Introduction
Plastics
have become an important component of our daily lives (Butbunchu and
Pathom-Aree 2019). They are made up of petrochemicals that are highly toxic and
known to persist in the environment for longer time. Plastic pollution is
increasing day by day. According to a UN Environment report (2018), nearly 200
million tonnes of plastic is being produced annually, which is approximately
equivalent to the weight of entire human population currently existing on the
planet earth. At the moment 9% of the plastic is recycled, 12% is incinerated
while 79% of it is dumped in a landfill or natural ecosystem (Geyer et al. 2017). One million plastic
drinking bottles are purchased every minute worldwide (Nace 2017). The
WWF-Pakistan started a campaign “Beat Plastic Pollution” with a focus of
creating awareness in public especially about using alternative to single-use
plastic for promoting green globe (The News International 2018). Plastic in the
marine environment exists in different forms as microplastic
(0.05–0.5 cm), mesoplastic (0.5–5 cm), macroplastic (5–50 cm) and megaplastic
(above 50 cm) which usually floats on the surface of sea until shredded down to
macroplastic (Bettler et al. 2017).
All these forms of plastic effectively pollute our biosphere by contaminating
the marine ecosystem, destroying fish population, entering the food chain,
clogging sewers, and becoming breeding places for mosquitoes (The News
International 2018). If current trend is not controlled, it may invade our
oceans more than fish by 2050 (Geyer et
al. 2017). Many countries are making policies to ban the use of plastics.
Scientists are making efforts to search suitable substitutes to replace
synthetic plastics (Jain and Tiwari 2015; Munir et al.
2015). Traditional plastics such as polypropylene (PP) and
polyethylene (PE) are in routine use due to their durability and elasticity.
Microorganisms including bacteria are known to produce
bioplastics or biodegradable plastics called as polyhydroxybutyrate (PHB). PHB
belongs to polyhydroxyalkanoate (PHA), a class of biopolymer which is polyester
in nature (Chaber et al. 2017). These
biopolymers are accumulated as intracellular storage granules inside the
bacteria. They have many advantages over traditional plastics as
biodegradability, non-toxicity and better physical as well as mechanical
properties (Bhagowati et al. 2015).
Many microorganisms are known to produce PHB including Halomonas (Hertadi et al.
2017), Pseudomonas, Bacillus and Alcaligenes species (Rehman et
al. 2015). It is reported that PHB accumulation inside cells is dependent
on carbon source (Bhagowati et al.
2015). The efforts for commercialization of PHB had been in focus since 1950s
when North-American Company W. R. Grace Co. marketed it (Barrett 2018). It has
been in the market by different names as Biopol®, Nodax®, Biogreen® and Biomer®
by different companies as Zeneca, Monsanto, Metabolix, Inc. (U.S.A.). Low
production efficiency of microbial strains, lack of suitable purification
method and high production cost (Singh et
al. 2011; Ivanov et al. 2015;
Munir et al. 2015) have limited its
industrial scale production. The 50% cost of substrate for PHB production makes
a difference of 12 times the cost of PP plastic (Castilho et al. 2009; Bhuwal et al.
2014). Thus, there is a high demand to search cost-effective substrate for PHB
production. Organic wastes consisting of fruit and vegetable left-overs are
generated all around the world on daily basis. Instead of dumping them in the
environment, their suitable use should be explored (Singh et al. 2013). This study includes the isolation, molecular
characterization of PHB producing bacterial strain from local
organic-contaminated environment and optimization of its complete PHB
(extracellular as well as intracellular) profile using agro-waste corncob.
Materials and Methods
Sample collection, isolation and purification of
bacterial strains and subsequent treatments
Soil
samples were collected from garbage of fruits and local vegetable market. The
temperature of the samples was 37°C whereas the pH ranged from 6 to 7.5.
Standard microbiological methods were used to isolate and purify bacterial
colonies from them. For this purpose, Luria Bertani (LB) agar medium (peptone
10 g, yeast extract 5 g, NaCl 5 g, agar 15 g and final volume made 1000 mL with
distilled water) was used. The cultural and morphological characterization of
bacterial colonies obtained was performed using colony morphology, Gram
staining and biochemical characterization (Cheesebrough 2001). All the
experiments were performed in triplicate following completely randomized design
using LB agar medium and incubation conditions of 37°C for 18–24 h.
Qualitative screening by staining methods
All the
bacterial colonies obtained (15) were screened for their PHBs production. On
the basis of results, only one bacterial colony was selected which showed
maximum PHB production. Further experiments were performed on the selected
bacterial strain.
Sudan black B staining: Fresh bacterial culture was used to prepare a smear
which was heat fixed on a glass slide. It was stained with Sudan black B (stain
was prepared by dissolving 0.3 g Sudan black B dye in 70% ethyl alcohol),
air-dried followed by washing with alcohol. Finally, 2–3 drops of
safranin were placed on the smear and observed under oil immersion. Lipid
granules confirming PHB granules were stained black (Collee et al. 1989).
Carbol fuchsin staining: Intracellular PHB was detected by this staining
technique. Fresh bacterial culture was heat fixed on a glass slide. It was
stained with carbol fuchsin stain for 45 sec. Dark color granules showed the
presence of intracellular PHB (Aneja 2001).
Nile blue A method: It was done
by the method of Spiekermann et al.
(1999). Nutrient agar medium (per 100 mL: beef extract 1 g, peptone 0.5 g, sodium
chloride 0.8 g, glucose 1 g and agar 1.5 g) plates were prepared containing 0.5
µg/mL Nile blue A reagent. The bacterial isolates were streaked on it
and incubated at 37°C for 24 h. The plates were observed on a UV illuminator
for orange fluorescence.
Quantitative analysis of PHB
Sodium dodecyl sulfate (SDS) method: It was used for the extraction of extracellular PHB.
Fresh culture was inoculated in 10 mL of LB broth and incubated at 37°C for 24
h. After 24 h, it was centrifuged at full speed for 15 min at room temperature.
The supernatant was shifted to a beaker while the pellet was air-dried, weighed
and stored in a refrigerator for next step. The SDS (5 g) was added to the
supernatant. It was mixed well in the form of a paste, followed by incubation
at 37°C for 1 h and heating at 121°C for 15 min. As it cooled down to room
temperature, it was centrifuged for 15 min at full speed. The upper layer was
shifted to a clean glass Petri plate and kept overnight at room temperature
(Kim et al. 2003).
Sodium hypochlorite and chloroform method: This method was used for the extraction of intracellular
PHB. In the pellet (saved in above mentioned step), chloroform and sodium
hypochlorite were added each as 13 µL of the pellet weight and mixed
gently by a micropipette. The mixture turned milky which was kept overnight at
room temperature. Next day, it was centrifuged at full speed for 15 min at room
temperature which resulted in three layers; the first was of aqueous
Na-hypochlorite, the second of cell debris and third layer of chloroform
containing PHB. The chloroform containing solution was further used by
pipetting into new eppendorf. In it, a 5X mixture of 70% methanol and 30% water
was added, mixed, centrifuged at full speed for 15 min and the chloroform was
allowed to evaporate at room temperature. The left-over pellet was of PHB.
Sulfuric acid was added to the pellet that converted it into crotonic acid,
which appeared as a brown colored precipitate (Chang et al. 1994; Singh and Parmar 2011).
Optimization of selected bacterial strain
Optimum
growth conditions of the selected bacterial strain were carried out using
different temperatures, pH and carbon sources.
Optimum temperature: For
determining it, 1% fresh inoculum was added to 10 mL autoclaved LB followed by
incubation at various temperatures (30ºC, 40ºC, 50ºC, 70ºC and 90ºC) for 24 h.
Optical density was read at 585 nm.
Optimum pH: For it,
the experiment was carried out at different pHs (5, 6, 7, 8 and 9). The LB
broth medium was prepared in different beakers and its pH was adjusted with HCl
or NaOH. The medium was dispersed into test tubes (triplicates), 10 mL each,
and autoclaved. Afterwards, 1% inoculum was taken and incubated at 37ºC. The
next day, OD was recorded at 585 nm.
Optimum carbon source: Various
carbon sources used were: glucose, sucrose, maltose, lactose and fructose. In
10 mL of the LB broth, 1% inoculum and 2% carbon source were added and then the
mixture was incubated at 37ºC for 24 h. Next day, OD was read at 585 nm.
Optimization of PHB production
Optimization
of PHB production was carried out using different organic wastes (orange peels,
banana peels, corncob, sugarcane left-over, potato peels and mixture of all
these). Glucose was used as a control.
Preparation of waste extracts: First of all, the respective material was taken, washed
with tap water, air-dried and cut into small pieces. They were boiled in 1000 mL
distilled water till one fourth of water was left behind. This leftover
solution was filtered and poured in clean sterilized glass bottle and placed in a refrigerator for further experimentation.
Optimization experiment: In 100 mL sterilized LB broth, 1% inoculum was added,
followed by addition of 2% agro-waste extract in each of properly labeled
flasks. It was incubated at 37°C for 24 h. The next day, 10 mL culture was
taken from it. The SDS and chloroform methods were performed. About 1 mL of the
same culture was used for protein profiling by SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) (Laemmli 1970).
Characterization of extracellular and intracellular PHB
Light microscopy: The
extracellular and intracellular PHBs were taken on a clean glass slide, stained
with crystal violet, and observed under 40 X.
Optical microscopy: For it,
extracellular and intracellular PHB samples were
mixed with chloroform separately. The mixture was
spread on a clean Petri dish and air-dried. It was observed using an optical
microscope.
Fourier transform infrared spectrophotometry (FTIR): The method described by Bhuwal et al. (2014) was followed with slight modifications. The purified
PHBs (2 mg both PHBs) were mixed with KBr separately and dried.
This mixture was subjected to FTIR spectrum using a Fourier Transform IR
spectrophotometer (Shimadzu, Japan).
Thermal gravimetric analysis (TGA): It was performed using 10 mg of PHBs in TGA instrument
(SDT Q600 V8.2 Build 100) calibrated with indium. The temperature was ramped at
a heating rate of 10°C/ min in air to a temperature (600°C) well above the
degradation temperature of the polymer (Bhuwal et al. 2014).
Differential scanning calorimetric (DSC) analysis: For it, 10 mg of extracellular and intracellular PHB
were used in standard aluminium pans. The experiments were performed using air.
The samples were heated, and temperature was increased at the rate of 10°C/min
(Bhuwal et al. 2014).
Molecular characterization of isolated strain
The 16S
rRNA sequencing of the selected bacterial strain was got performed from
Macrogen® South Korea.
Statistical analysis
All
experiments were run in triplicate following completely randomized design.
Three reading were taken, their mean and standard error of the means were
calculated. The significance of the data (P
≤ 0.05) was checked using SPSS v.17.0.
Results
Isolation, screening and selection of PHB producing
bacterial strains
Of 10 soil
samples, 35 bacterial isolates were obtained. The staining methods revealed
only six isolates positive for PHB. The bacterial isolate, named SS-1.9, was
selected as the best producer of PHB out of six isolates on the basis of
results obtained from the SDS and chloroform methods. The colonies showed
irregular, flat elevation, creamy shiny center but transparent at borders which
were irregular (rhizoidal). According to morphological, biochemical and
molecular characterization, SS-1.9 was found to be Bacillus licheniformis. The sequence was submitted to the Table 1: Quantification of total, extracellular and intracellular PHB of B. licheniformis-MK656314
|
Features |
DCW (g/L) |
PHB accumulation (%) |
|
Total |
22.31 |
59.83 |
|
Extracellular PHB |
9.87 |
44.24 |
|
Intracellular PHB |
3.48 |
15.59 |
Table 2: Extracellular and
intracellular PHB yield per 10 mL bacterial culture using different organic
wastes
|
Sr. No. |
Organic wastes utilized |
PHB obtained (weight in grams per 10 mL
culture) |
|
|
Extracellular |
Intracellular |
||
|
1. |
Orange peels |
3.028 |
0.123 |
|
2. |
Potato peels |
1.428 |
0.065 |
|
3. |
Banana peels |
2.710 |
0.171 |
|
4. |
Sugar-cane leftover |
2.670 |
0.209 |
|
5. |
Corncob |
7.909 |
0.524 |
|
6. |
Mixture of all wastes |
1.995 |
0.188 |
|
7. |
Glucose |
1.738 |
0.150 |
NCBI
GenBank to obtain accession number which was registered as MK656314.
Quantification of PHB
The
quantification of extracellular and intracellular B. licheniformis per dry cell weight (DCW) in g/L is given in Table
1.
Optimization of growth conditions
The optimum
growth conditions of B. licheniformis-MK656314
were found to be 40°C, pH 7 and glucose as carbon source.
Optimization of PHB production
Of various
organic wastes used, corncob was found to be an optimum carbon source for
extracellular as well as intracellular PHB production (Fig. 1a–b and 2 and Table
2).
Protein studies
Different
organic wastes caused the expression of different proteins by B. licheniformis. Their expression
patterns as observed using SDS-PAGE are shown in Fig. 3.
Characterization of extracellular and intracellular PHB
Light microscopy
The
extracellular and intracellular PHB appeared as black structures in light
microscopy (Fig. 4a–b).
Optical microscopy
The surface
of extracellular and intracellular PHBs appeared porous (Fig. 4c–d).
FTIR
%2035-42_files/image002.png)
Fig. 1: Optical density
showing the growth of cells at 587 nm and extracellular and intracellular PHB
production (%) in the presence of (a)
glucose, and (b) corncob as carbon
sources
%2035-42_files/image004.png)
Fig. 2: Comparison of extracellular and intracellular PHB
accumulation (%) in the presence of glucose and corncob
The
spectrums of both extracellular and intracellular PHB are shown in Fig. 5. For
characterization of PHB, the FTIR spectrum of extracellular PHB showed the
presence of peaks at 3446.08 cm-1, 2916.58 cm-1, 1656.29
cm-1, 1467.63 cm-1 and 1216.85 cm-1 which
correspond to hydroxyl (-OH) stretching, aliphatic (C-H) stretching, carboxylic
(C=O) stretching, methyl (CH3) stretching and again aliphatic (C-H)
stretching, respectively. The FTIR analysis of B. licheniformis intracellular PHB showed the peaks at 3364.97 cm-1,
2923.92 cm-1, 1633.77 cm-1 and 1077.81 cm-1
corresponding to hydroxyl (–OH) stretching, ester (CHO) stretching, carboxylic
(C=O) stretching and again ester (CHO) stretching, respectively.
TGA/DSC
%2035-42_files/image006.png)
Fig. 3: Protein bands of B. licheniformis-MK656314 when grown in
the presence of different organic wastes. The labeling of the wells is as
follows: 1 = orange peel, 2 = potato peel, 3 = banana peel, 4 = mixture of all
wastes, 5 = glucose, 6 = corncob, 7 = sugarcane and 8 = protein ladder
%2035-42_files/image008.png)
Fig. 4: Light microscopy of (a)
extracellular, and (b) intracellular
PHB. Optical microscopy of (c)
extracellular and (d) intracellular
PHB
The TGA of
the sample was performed to check its thermal stability at heating rate of 10°C
per min in the temperature range of 0–600°C. The results (Fig. 6) showed that the
decomposition of the sample started at 25°C and continued till 200°C. During
this initial decomposition process, weight loss occurred in extracellular and
intracellular PHB, which may have been due to loss of water and gaseous
components from the sample. Extracellular PHB shows little stability up to
214.64°C and then weight loss occurred. At 299.24°C, the amount of the sample
remained was 37.33%. As far as intracellular PHB was
concerned, it showed little stability up to 171.07°C. Finally, at 290.16°C, the
sample amount remained 58.12% by weight (Fig. 6).
Similarly, DSC of both samples showed a positive heat flow during sample
decomposition, which represents endothermic nature of the decomposition reaction.
Tg, Tc and Tm of both PHBs are given in Table 3.
%2035-42_files/image010.png)
Fig. 5: FTIR spectra of
extracellular and intracellular PHB of B.
licheniformis-MK656314
%2035-42_files/image012.png)
Fig. 6: DSC-TGA thermograms of extracellular and intracellular
PHB of B. licheniformis- MK656314
Table 3: Differential
scanning calorimetric (DSC) properties of extracellular and intracellular PHBs
|
Properties |
Tg (°C) |
Tc (°C) |
Tm (°C) |
|
Extracellular PHB |
102.2 |
194.72 |
355.08 |
|
Intracellular PHB |
170.0 |
80.0 |
207.1 |
%2035-42_files/image014.png)
Fig. 7: Hypothetical model of secretion of extracellular PHB
outside cytosol after being synthesized intracellularly
Discussion
Bacterial
plastics as an alternative to petroleum-based plastics are studied by different
researchers (Arikan and Bilgen 2019). PHB producing B. licheniformis was successfully isolated from local environment.
The 22.311 g/L B. licheniformis cells
(DCW) were found to secrete 9.87 g/L extracellular and 3.48 g/L intracellular
PHB. As PHB is an intracellular lipid granule, the question arises as to
whether it is intracellular, then how it is released outside the cytosol. It
can be explained by considering the role of calcium (Ca2+) ions that
are known to play a myriad of significant physiological roles in bacteria like
host pathogen interactions, virulence, chemotaxis, cell differentiation and
membrane transport (Dominguez 2018). The hypothetical model of secretion of
extracellular PHB outside the cytosol is shown in Fig. 7. PHB binds to
polyphosphates (PP) present in the cytosol and forms a PHB-PP complex (Ripoll et al. 2004). This structure then binds
to Ca2+ ions and is exported out via ATPase channels. Intracellular
PHBs exist in amorphous “rubbery” form, whereas extracellular PHBs are in
amorphous crystalline form (Handrick et
al. 2004). Both PHBs production was enhanced by using corncob as a carbon
source in the medium (Table 2). The production of PHB can be related with the
corncob as a carbon source because it contains xylose and arabinose (Pointner et al. 2014). B. licheniformis prefers xylose and arabinose (Mota et al. 2002) over glucose (Scheler and
Hillen 1994) for PHB production. Our study is in partial agreement with Rehman et al. (2016) who reported glucose and
fructose as preferred carbon sources for B.
cereus NRRL-B-3711 for PHB production. The positive effect of xylose on PHB
production was observed by Singh et al.
(2011). Interestingly, by using corncob, protein bands of 34 kDa, 29.5 kDa,
29.3 kDa, 28 kDa, 27 kDa and 17 kDa were obtained. It showed more protein
expression as compared to that of glucose where only two bands of 29 kDa and 27
kDa were obtained. A detailed study of these protein bands can help us to
establish their positive correlation with PHB production. At the moment,
appropriate literature does not exist which can support our findings. Both
extracellular and intracellular PHBs were same, i.e., black membranous
structure when observed under the light microscope (Fig. 4 a–b). The optical
microscopy revealed porous topology of both PHBs (Fig. 4 c–d). Râpă et al. (2011) also reported porous PHB.
In contrast, Rehman et al. (2016)
found a smooth film without any crack which is indicative of brittleness of PHB
material. Porous PHB is in limelight with respect to bone tissue engineering
for addressing small bone defect replacement (Tan et al. 2016; Senatov et al.
2017; Petrovova et al. 2019). The
confirmation of PHB was performed by FTIR spectra of extracellular and
intracellular PHB. In this study, FTIR spectra were recorded in the range of
4000–500 cm-1.
The presence of C=O group was the confirmation of PHB (Bhuwal et al. 2014; Bhagowati et al. 2015; Rehman et al. 2016; Hertadi et al.
2017) which was in agreement with our study (Fig. 5). According to TGA/ DSC
results, the endothermic nature of both types of PHBs showed that it could
absorb heat (Fig. 6 and Table 3). Higher Tm revealed that microcrystals of PHB
molecules pack more tightly in perfect structures. Tm obtained here is more
than that (109.4°C) reported by Bhagowati et
al. (2015) and (120°C) Singh et al.
(2013). Higher Tm showed the thermal characterization of PHB as thermostable
biopolymer (Rehman et al. 2016).
Conclusion
B. licheniformis-MK656314
was found to produce PHBs whose production can be enhanced by using corncob
which is agro-waste material. Further characterization of PHBs by NMR and XRD
can help us in establishing its potential applications in industry.
References
Aneja KR (2001). Experiments in Microbiology, Plant Pathology, Tissue Culture and Mushroom
Production Technology, New Age Inernational Limited, New
Delhi, India
Arikan EB, HD Bilgen (2019). Production of bioplastics from potato peel
waste and investigation of its biodegradability. Intl Adv Res Eng J 03:93‒97
Barrett A (2018). The
history and most important innovations of bioplastics. Bioplastics
Chronicles. Webpage accessed on March 30, 2019.
(https://bioplasticsnews.com/2018/07/05/history-of-bioplastics/)
Bettler MCM, MA Ulla, AP Rabuffetti, N Garello (2017).
Plastic pollution in freshwater ecosystems: Macro, meso- and microplastic
debris in flood plain lake. Environ
Monitor Assess 189:580‒592
Bhagowati P, S Pradhan, HR Dash, S Das (2015).
Production, optimization and characterization of polyhydroxybutyrate, a
biodegradable plastic by Bacillus
spp. Biochem Mol Biol 79:1454‒1463
Bhuwal
AK, G Singh, NK Aggarwal, V Goyal, A Yadav (2014). Poly-β-hydroxybutyrate
production and management of cardboard industry effluent by new Bacillus spp. NA10. Bioresour Bioproc 1; Article 9
Butbunchu N, W Pathom-Aree (2019). Actinobacteria as
promising candidatefrom polylactic acid type bioplastics degradation. Front Microbiol 10; Article 2834
Castilho LR, DA Mitchell, DMG Freire (2009). Production
of polyhydroxyalkanoates (PHAs) from waste materials and byproducts by
submerged and solid-state fermentation. Bioresour
Technol 100:5996‒6009
Chaber P, M Kwiecieʼn, M Zięba, M Sobota, G
Adamus (2017). The heterogenous selective reduction of PHB as a useful method
for preparation of oligodiols and surface modification. RSC Adv 7:35096‒35104
Chang Y, S Hahn, B Kim, H Chang (1994). Optimization of
microbial poly (3-hydroxybutyrate) recovery using dispersions of sodium
hypochlorite solution and chloroform. Biotechnol
Bioeng 44:256‒261
Cheesebrough M (2001). Biochemical tests to identify
bacteria. In: District Laboratory Practice in Tropical Countries, pp:63‒70. Cambridge University press, Cambridge,
UK
Collee JG, JP Duguid, AG Fraser, BP Marmion (1989). Mackie and McCartney Practical Medical
Microbiology, 3rd edn, pp:54‒55. Churchill Livingstone Publisher,
Edinburgh, UK
Dominguez DC (2018). Calcium
Signaling in Prokaryotes, pp:89‒106. IntechOpen, London
Geyer R, JR Jambeck, KL Law (2017). Production, use, and
fate of all plastics ever made. Sci Adv 3; Article e1700782
Handrick R, S Reinhardt, P Kimmin, D
Jenrossek (2004). The
“intracellular” poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a
periplasm-located protein with specificity for native PHB and with structural
similarity to extracellular PHB depolymerases. J Bacteriol 186:7243‒7253
Hertadi R, K Kurnia, W Falahudin, M Puspasari (2017).
Polyhydroxybutyrate (PHB) production by Halomonas
elongata BK AG18 indigenous from salty mud crater at central Java
Indonesia. Malays J Microbiol 13:26‒32
Ivanov V, V Stabnikov, Z Ahmed, S Doberenko, A Saliuk (2015).
Production and applications of crude polyhydroalkanoate-containing bioplastic
from the organic fraction of municipal solid waste. Intl J Environ Sci Technol 12:725‒738
Jain R, A Tiwari (2015). Biosynthesis of planet friendly
bioplastics using renewable carbon source. J
Environ Health Sci Eng 13:11‒15
Kim M, KS Cho, HW Ryu, EG Lee, YK Chang (2003). Recovery
of poly 3-hydroxybutyrate from high cell density culture of Ralstonia eutropha by direct addition of
sodium dodecyl sulfate. Biotechnol Lett
25:595‒598
Laemmli UK (1970). Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 227:608‒685
Mota LJ, P Tavares, I Sá-Nogueira (2002). Mode of action
of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis. Mol
Microbiol 33:476‒489
Munir S, S Iqbal, N Jamil (2015). Polyhydroxyalkanoates
(PHA) production using paper mill wastewater as carbon source in comparison
with glucose. J Pure Appl Microbiol
9:1‒8
Nace T (2017). We’re
now at a million plastic bottles per minute-91% of which are not recycled.
Forbes. Website accessed on March 30, 2019.
(https://www.forbes.com/sites/trevornace/2017/07/26/million-plastic-bottles-minute-91-not-recycled/#3c35f257292c)
Petrovova E, M Giretova, A Kvasilova, O Benada, J Danko,
L Medvecky (2019). Preclinical alternative model for analysis of porous
scaffold biocompatibility in bone tissue engineering. Altern Anim Exp 36:121‒130
Pointner M, P Kuttner, T Obrlik, A Jäger, H Kahr (2014).
Composition of corncobs as a substrate for fermentation of biofuels. Agron Res 12:391‒396
Râpă M, ME Popa, E Grosu, M Geicu, P Stoica (2011).
Evaluation of the biodegrading action of the Penicillium spp. on some composites based on PHB. Roman Biotechnol Lett 16:9‒18
Rehman AU, A Aslam, R Masood, MN Aftab, R Ajmal, IU Haq
(2016). Production and characterization of a thermostable bioplastic
(poly-β-hydroxybutyrate) from Bacillus
cereus NRRL-B-3711. Pak J Bot
48:349‒356
Rehman RA, AQ Rao, Z Ahmed, A Gul (2015). Selection of
potent bacterial strain for over-production of PHB by using low cost carbon
source for eco-friendly bioplastics. Adv
Life Sci 3:29‒35
Ripoll C, V Norris, M Tellier (2004). Ion condensation
and signal transduction. BioEssays 26:549‒557
Scheler A, W Hillen (1994). Regulation of xylose
utilization in Bacillus licheniformis:
Xyl repressor-xyl-operator interaction studied by DNA modification protection
and interference. Mol Microbiol 13:505‒512
Senatov F, N Anisimova, M Kiselevskiy, A Kopylov, V
Tcherdyntsev, A Maksimkin (2017). Polyhydroxybutyrate/ hydroxyapatite highly
porous scaffold for small bone defects replacement in the non-load bearing
parts. J Bionic Eng 14:648‒658
Singh G, A Kumari, A Mittal, A Yadav, NK Aggarwal
(2013). Poly β-hydroxybutyrate production by Bacillus subtilis NG220 using sugar industry waste water. BioMed Res Intl 2013:1‒10
Singh G, A Mittal, A Kumari, V Goel, NK Aggarwal, A
Yadav (2011). Optimization of poly-β-hydroxybutyrate production from Bacillus species. Eur J Biol Sci 3:112‒116
Singh P, N Parmar (2011). Isolation and characterization
of two novel polyhydroxybutyrate (PHB)-producing bacteria. Afr J Biotechnol 10:4907‒4919
Spiekermann B, BHA Rehm, R Kalscheuer,
D Baumeister, A Steinbüchel (1999). A sensitive, viable-colony staining method using Nile red
for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and
other lipid storage compounds. Arch
Microbiol 171:73‒80
Tan WL, NN Yaakob, AZ Adidin, MA Bakar, NHHA Bakar (2016).
Metal chloride induced formation of porous polyhydroxybutyrate PHB films: Morphology,
thermal properties and crystallinity. In:
IOP Conference Series: Materials Science and Engineering, Vol. 133, pp:1‒11. IOP Publishing, Bristol, UK
The News International (2018). WWF-Pakistan Holds Campaign to Create Awareness of Plastic Pollution.
Available at https://www.thenews.com.pk/print/355588-wwf-pakistan-holds-campaign-to-create-awareness-of-plastic-pollution (Accessed 18 March 2019)
UN Environment Report (2018). Banning Single-use Plastic:
Lesson and Experience form Countries. Available at: https://www.
unenvironment.org/interactive/beat-plastic-pollution/ (Accessed
18 March 2019)