonyl) and –COO (ester)

group. The band at 1057–1289 cm–1 corresponds to C–O bonding. These results resemble

closely with the previous reported literature (Hu et al., 2013; Alarfaj et al., 2015; Brinda Devi

et al., 2015; Altaee at al., 2016).

XRD: The X–ray diffractograms of the PHB synthesized from pentose and hexose–rich

hydrolyzates of both biomasses are shown in Fig. S.5B in the Supplementary Material

provided with this paper. Four major peaks in the X–ray diffractograms shown in Fig. S.5B

and their corresponding 2θ values are: (020) at 13.5°, (110) at 16.9°, (111) at 25.5° and (130)

at 27.1°. The most intense peak (020) at 2θ = 13.5° indicates crystalline nature of the

polymer. Skrbic and Divjakovic (1996) have proposed that PHB molecules adopt the regular

helicoidal conformation with two antiparallel chains in rhombic unit cell within the

crystalline domain. The WAXS diffractogram of PHB biopolymer at 30°C reported by Skrbic

and Divjakovic (1996) shows strong peaks corresponding to (020), (110) and (111) planes.

The X–ray diffractogram in Fig.S.5B matches closely with previously reported studies by

Galego et al. (2000), Oliveira et al. (2007), and Brinda Devi et al. (2015). Comparing
between the two types of hydrolyzates, the PHB from hexose–rich hydrolyzate has higher

crystallinity, as indicated by the sharper peaks. Higher crystallinity imparts the PHB higher

resistance for thermal degradation, as revealed from TGA and DTG analysis explained later.

TGA: The TGA profile of PHB synthesized from the two biomasses are shown in Fig. 1. For

all biopolymers synthesized from pentose and hexose rich hydrolysate, the TGA curves show

two types of weight loss. The initial stage of weight loss (in the temperature range of 120°–

130°C) corresponds to evaporation of physically adsorbed solvents on the polymer. The

degradation of the polymer commences with melting in temperature range of 170°–180°C. As

per the previous reports (Hablot et al., 2008), thermal degradation of PHB occurs rapidly near

melting point due to random chain scission process (involving cleavage of C=O and C–O

bonds in ester moieties by β–scission). The second step weight loss occurs with further rise in

temperature to ~250°C, hydrolysis also contributes to degradation (in addition to chain

scission) resulting in formation of crotonic acid. For PHB derived from pentose–rich

hydrolyzates degradation is essentially complete till ~275°C. The PHB synthesized from

hexose–rich hydrolyzates show higher stability with great residual mass at temperature >

350°C. Table 4 provides the quantitative details of weight loss verses temperature data of the

PHBs synthesized from two biomasses. It could be inferred form Table 4 that degradation

temperature corresponding to 10% weight loss (T10%) of PHB shows a wide range of 220°–

278°C. Among the two biomasses, PHB derived from P. hysterophorus are seen to have

higher thermal stability indicated by relatively higher T10% temperatures. Nonetheless, all

biogenic PHBs have higher T10% temperature that the standard PHB. For higher degradation,

corresponding to T50% temperature, the PHBs derived from hexose–rich hydrolyzate show

greater stability. The T50% temperatures of ~365°C for hexose–derived PHB from both

biomasses are significantly higher than pentose derived PHB. This is also corroborated by

greater residual weight for higher temperature (≥ 350°C) seen in the TGA profile of the
hexose derived PHBs, as noted earlier. As noted in the XRD analysis, the PHB synthesized

from fermentation of hexose–rich hydrolyzate of both biomass have higher crystallinity due

to which their resistance for thermal degradation increases. A plausible cause underlying this

result could be formation of large molecular weight polymer from hexose–sugars which has

greater resistance to thermal decomposition at high temperature. Moreover, small quantities

of residual lignin left in the cellulose–rich biomass subjected to enzymatic hydrolysis may

also appear in the form of high molecular weight aromatic impurities in the PHB matrix. The

residual mass at high temperature in the thermogravimetric analysis may comprise of these

impurities as well. Further investigation is necessary in this regard.

DTG: The differential thermogravimetric (DTG) curves for PHB biopolymer synthesized

from the two biomasses are shown in Fig. 2A. DTG curves are essentially are the temperature

derivatives of the weight loss of the polymer sample (or the TGA curve). The peaks of the

DTG curve essentially indicate the temperature corresponding to maximum degradation rates

of the polymer matrix. In other words, the peak of the DTG curve indicate the thermal

stability of the PHB. It could be inferred from Fig. 2A, that PHB synthesized from hexose–

hydrolyzate has higher temperature corresponding to maximum degradation rate; as

compared to pentose hydrolyzate derived PHB. This essentially indicates higher thermal

stability or greater resistant to thermal degradation of PHB derived from hexose–rich

hydrolyzate. This result can also be explained on the basis of presence of high molecular

weight polymer or residual lignin impurities in the PHB derived from hexose–rich

hydrolyzate of both biomasses.

DSC: The DSC analysis is a measure of assessing the molecular mobility of the polymer

chains. The molecular mobility is manifested in terms of the glass transition temperature,

which is an important characteristics of the polymer. The glass transition temperature is

represented by the point of inflection in the DSC curve between onset and offset
temperatures. The DSC curves of the PHB synthesized from two biomasses are shown in Fig.

2B. It could be seen that hexose–derived PHB has relatively lower glass transition

temperature than pentose–derived PHB. A possible explanation for this result could be given

on the basis of basic structure of PHB. As revealed by XRD analysis, the hexose derived

PHB has better crystallinity (as indicated by sharper peaks), which essentially indicates more

ordered arrangement of polymer chains. The initial movement of the polymer chains leading

to lower glass transition temperatures. On the other hand, higher amorphous (or random)

nature of pentose derived PHB may hinder initial movement of polymer chains resulting in

higher glass transition temperature. Despite this discrepancy, the melting point of the PHB

synthesized from hexose–rich and pentose–rich hydrolyzates of both biomasses are almost

similar (in the range of 173° to 177°C). Glass transition temperatures reported in literature for

PHB (obtained from different substrate) have wide variation. Alatee et al. (2016) have

reported Tg of 2.79°C for PHB synthesized from crude palm kernel oil. Sandhya et al. (2013)

have reported Tg = 10°–15°C for PHB synthesized from fermentation of paddy straw.

4. Conclusion

This study has demonstrated potential process of synthesis of biopolymer PHB from

two ubiquitous invasive weeds, viz. P. hysterophorus and E. crassipes. The characterization

of PHB revealed significant differences in thermal properties of pentose– and hexose–derived

PHBs. PHB from pentose–rich hydrolyzate had higher glass transition temperature while

PHB from hexose–rich hydrolyzate had higher maximum thermal degradation temperature.

Probable causes underlying this difference are: (1) variation in molecular weight, (2)

variation in extent of crystallinity, and (3) presence of impurities in polymer matrix.

Nonetheless, thermal properties of as–synthesized PHB are comparable or better than

commercial PHB.
Supplementary material

The following supplementary material is provided with this paper: (1) 1H and 13C NMR

spectra, FTIR spectra, and X-Ray diffractograms of PHB produced by R. eutropha by

fermentation of hexose and pentose–rich (or enzymatic) hydrolysate of P. hysterophorus and

E. crassipes. (2) Summary of literature on PHAs/ PHB production from economic

fermentation substrates

Acknowledgement

Authors acknowledge access to analytical facilities provided by Central Instruments Facility

(CIF), I.I.T. Guwahati. Use of X–ray diffraction facility (FIST Grant No. SR/FST/ETII–

028/2010, Department of Science and Technology, Govt. of India) at Department of

Chemical Engineering, I.I.T. Guwahati is also acknowledged.

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Figure 1. Thermogravimetric (TGA) curves of PHB produced from microbial fermentation
of pentose– and hexose–rich hydrolyzates from different biomasses. Notation: PH: P.
hysterophorus, WH: E. crassipes (water hyacinth), G: hexose rich (or enzymatic)
hydrolysate, X: pentose rich (or acid) hydrolysate
 (A) (B)

Figure 2. Analysis of thermal properties of PHB produced from microbial fermentation of pentose– and hexose–rich hydrolyzates
from different biomasses. (A) Differential Thermogravimetric (DTG) curves. (B) Differential Scanning Calorimetric (DSC)
curves. Notation: PH: P. hysterophorus, WH: E. crassipes (water hyacinth), G: hexose rich (or enzymatic) hydrolysate, X: pentose
rich (or acid) hydrolysate
 Table 1. Summary of PHB production from fermentation of hydrolyzates of two biomasses
by R. eutropha.

PHB yield
PHB
Type of DCW PHB content (g PHB/ g
Biomass production
hydrolyzate (g/L) of DCW (%) raw
(g/L)
biomass)
Pentose– 2.93 ± 0.13 0.24 ± 0.02 8.03 ± 0.17 6.85 × 10–3
rich
P. hysterophorus hydrolyzate
Hexose–rich 3.35 ± 0.15 0.60 ± 0.01 17.93 ± 0.51 17.59 × 10–3
hydrolyzate
Pentose– 3.70 ± 0.05 0.30 ± 0.01 8.11 ± 0.38 7.16 × 10–3
rich
E. crassipes hydrolyzate
Hexose–rich 4.44 ± 0.12 0.96 ± 0.07 21.62 ± 1.02 36.41 × 10–3
hydrolyzate
Table 2. Chemical shift signals obtained from NMR spectra of PHB synthesized by microbial
fermentation of pentose and hexose rich hydrolyzates of different biomasses

Chemical Shift (ppm)

1 13
H NMR spectrum C NMR spectrum

Pentose–rich Hexose–rich Pentose–rich Hexose–rich

C atom hydrolyzate hydrolyzate hydrolyzate hydrolyzate

Biomass: P. hysterophorus

–CH 5.25 5.25 67.81 67.82

–CH2 2.61, 2.59, 2.17, 2.61, 2.58, 2.47, 40.98, 31.12 40.98, 29.90,
2.58,2.48, 2.45 2.45, 2.16 29.56

–CH3 1.26 1.26 19.95 19.96

–C=O 169.35 169.36

RCOOR 3.48 3.47, 3.59 51.05

Biomass: E. crassipes

–CH 5.26, 5.18 5.24 67.81 67.81

–CH2 2.62, 2.59, 2.55, 2.61, 2.58, 2.48, 40.97, 31.12, 40.97, 31.12,
2.48, 2.46 2.45, 2.16 29.88 29.87

–CH3 1.16, 1.25, 1.27 1.36, 1.26, 1.15 19.95 19.95

–C=O 169.35 169.36

RCOOR 3.47, 3.58 3.47 51.02 51.03

Table 3. Assignment of band positions in IR spectra of PHB obtained from microbial
fermentation of hydrolyzates of the biomasses of P. hysterophorus and E. crassipes

Group P. hyserophorus E. crassipes

or
moiety Pentose–rich Hexose–rich Pentose–rich Hexose–rich
hydrolyzate hydrolyzate hydrolyzate hydrolyzate

–CH 2976, 2934 2975, 2932 2923, 2852 2975, 2933

–C=O 1729 1727 1745 1726

–C–O 1288, 1058 1285, 1057 1278, 1062 1289, 1058

Table 4. Summary of TGA analysis of PHB obtained from different fermentation substrates

Biomass: P. hysterophorus

Sample T5% (oC) T10% (oC) T50% (oC)

Pentose–rich hydrolyzate 274 278 289

Hexose–rich hydrolyzate 210 253 365

Biomass: E. crassipes

Pentose–rich hydrolyzate 213 220 239

Hexose–rich hydrolyzate 207 244 366.5

Standard PHB 212.3 217.35 236.35

Abbreviations: TX% – temperature corresponding to X% weight loss
Graphical Abstract
RESEARCH HIGHLIGHTS

 Synthesis of PHB from two invasive weeds, viz. P. hysterophorus and E. crassipes

 Separate fermentation of pentose and hexose–rich hydrolyzates by Ralstonia eutropha

 PHB content of dry cell mass = 8.1–21.6% w/w

 PHB yield = 6.85×10–3 – 36.41×10–3 % w/w raw biomass

 Glass transition temp. = –9o to 9oC, Maximum thermal degradation temp. = 370o to 389oC