Bioresource Technology 257 (2018) 92–101

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

Engineering of artificial microbial consortia of Ralstonia eutropha and T

Bacillus subtilis for poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
copolymer production from sugarcane sugar without precursor feeding
Shashi Kant Bhatiaa,b, Jeong-Jun Yoonc, Hyun-Joong Kima, Ju Won Honga, Yoon Gi Honga,
⁎
Hun-Seok Songa, Yu-Mi Moona, Jong-Min Jeona, Yun-Gon Kimd, Yung-Hun Yanga,b,
a
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, South Korea
b
Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 05029, South Korea
c
Intelligent Sustainable Materials R&D Group, Korea Institute of Industrial Technology (KITECH), Chonan 31056, South Korea
d
Department of Chemical Engineering, Soongsil University, Seoul 06978, South Korea

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: Ralstonia eutropha is a well-known microbe reported for polyhydroxyalkonate (PHA) production, and unable to
Ralstonia eutropha utilize sucrose as carbon source. Two strains, Ralstonia eutropha H16 and Ralstonia eutropha 5119 were co-
Polyhydroxyalkonate cultured with sucrose hydrolyzing microbes (Bacillus subtilis and Bacillus amyloliquefaciens) for PHA production.
Co-culture Co-culture of B. subtilis:R. eutropha 5119 (BS:RE5) resulted in best PHA production (45% w/w dcw).
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
Optimization of the PHA production process components through response surface resulted in sucrose: NH4Cl:B.
Consortia
subtilis: R. eutropha (3.0:0.17:0.10:0.190). Along with the hydrolysis of sucrose, B. subtilis also ferments sugars
into organic acid (propionic acid), which acts as a precursor for HV monomer unit. Microbial consortia of BS:RE5
when cultured in optimized media led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P
(3HB-co-3HV) with 66% w/w of dcw having 16 mol% HV fraction. This co-culture strategy overcomes the need
for metabolic engineering of R. eutropha for sucrose utilization, and addition of precursor for copolymer pro-
duction.

⁎
Corresponding author at: Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, South Korea.
E-mail address: seokor@konkuk.ac.kr (Y.-H. Yang).

https://doi.org/10.1016/j.biortech.2018.02.056
Received 15 December 2017; Received in revised form 12 February 2018; Accepted 13 February 2018
Available online 15 February 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
S.K. Bhatia et al.
1. Introduction
Polyhydroxyalkonates (PHA) has immense industrial potential, as it
has many applications in tissue engineering, drug delivery, and
packaging (Bhatia et al., 2016a; Lim et al., 2017; Panith et al., 2016).
Various microbes are able to produce PHA under environmental and
nutritional stress conditions, utilizing different carbon sources (Chen
et al., 2017; Juengert et al., 2017; Vjayan and Vadivelu, 2017). Poly(3-
hydroxybutyrate) (P(3HB)) is the most common type of PHA naturally
accumulated by microbes, but has limited application, due to its ri-
gidity, brittleness, and low thermal stability (Czerniecka-Kubicka et al.,
2017; Obruca et al., 2016). Incorporation of various monomers in P
(3HB) can improve its physical properties, so production of copolymer
is a topic of current research interest. Copolymer P(3-hydroxybutyrate-
co-3-hydroxyvalerate) P(3HB-co-3HV) is superior in properties as
compared to P(3HB) due to its low melting point and level of crystal-
linity (Biernacki et al., 2017; Park et al., 2015). Different types of
polymers can be produced by using a precursor feeding approach, or
metabolic engineering of the microbe (Bhatia et al., 2015b; Srirangan
et al., 2016). Acetic acid promotes 3HB production, propionate, and
valerate plays a role in 3-hydroxyvalerate (3HV) synthesis in copolymer
P(3HB-co-3HV), whereas butyrate acts as a precursor for hydro-
xyhexonate (HHx) unit in P(3-hydroxybutyrate-co-3-hydro-
xyhexanoate) (P(3HB-co-3HHx)) (Bhatia et al., 2015a; Jeon et al., 2014,
2017). The addition of all these external precursors in fermentation
media adds to the cost.
Ralstonia eutropha is a well-known microbe for polyhydroxyalkonate
production with higher productivity, but has limited carbon source
utilization range (fructose, N-acetylglucosamine, gluconate, and a few
organic acids etc.), and able to produce only P(3HB) (Bhatia et al.,
2017b; Sichwart et al., 2011). Culture media components have greater
influence on PHA production cost, so we wanted to extend R. eutropha’s
capability to utilize other cheaper carbon sources, and produce copo-
lymer without addition of any precursor. Sucrose is a cheaper carbon
source, and readily available in nature, as it comprises 90% of the total
sugars in sugar cane (Zabed et al., 2014). However, wild type of R.
eutropha is not able to utilize sucrose as a carbon source (Park et al.,
2015). As an solution, sucrose hydrolysis into its free sugar (glucose and
fructose) can be achieved by using invertase enzyme, but have limita-
tion as it adds into cost when applied to large scale (Kehlbeck et al.,
2014). Use of microbial consortia can be an option where one microbial
strain can hydrolyze sucrose into free sugars, and these sugars further
utilized by R. eutropha 5119 for PHA production. There are many ex-
amples of mixed and co-culture techniques to improve carbon utiliza-
tion range and fermentation productivity (Bhatia et al., 2015c; Dwidar
et al., 2013; Morgan-Sagastume et al., 2015). Different types of wild
and engineered microbes can be co-cultured to construct natural, syn-
thetic, or semisynthetic microbial consortia (Bhatia et al., 2017a,d). Wu
et al. reported a microbial consortium for butanol production, where
one partner Bacillus cereus scavenges the available oxygen, and provides
a micro anaerobic environment for Corynebacterium acetobutylicum
growth and butanol production (Wu et al., 2016). To increase carbon
source utilization Xin et al. constructed a microbial consortium of
Kluyvera sp. strain OM3 and Clostridium sp. strain BOH3 where the first
strain hydrolyzes xylan and process resulted in increased production of
butanol (Xin and He, 2013).
In this study, we have demonstrated a novel approach for copolymer
P(3HB-co-3HV) production from sucrose without any precursor addi-
tion, by using a co-culture of PHA-accumulating R. eutropha 5119 and
sucrose-hydrolyzing Bacillus subtilis. B. subtilis hydrolyzes sucrose into
glucose and fructose, and also ferments these sugars into organic acids
(acetic acid and propionic acid). R. eutropha 5119 utilize free sugars and
organic acid for growth and P(3HB-co-3HV) production. Thus, this co-
culture process presents a new approach for copolymer production from
sucrose avoiding addition of costly precursors (propionic acid required
for 3HV monomer unit), and an enzyme (invertase) required for sucrose
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Bioresource Technology 257 (2018) 92–101
hydrolysis.
2. Material and methods
2.1. Chemicals
All the chemicals for media were purchased from BD Difco labora-
tories (Becton-Dickinson, Franklin Lakes, NJ, USA). Reagents for GC,
HPLC and GC–MS analysis (chloroform, methanol, and other deriva-
tizing agent) and fatty acid methyl esters (FAMEs) standard (RM-5 and
Rapeseed mix) were purchased from Sigma-Aldrich (St. Louis, MO,
USA).
2.2. Microorganism, media, and culture condition
Various microorganisms used in this study, i.e. Ralstonia eutropha
H16, Ralstonia eutropha 5119, Bacillus subtilis, and Bacillus amylolique-
faciens, were procured from the Korean Collection for Type Culture
(KCTC). All strains were maintained on LB agar plate, and stored at
4 °C. Luria Bertani (LB) was used as media to prepare seed culture. Seed
culture was prepared by inoculating a loop full of bacterial culture in
5 mL LB media, and incubating at 30 °C for overnight.
For PHA production, M9 minimal media, 5× (g/L, Na2HPO4 (33.9),
KH2PO4 (15), NH4Cl (5), NaCl (2.5) and sucrose (1%) was used. The
microbes were cultured individually (0.1 mL seed culture), and for the
consortia in combination of two, using 1:1 ratio seed culture, respec-
tively, thereby keeping the final inoculum volume constant (0.1 mL).
The microbes were cultured in 10 mL scale tubes having a total capacity
of 25 mL at 30 °C, with agitation at 160 rpm for 72 h, after which an
analysis was done for the PHA production and composition.
2.3. Analytical methods
Gas chromatography method with slight modification of the already
reported method was used to analyze the PHA production and com-
position (Braunegg et al., 1978). On completion of growth, microbial
culture was centrifuged at 10,000g to separate cells from the super-
natant. Cell pellets were washed with deionized water two times, sus-
pended in the minimum volume of deionized water, and subjected to
lyophilization overnight. For methanolysis, approximately 10 mg of
freeze-dried cells from each experiment were weighed, and placed in
Teflon-stoppered glass vials, and 1 mL chloroform and 1 mL methanol/
sulphuric acid (85: 15 v/v) were added to the vials. The vials were
heated at 105 °C for two hours for methanolysis reaction. On comple-
tion of the reaction, the vials were cooled to room temperature, and
0.5 mL distilled water was added. The reaction mixture was vertexed
for a few seconds, and allowed to separate into two layers. The lower
layer was carefully collected, and added into an eppendorf tube con-
taining Na2SO4. Samples were filtered by 0.22 mm Millex-GP syringe
filter unit, and subjected to GC analysis. A 2 µL portion of the organic
phase of these samples was then injected into a gas chromatograph
(Agilent, Santa Clara, CA) equipped with a fused silica capillary column
(Supelco SPB-5, 30 m × 0.32 mm, i.d. 0.25 µm film), with hydrogen as
the carrier gas. The inlet of the gas chromatograph was maintained at
250 °C. The oven was held at 80 °C for 5 min, heated to 220 °C at
20 °C min−1, and then held at 220 °C for 5 min. Peak detection was
performed by a flame ionization detector, which was maintained at
300 °C.
An HPLC system equipped with a Bio-Rad Aminex HPX-87H column
(Bio-Rad Co., Hercules, CA, USA) was used to analyze the components
of various sugars and metabolite (Bhatia et al., 2017c,b). A mobile
phase of 5 mM H2SO4 at a flow rate of 0.6 mL/min was used, and the
column temperature was maintained at 50 °C.
S.K. Bhatia et al.
2.4. Selection of microbes for consortia construction
Microbial consortia were constructed that had the potential to hy-
drolyze sucrose, ferment free sugars and accumulate PHA. While PHA-
producing microbe R. eutropha is unable to utilize sucrose, this strain
can be co-cultured with other sucrose-hydrolyzing compatible mi-
crobes. For sucrose hydrolysis, two bacterial strains, i.e., Bacillus subtilis
(BS) and Bacillus amyloliquefaciens (BA), were used. To utilize free su-
gars and produce PHA, a well reported fructose-utilizing R. eutropha
H16 (RE) and glucose/fructose-utilizing strain R. eutropha 5119 (RE5)
were used. All microbes were investigated individually (B. subtilis, B.
amyloliquefaciens, R. eutropha H16, and R. eutropha 5119), as well as in
co-culture form (B. subtilis:R. eutropha H16 (BS:RE), B. subtilis:R. eu-
tropha 5119 (BS:RE5), B. amyloliquefaciens:R. eutropha H16 (BA:RE),
and B. amyloliquefaciens:R. eutropha 5119 (BA:RE5)), to study sucrose
utilization and PHA accumulation. Preculture was prepared as men-
tioned in the above section, and minimal medium was used as pro-
duction media, with 1% sucrose as carbon source. Microbes were cul-
tured for 72 h, and on completion of growth, all samples were analyzed
for growth, PHA accumulation, and residual sugars. Microbial combi-
nations that showed better sucrose hydrolyzing and PHA accumulation
potential were used for further study.
2.5. Utilization of different carbon source by selected consortia
Sucrose is a disaccharide that is composed of glucose and fructose.
All these three sugars (sucrose, glucose, and fructose) were investigated
for biomass production and PHA accumulation. For consortium BS:RE5,
sucrose (1%) was used as carbon source, and for pure culture of R.
eutropha 5119, sucrose (1%), glucose (1%) and fructose (%) were used.
Microbes were cultured for 72 h at 30 °C. Samples were collected at 24 h
intervals, and analyzed for sugar consumption, microbial growth, PHA
production, and copolymer composition.
2.6. Response surface design for media optimization
The main objective of this study was to engineer a microbial con-
sortium that was able to hydrolyze and utilize sugarcane-derived su-
crose as a carbon source for PHA production. Sugar concentration, ni-
trogen source, and microbial ratio in the consortium are the main
factors that can affect biomass production and PHA production in a
microbial consortium. Sucrose, NH4Cl, B. subtilis, and R. eutropha 5119
are the four major components necessary for PHA production in con-
sortia. To find out the interaction effect of various factors, i.e. sucrose:
NH4Cl: B. subtilis: R. eutropha 5119 on consortia growth and PHA ac-
cumulation, a central composite experiment was designed using
Minitab 17. A thirty-one set of experiments was performed using var-
ious predicted combinations (Table 1). Experiments were performed at
10 mL scale in M9 medium using a stock concentration of sucrose
(20%), NH4Cl (10%), and B. subtilis and R. eutropha 5119 seed culture.
All the four components were added in the designed ratio. Regression
analysis using ANOVA was performed, and model fitting methods ap-
plied for data analysis. Contour surface plots were created to determine
the interaction effects of all the components on biomass production,
PHA accumulation, and its composition. The combination of predictor
settings that optimized the fitted response was used to verify the model.
2.6.1. Verification of the model of PHA production using sucrose
A numerical optimization method was applied to predict the value
of different variables of sucrose: NH4Cl: B. subtilis: R. eutropha 5119 for
the desired response. The high and low values were adjusted for all
responses using optimal parameter settings, as recommended by the
statistical software (Minitab 17) to obtain the most suitable responses.
Microbial consortia were cultured in M9 media with an optimized
concentration of sucrose:NH4Cl:B. subtilis:R. eutropha 5119
(3:0.17:0.10:0.19) at 10 mL scale. Consortia were cultured in the
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Bioresource Technology 257 (2018) 92–101
optimized media up to 72 h, and samples were collected at 24 h inter-
vals, and analyzed for biomass production and PHA accumulation.
2.7. Structure analysis of copolymer
For copolymer analysis after the completion of growth, the pro-
duction culture was centrifuged at 11,200g for 10 min, and cells were
suspended and washed with distilled water twice, then subjected to
lyophilization. Lyophilized cell (0.6g) powder was suspended in 20 mL
of chloroform and 20 mL of a sodium hypochlorite solution (30%, pH
12.15). The suspended culture was incubated at 30 °C for 150 min, and
after incubation, the mixture was centrifuged (4000×g; Hanil, Seoul,
Korea) for 20 min at 30 °C. Three distinct phases were formed after the
centrifugation. The bottom chloroform phase was carefully collected,
and the PHA was recovered by an (80% methanol) precipitation and
filtration method (Saratale and Oh, 2015). The resulting white pre-
cipitate was air dried, and used for 1H NMR study. For NMR analysis, 20
mg sample was dissolved in high purity deuterochloroform (CDCl3),
and NMR spectra were recorded by Bruker Avance II 500 spectrometry
(Bruker Co., Billerica, MA) at the National Center for Inter-University
Research Facilities (NCIRF), Seoul National University, Seoul, Korea.
The 1H spectra was observed at 500 Mhz at room temperature.
2.8. Effect of medium pH on PHA accumulation and organic acid (acetic
acid and propionic acid) content
R. eutropha is generally able to produce P(3HB) as the major
polymer, when cultured with simple sugars fructose and other carbon
source (Volova and Kalacheva, 2005). Use of BS:RE5 consortia with
sucrose results in copolymer P(3HB-co-3HV) production. Propionic acid
act as a main precursor for 3HV monomer unit, and by varying its
concentration content of 3HV fraction in P(3HB-co-3HV) copolymer can
be changed. Effect of medium pH was studied on consortia growth, PHA
accumulation, HV fraction and production of various organic acid. Pure
culture (BS) and consortia (BS:RE5) were cultured in optimized media
at different pH (5–9), and analyzed for biomass, PHA content and
various metabolites (acetic acid and propionic acid).
2.9. Microbial consortia compatibility and population dynamics
2.9.1. Compatibility test of microbes
Pure culture of B. subtilis and R. eutropha 5119 and consortia BS:RE5
were cultured in optimized media for 72 h at 30 °C, and observed for
growth. Samples from microbial consortia (BS:RE5) culture were col-
lected at intervals of 24 h, and subjected to different dilution for 24 h
(10–4), 48 h (10–6), and 72 h (10–8), and pour plated on LB agar plate.
Plates were incubated at 30 °C for 48 h. Both B. subtilis and R. eutropha
have different colored and morphology colony, and can be easily dif-
ferentiated.
2.9.2. PLFA analysis for population dynamics
Different microbial groups have different phospholipid-derived fatty
acids (PLFA), which can be used as a marker to study microbial com-
munities. The Gram-positive bacteria are represented by i15:0, a15:0,
i16:0, i17:0, and a17:0, and Gram-negative bacteria by the cyclopro-
pane fatty acids cy17:0, 16:1ω7, 18:1ω7, and 17:1ω9 (Moore-Kucera
and Dick, 2008). PLFA analysis can be used to study the population
dynamics of the consortia B. subtilis: R. eutropha 5119, as these are
Gram-positive and Gram-negative bacteria, respectively. PLFA extrac-
tion was performed using a 10 mL consortium culture, which was
centrifuged at 4000g for 10 min at 4 °C to remove the media compo-
nents, and the cell pellet was further washed with ion-free water two
times. The cell pellet was freeze-dried overnight, and further subjected
to PLFA extraction by adding 1 mL chloroform. The chloroform phase
was collected, reduced in volume by rotary evaporation, and fractioned
by chromatography on silicic acid (mesh size: 200–400). The neutral
S.K. Bhatia et al. Bioresource Technology 257 (2018) 92–101

Table 1
Experimental design points selected by the response surface methodology (RSM) using variables sucrose, NH4Cl, B. subtilis, R. eutropha 5119 and their subsequent effects on biomass (g
dcw/L), PHA production (g/L), growth yield coefficient (Yx/s g dcw/g sucrose), PHA yield coefficient (Yp/s g/g sucrose), PHA accumulation (% w/w of dcw), and HV mol%.

Sucrose NH4Cl B. subtilis R. eutropha Biomass PHA Yx/s Yp/s PHA HV

(%) (%) (% v/v) (% v/v) (g dcw/L) (g/L) (g/g) (g/g) (%) (mol%)

5 0.0 0.0 0.2 0.00 0.00 0.00 0.00 0.00 0.00

3 0.1 0.1 0.1 3.28 2.28 0.11 0.08 69.60 19.92
3 0.1 0.1 0.1 3.21 2.24 0.11 0.07 70.00 18.28
3 0.1 0.1 0.1 3.20 2.12 0.11 0.07 66.32 16.16
1 0.2 0.0 0.0 0.00 0.00 0.00 0.00 0.00 0.00
3 0.1 0.1 0.0 1.50 0.00 0.05 0.00 0.00 0.00
0 0.1 0.1 0.1 0.00 0.00 0.00 0.00 0.00 0.00
5 0.2 0.0 0.2 0.00 0.00 0.00 0.00 0.00 0.00
1 0.0 0.0 0.2 0.00 0.00 0.00 0.00 0.00 0.00
3 0.1 0.3 0.1 3.60 1.36 0.12 0.05 37.83 17.07
5 0.0 0.2 0.0 0.00 0.00 0.00 0.00 0.00 0.00
3 0.0 0.1 0.1 0.00 0.00 0.00 0.00 0.00 0.00
3 0.1 0.1 0.1 3.07 2.13 0.10 0.07 69.53 19.19
3 0.1 0.1 0.1 3.31 2.21 0.11 0.07 66.90 14.96
5 0.0 0.2 0.2 0.00 0.00 0.00 0.00 0.00 0.00
3 0.3 0.1 0.1 3.75 1.36 0.13 0.05 36.52 18.69
3 0.1 0.1 0.1 3.22 2.17 0.11 0.07 67.55 18.84
3 0.1 0.1 0.1 3.45 2.30 0.12 0.08 66.89 15.56
3 0.1 0.1 0.3 3.19 1.78 0.11 0.06 55.83 20.10
3 0.1 0.0 0.1 0.00 0.00 0.00 0.00 0.00 0.00
7 0.1 0.1 0.1 2.09 1.57 0.03 0.02 75.17 8.78
1 0.2 0.0 0.2 0.00 0.00 0.00 0.00 0.00 0.00
1 0.0 0.2 0.2 0.00 0.00 0.00 0.00 0.00 0.00
1 0.2 0.2 0.2 2.89 1.37 0.30 0.14 47.45 18.16
5 0.2 0.2 0.0 1.20 0.00 0.02 0.00 0.00 0.00
5 0.0 0.0 0.0 0.00 0.00 0.00 0.00 0.00 0.00
5 0.2 0.2 0.2 3.67 1.50 0.07 0.03 40.88 10.82
1 0.2 0.2 0.0 1.40 0.00 0.14 0.00 0.00 0.00
5 0.2 0.0 0.0 0.00 0.00 0.00 0.00 0.00 0.00
1 0.0 0.0 0.0 0.00 0.00 0.00 0.00 0.00 0.00
1 0.0 0.2 0.0 0.00 0.00 0.00 0.00 0.00 0.00

lipids and glycolipids were eluted with chloroform (5 mL) and acetone
(5 mL), respectively, and discarded. The polar lipids were eluted with
(5 mL) methanol, and collected in glass test tubes. The solvent was
evaporated under a stream of oxygen-free nitrogen, and the residue
subjected to alkaline methanolysis by vortexing for 30 s in a mixture
(2 mL) of 0.56% (w/v) KOH in dried methanol (BDH) and a toluene/
methanol solution [1:1 (v/v)], followed by incubation at 37 °C for
30 min. After cooling, the mixtures were neutralized with 0.3 mL of 1 M
acetic acid, and fatty acid methyl esters (FAMEs) were extracted using 2
mL of hexane and water. The upper layer was collected, and after
evaporation of the solvent under oxygen-free nitrogen, the FAMEs were
resuspended in chloroform. The FAMEs were quantified by GC–MS, as
mentioned in the above Section. The ratio of the B. subtilis and R. eu-
tropha 5119 was calculated from the PLFA marker i.e. hexadecanoic
acid, 15-methyl, methyl ester, and cyclopropaneoctonoic acid, 2-hexyl-,
methyl esters concentration, which were present in the B. subtilis and R.
eutropha 5119, respectively, and the standard used was a corresponding
pure culture PLFA amount of B. subtilis and R. eutropha 5119.
3. Results and discussion
3.1. Engineering of synthetic microbial consortia
Different microbes, individually, as well as in combination, were
screened for PHA production, using sucrose as carbon source. When
cultured individually, B. subtilis and B. amyloliquefaciens were able to
utilize sucrose as a carbon source, and no PHA accumulation was ob-
served, as these strains have no capability to accumulate PHA. Low
biomass production was observed in the case of B. subtilis, while B.
amyloliquefaciens grew rapidly, and higher biomass production was
observed (Fig. 1 (a)). No pure culture of either R. eutropha H16 or R.
eutropha 5119 was able to grow, as these have no capability to hydro-
lyze sucrose, and no free sugars were observed in their broth. On the
other hand, all four types of consortia were able to utilize sucrose for
growth and PHA accumulation. Maximum biomass production was
observed in consortium BA:RE5 (2.65 g dcw/L), followed by the other
consortia in the order BS:RE5 > BA:RE > BS:RE (Fig. 1 (a)). No PHA
production was analyzed in the case of pure culture, as neither Bacillus
strain is able to accumulate PHA, and both R. eutropha strains failed to
grow, as these were not able to utilize sucrose. Maximum PHA pro-
duction was observed in the case of consortium BS:RE5 (45% w/w of
dcw), followed by the other consortia in the order BA:RE5 > BS:RE >
BA:RE (Fig. 1 (b)). The accumulated PHA in all consortia was identified
as copolymer P(3HB-co-3HV) using GC analysis.
In residual sugar analysis, 1.8 and 0.7 g/L residual sucrose was
observed in the fermented broth of B. subtilis and B. amyloliquefaciens,
respectively (Fig. 2). There was an accumulation of glucose (2.7 g/L)
and fructose (3.8 g/L) recorded in the case of B. subtilis, while in the
case of B. amyloliquefaciens, 2.1 and 3.3 g/L glucose and fructose ac-
cumulation was observed, respectively (Fig. 2). In the case of R. eu-
tropha H16 and R. eutropha 5119, no free sugars were analyzed, as these
strains are not able to hydrolyze sucrose. In consortia of B. subtilis with
R. eutropha and R. eutropha 5119, sucrose was hydrolyzed with a higher
rate in BS:RE5 as compared to BS:RE, as only 1.1 and 0.7 g/L residual
sucrose was observed, respectively. Similarly, in consortium BA:RE5,
the sucrose hydrolysis rate was higher than in consortium BA:RE. In
consortia BS:RE and BA:RE broth, more glucose accumulated, as com-
pared to BS:RE5 and BA:RE5, as R. eutropha H16 strain is not able to
utilize glucose. While in the case of BS:RE5 and BA:RE5, decreases in
glucose level indicate that R. eutropha 5119 strain is able to utilize
glucose along with fructose as a carbon source (Fig. 2). This study
shows B. amyloliquefaciens is able to perform sucrose hydrolysis with a
higher rate, but decrease in free sugar content in its broth shows it
ability to consume free sugars more rapidly. These results suggest that
B. subtilis is a more appropriate microbe to construct consortia in
comparison with B. amyloliquefaciens, as it has higher sucrose

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S.K. Bhatia et al.
Fig. 1. Engineering of synthetic microbial consortia for PHA production, (a) growth and biomass production in pure culture and microbial consortia on sucrose, and (b) PHA production
in pure culture and consortia.
hydrolyzing activity, and low consumption rate of free sugars. R. eu-
tropha 5119 is able to utilize both glucose and fructose as carbon
source, and its consortium with B. subtilis, i.e. BS:RE5, proved the best
consortium for PHA production from sucrose. Consortium BS:RE5 re-
sulted in optimal biomass and maximum PHA production, and was used
for further experiments.
3.2. Utilization of different carbon source
The carbon source utilization capacity of R. eutropha 5119 was
studied against sucrose, glucose, and fructose, while consortium BS:RE5
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Bioresource Technology 257 (2018) 92–101
was studied against sucrose. R. eutropha 5119 is unable to utilize su-
crose and failed to grow, while it can utilize glucose and fructose effi-
ciently as a carbon source for growth and PHA accumulation. Biomass
production kept on increasing up to 72 h, and after that, little change in
biomass production was observed, in the case of glucose and fructose.
Maximum biomass production, i.e. 2.82 and 2.66 g dcw/L was observed
at 72 h in glucose and fructose, respectively (Fig. 3 (a)). In the case of
BS:RE5 consortium, where B. subtilis hydrolyzes sucrose into glucose
and fructose, which are further utilized by both bacteria, maximum
biomass (2.68 g dcw/L) production was recorded at 72 h. PHA pro-
duction is associated with biomass, and kept on increasing up to 72 h;
S.K. Bhatia et al. Bioresource Technology 257 (2018) 92–101

Fig. 2. Sucrose hydrolysis potential of pure culture and different consortia. The figure
shows the residual sucrose after fermentation, and free sugars (glucose and fructose)
accumulated during the fermentation.

after that, almost no significant increase in PHA content was recorded

in both glucose (60.8% w/w of dcw) and fructose (62.6% w/w of dcw).
In the case of consortium BS:RE5, 55% w/w of dcw PHA content was
recorded at 72 h (Fig. 3 (b)). These results showed that sucrose can be
used as carbon source, as almost the same amounts of PHA accumula-
tion and productivity were recorded using sucrose, in comparison to
glucose and fructose. In the case of pure culture, the PHA accumulated
is composed of hydroxybutyrate (HB) as the major component, having
HV content of less than 5 mol%, while the case of consortium culture
PHA copolymer, i.e. poly(3HB-co-3HV) has a HV fraction of about 13
mol% (Fig. 3 (c)).

3.3. Response surface design for media optimization

PHA accumulation and its composition are affected by the carbon

and nitrogen source concentrations (Cui et al., 2017). To determine the
correlation effect of sucrose, NH4Cl, B. subtilis, and R. eutropha 5119 on
PHA production, response surface design using Minitab 17 was per-
formed. Consortia BS:RE5 was grown for 72 h in M9 media, with ad-
ditional components (sucrose and NH4Cl) in different ratios (Table 1).
On completion of growth, samples were analyzed for biomass produc-
tion, PHA accumulation, and its composition.
The contour plots of sucrose, NH4Cl, BS and RE5 were generated,
using Minitab 17 modelling software to study the interaction effect of
various responses. Analysis of variance (ANOVA) was applied on var-
ious responses, and a low p value (< 0.05) indicates the significance of
the designed model. The correlation coefficient (r) and adjusted coef-
ficient (R2) value were > 94%, which showed the high significance of
the experiments. The contour plot analysis indicates that all the com-
ponents have a role in biomass production (Fig. 4 (a)), while sucrose Fig. 3. Growth profile (a) PHA production, (b) 3HV fraction, (c) analysis of R. eutropha
and ammonia require optimum concentration for biomass production, 5119 cultured on sucrose, glucose and fructose, and consortium BS:RE5 on sucrose.
above and below which decrease in biomass production was recorded.
PHA production goes on increasing up to a certain concentration of was recorded as 0.3 g dcw/g sucrose and 0.14 g PHA/g sucrose, re-
ammonia and sucrose, then starts to decrease (Fig. 4 (b)). B. subtilis and spectively at sucrose:NH4Cl:BS:RE5 (1:0.2:0.2:0.2). Yx/s increase as the
R. eutropha 5119 concentrations play important roles; in the absence of concentration of carbon and nitrogen source increase together, while
B. subtilis, no PHA production was observed, as R. eutropha 5119 alone Yp/s increase with carbon source concentration, but increase in nitrogen
was not able to utilize sucrose; similarly, at higher B. subtilis con- concentration has a negative effect on PHA accumulation. The HV
centration, decrease in PHA content was observed, as most of the sugars fraction of copolymer P(3HB-co-3HV) was also affected by various
were utilized by it. The carbon source concentration had a significant factors, and it varies in the range (0–21) mol% under different sets of
effect on the growth yield coefficient (Yx/s) and PHA yield coefficient conditions (Fig. 4 (c), Table 1). Based on the results from the
(Yp/s) as showed in the table. 1. The maximum value of Yx/s and Yp/s

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S.K. Bhatia et al. Bioresource Technology 257 (2018) 92–101

Fig. 4. Contour plots for the interactive effect of sucrose: NH4Cl: B. subtilis: R. eutropha 5119 (a) biomass production (g dcw/L), and (b) PHA content (% w/w of dcw) and 3HV fraction
(mol%).

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S.K. Bhatia et al.
Fig. 5. Effect of medium pH on (a) B. subtilis organic acid (acetic acid and propionic acid) production, and (b) consortia BS: RE5 growth, PHA accumulation and 3HV fraction.
sucrose:NH4Cl:B. subtilis:R. eutropha 5119 response surface metho-
dology analysis experiments, it was clear that along with sucrose and
nitrogen source, the ratio of B. subtilis to R. eutropha 5119 is important
to produce the copolymer of desired composition.
For maximum biomass production and PHA production with op-
timum HV fraction, a numerical optimization method in Minitab 17 was
applied to predict the value of different variables for sucrose, NH4Cl, B.
subtilis, and R. eutropha 5119. To verify the design, we chose a model to
produce biomass (3.5 g dcw/L) with PHA content (65% w/w of dcw)
and HV content (15 mol%). The composite desirability (D) and in-
dividual desirability (d) values were close to 1.000, which confirms the
suitability of the model. The optimum value for sucrose: NH4Cl: BS: RE5
was predicted as (3.0:0.17:0.1:0.19)%. The results obtained using pre-
dicted response, verify the model with the highest degree of accuracy
(> 90%), and indicates that the model is valid under the tested con-
ditions. Experiments were performed using the predicted values of
various responses, and biomass production (3.7 g dcw/L), with PHA
content 63% w/w of dcw, and HV fraction 16 mol% was achieved. Park
et al. engineered R. eutropha for sucrose utilization by expressing b-
fructofuranosidas (sacC) from Mannheimia succiniciproducens MBEL55E,
and used this strain for copolymer poly(3-hydroxybutyrate-co-lactate)
[P(3HB-co-LA) production (Park et al., 2015). The polymer extracted
from the consortium BS:RE5 was analyzed for its chemical structure
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Bioresource Technology 257 (2018) 92–101
using 1H NMR and confirmed as copolymer P(3HB-co-3HV).
3.4. pH effect on copolymer production and organic acid (acetic acid and
propionic acid) content
Medium pH affects the microbial growth, PHA accumulation and
propionic acid to acetic acid ratio (Seshadri and Mukhopadhyay, 1993).
The ratio of propionic acid and acetic acid was higher at lower pH
(4.4:1), but propionic acid productivity was lower (0.14 g/L). Above pH
6, propionic acid to acetic acid ratio decrease (1.1:1), and higher pro-
pionic acid productivity was observed (0.21 g/L). pH 7 was observed as
optimum pH for propionic acid (0.2 g/L) and acetic acid (0.28 g/L)
production in BS culture (Fig. 5a). Consortia BS:RE5 able to grow at pH
(5–9) and maximum growth was achieved at pH 7 i.e. 3.5 g dcw/L
(Fig. 5b). PHA content increased with the increase of pH and maximum
PHA accumulation (66% w/w of dcw) was recorded at pH 7. Maximum
3HV mol% was observed at pH 5 (23%), and it decreases with the
further increase of pH. Optimum pH for consortia BS:RE5 growth (3.5 g
dcw/L) was pH 7, as maximum PHA accumulation (66%) with 16 mol%
3HV content was recorded at this pH. This co-culture approach results
in P(3HB-co-3HV) production from sucrose without addition of any
precursor, while to produce copolymer P(3HB-co-3HV), other re-
searchers used engineered E. coli strain fed with propionate as precursor
S.K. Bhatia et al. Bioresource Technology 257 (2018) 92–101

Fig. 6. Consortium population dynamics of BS:RE5 using PLFA: (a) change in B. subtilis and R. eutropha 5119 population with time course, (b) PLFA marker of B. subtilis, and (c) PLFA
marker of R. eutropha 5119 used for population dynamics study.

for HV monomer unit (Bhatia et al., 2015a; Jeon et al., 2017). 2002). Consortium BS:RE5 was cultured for 72 h, and samples were
collected at 24 h intervals. For population study in consortium BS:RE5,
PLFA marker hexadecanoic acid, 15-methyl, methyl ester, and cyclo-
3.5. Microbes compatibility and consortia population dynamics
propaneoctonoic acid, 2-hexyl-, methyl esters were selected as PLFA
markers for B. subtilis and R. eutropha 5119, respectively. The B. subtilis
Pure culture of R. eutropha 5119 did not show any growth in opti-
and R. eutropha ratio in consortium BS:RE5 was calculated using the
mized media, as it was unable to utilize glucose; B. subtilis showed
PLFA profile of pure culture (Fig. 6). The pure culture of B. subtilis has
growth in optimized media, while a tremendous increase in the growth
hexadecanoic acid, 15-methyl, methyl ester as PLFA marker (20 µg/g
of BS:RE5 consortium was observed. The success of a microbial con-
dcw), while R. eutropha 5119 has cyclopropaneoctonoic acid, 2-hexyl-,
sortium depends on the compatibility of microbes to grow together, and
methyl esters PLFA marker (31 µg/g dcw). At 24 h, the B. subtilis and R.
their interaction with each other. Microbial consortium BS:RE5 samples
eutropha ratio was 83:17, which at 48 h was changed to 33:67. The final
were collected at intervals of 24 h, and pour plated onto agar plate. B.
ratio at 72 h was analyzed as 15:85 in consortium BS:RE5 (Fig. 6). PLFA
subtilis and R. eutropha 5119 were found as highly compatible with each
analysis results supported the dilution plate method results, as both
other, and grew together without affecting each other’s growth, as there
studies showed that on starting, B. subtilis was the dominant microbe,
was no zone of inhibition observed on the plate. The pour plate dilution
and then as the free sugars became available, R. eutropha 5119 grew
study showed that at 24 h, B. subtilis was the dominant microbe, as it is
rapidly, and become dominant in the consortium.
able to use sucrose as carbon source; thereafter, as free sugars become
available, R. eutropha 5119 started to grow, and became the dominant
organism at 48 h and 72 h. 4.0. Conclusion
The population dynamics of microbial consortium BS:RE5 was stu-
died using PLFA marker, as this method was previously used for the R. eutropha 5119 is not able to utilize sucrose, and this study de-
community profiling of microbes (Bhatia et al., 2015c; Wilkinson et al., monstrates that co-culture of R. eutropha 5119 with B. subtilis can be

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used for PHA production from sucrose. Along with sucrose utilization,
this co-culture system also offers the advantage of copolymer P(3HB-co-
3HV) production, as the precursor required (propionate) for its
monomer unit HV is provided by B. subtilis. The consortium culture
technique can be further extended to utilize other carbon sources, and
to produce copolymers of varying composition. There is a need to de-
velop a technology that can help to understand the compatibility of
various microbes, so different consortia of commercial importance can
be constructed.
Acknowledgement
The authors acknowledge the KU Research Professor Program of
Konkuk University, Seoul, South Korea, for providing financial support
to Dr. Shashi Kant Bhatia. This study was also supported by the
National Research Foundation of Korea (NRF), funded by the Ministry
of Education (NRF-2015M1A5A1037196, 2017R1D1A1B03030766),
Research Program to solve social issues of the National Research
Foundation of Korea (NRF) funded by the Ministry of Science and ICT
(2017M3A9E4077234,), and Korea Institute of Energy Technology
Evaluation and Planning (KETEP), and the Ministry of Trade, Industry &
Energy (MOTIE) of the Republic of Korea (No. 20163010092150).
Consulting service from the Microbial Carbohydrate Resource Bank
(MCRB, Seoul, Korea) was kindly appreciated.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.biortech.2018.02.056.
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