International Biodeterioration & Biodegradation 181 (2023) 105615

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Degradation profiling of in-vitro-produced polyhydroxyalkanoate

synthesized by the soil bacterium Bacillus sp. PhNs9 under
different microenvironments
Pritam Bajirao Patil, Debapriya Sarkar, Kasturi Poddar, Ji-Dong Gu, Angana Sarkar *
Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India

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

Keywords: Polyhydroxyalkanoates have been proven to be one of the best alternatives for synthetic plastics due to the
Polyhydroxyalkanoates similarity in their properties. The current study is focused on the natural degradation of the in vitro-produced
Biopolymer PHA by the strain Bacillus sp. PhNs9. Different microenvironments were created to mimic different terrestrial and
Natural degradation
aqueous ecosystems. Maximum degradation of 98.62% was obtained in the soil while, in the case of aqueous
Microenvironments
systems, maximum degradation of 89.75% was found in the sewage wastewater in 30 days. The degradation
profile of PHA was studied at different temperatures, pH, and moisture contents to ensure the degradation of
produced PHA in different geographical locations having different environmental conditions. Different equations
were obtained through a polynomial fitting for the prediction of degradation percentage at different conditions.
The films were characterized using FTIR, Raman spectroscopy, drop shape analysis, and XRD which confirmed
the decrease in both hydrophobicity and crystallinity of the films after degradation. This study established the
natural degradation of the PHA produced by Bacillus sp. PhNs9 which could be commercialized without concerns
about its accumulation in the environment.

1. Introduction through different modes enter into the food chain and get accumulated
at different tropic levels causing health hazards to many organisms.
The increased commercial usage of petroleum-based plastics in the Several reports had established the hazardous effects of microplastics on
past few decades has raised concerns regarding the pollution caused by human health (Patil et al., 2022b; Gu, 2021a,b).
them. Global plastic production has reached 460 million tons while the Bioplastic includes plastic derived from bio-based renewable mate­
waste generated through it has reached 350 million tons of which only rials like cellulosic biomass, bacterial biomass, vegetable oils, and
9% is being recycled, 19% is being incinerated while other 50% ends up starches (Coppola et al., 2021). The main advantage of bioplastics over
in landfills. The rest of the plastic waste goes unaccounted for and gets synthetic plastics is their biodegradability which ensures proper waste
accumulated at uncontrolled dump yards and finally into oceans due to management (Rosenboom et al., 2022; Gao and Gu, 2021). Bioplastics
its non-biodegradable nature (Gu, 2017, 2021; Gu, 2021a,b). Synthetic can be divided into different types based on the material they are
plastics also accounts for 4% of greenhouse gas emission (OCED Global derived from. For example, polysaccharide-based, protein-based,
Plastics Outlook 2022). The plastics that get degraded over many years plant-based, and bacterial-based. Examples of polysaccharides used in
because of different mechanical stresses like heat, sunlight, the salinity the production of bioplastics are starch, cellulose, lignin, chitosan,
of the seawater, shear stresses, etc. turn into microplastics (Gu, 2003). alginate, and fatty acids (Coppola et al., 2021; George et al., 2021).
Due to their micro size, microplastics leach into the environment un­ While protein-based bioplastics are derived from raw materials like
knowingly. Some microplastics are made intentionally to be used in casein and gluten. Bacterial biomass-based bioplastics include polylactic
different fields like personal care products, and packaging industries. It acid (PLA) and polyhydroxyalkanoates (PHA), both of which are
has been reported that personal care products have been a potential considered aliphatic polyesters of fatty acids. The properties of the
source of microplastic release (Bashir et al., 2021). These microplastics bioplastics depend on the monomer type, production process, and

* Corresponding author.
E-mail addresses: patilpritam26597@gmail.com (P.B. Patil), deb.apn@gmail.com (D. Sarkar), kasturi.p93@gmail.com (K. Poddar), jdgu.ibb@gmail.com
(J.-D. Gu), sarkar.angana@gmail.com, sarkara@nitrkl.ac.in (A. Sarkar).

https://doi.org/10.1016/j.ibiod.2023.105615
Received 25 February 2023; Received in revised form 10 April 2023; Accepted 22 April 2023
Available online 9 May 2023
0964-8305/© 2023 Published by Elsevier Ltd.
P.B. Patil et al. International Biodeterioration & Biodegradation 181 (2023) 105615

number of carbons in the aliphatic chains (Li et al., 2022; Shlush and produced PHA can be degraded in every ecosystem and at different at­
Davidovich-Pinhas, 2022). mospheric conditions and to ensure that it does not accumulate at any
PHA is a class of bioplastics produced by different microbes like level in the environment.
bacteria, fungi, and actinomycetes under carbon-rich and nutrient (ni­
trogen, phosphorus, oxygen) stressed environments (Raza et al., 2018; 2.2.1. In soil
Morgan-Sagastume et al., 2014). These are produced as water-insoluble For studying the degradation of films, the soil was obtained from the
storage granules inside the bacterial cells to survive under garden of VS hall of residence, NIT Rourkela. The moisture content of
nutrient-limiting conditions. Structurally these are methyl esters of the soil was obtained using a standard method, briefly, the collected soil
long-chain fatty acids, hence the properties like hydrophobicity, melting was weighed and then dried in the hot air oven at 110 ◦ C till a constant
point, glass transition temperature, crystallinity, etc. Vary according to weight was obtained. The difference in the weight of soil before and
the type of PHA being produced (Volant et al., 2022; Syed Mohamed after drying was considered as the moisture content (O’Kelly, 2004). The
et al., 2022). Although these are biodegradable in nature, the extent of collected soil was poured into containers with dimensions of 6 cm × 6
biodegradability depends on the type of PHA being produced and used cm × 20 cm (W × L × H). For the degradation experiment, 5 similar
for commercial applications (Mangaraj et al., 2019). There were setups were created and preweighed PHA films were buried in the soil at
different studies reported on the degradation of commercially available a height of 10 cm from the surface and kept for incubation at room
PHAs. Volova et al. studied the degradation of 2 types of commercially temperature, varying between 28 and 32 ◦ C, under sunlight to mimic the
available PHAs namely polyhydroxy butyrate (PHB) and 3-polyhydroxy natural conditions. PHA films were removed at regular intervals of 6
butyrate valerate (PHBV). It was observed that the degradation was days from the respective containers so that the maximum incubation
mainly dependent on the production method and shape of the polymer time would be of 30 days. Each removed film was washed with 70%
product revealing that the films were more prone to degradation ethanol and then with distilled water to remove the soil particles, bac­
compared to solid objects (Volova et al., 2011). In a study conducted by terial biofilms, and other impurities present on the surface. Washed films
Voinova et al., PHBV films were subjected to natural degradation in two were then dried and weighed to find out the difference in the weight of
aqueous systems having different ecosystems. It was observed that under the film before and after incubation.
anaerobic conditions, the rate of degradation was slow compared to
aerobic (Voinova et al., 2008). 2.2.2. In different types of water
Although different studies are present on the degradation of Different water samples having different ecosystems were collected.
commercially available PHAs, a limited number of studies considered The river water from the Koyal River, Rourkela was considered as a
the degradation of the films prepared from in-vitro-produced PHA. Also, representative of freshwater systems. The Municipality wastewater/
in most of the reported studies, only aqueous ecosystems were consid­ Sewage water collected from Naga Pond at NIT Rourkela as a repre­
ered with a limited number of variations in the environmental param­ sentative of wastewater. Tap water was collected as a representative of
eters. The current study involves the natural degradation of the film potable/treated water. While seawater was collected from the Bay of
synthesized from the PHA produced by the soil isolate Bacillus sp. PhNs9. Bengal, Puri, Odisha as a representative of the marine environment.
in different microenvironments created to mimic different geographical Different sources of water were selected to study the degradation profile
conditions. Also, the degradation was carried out in different microen­ of produced PHA in every ecosystem. Five different setups were pre­
vironments created to mimic the environmental conditions of different pared, each containing different containers for a different type of water.
ecosystems. Moreover, polynomial fitting was performed to predict the Then the preweighed PHA films were introduced in 500 mL of each
degradation percentage at different locations. This study is a pivotal step water and incubated at room temperature, varying between 28 and
in the process of commercialization of the PHA produced by Bacillus sp. 32 ◦ C, with natural sunlight to mimic the natural environmental con­
PhNs9 as an eco-friendly alternative to synthetic plastic. ditions. Films were removed at regular intervals of 6 days, washed
thoroughly with 70% ethanol, and further with distilled water to remove
2. Materials and method the dirt and bacterial biofilms. Further, the films were dried and
weighed to find out the loss in weight after degradation.
2.1. Production and preparation of PHA film
2.2.3. Effect of temperature
PHA was produced using previously isolated soil bacterium Bacillus To ensure the degradation of produced PHA at every geographical
sp. PhNs9 in glucose medium. The produced PHA was then extracted location, the films were studied for degradation at different tempera­
using the chloroform extraction method and purified using repeated tures in both soil and water. Although the average atmospheric tem­
dissolution in chloroform (Patil et al., 2022a; Colak and Güner, 2004). perature at different locations ranges from 10 ◦ C to 60 ◦ C, the maximum
After purification, films were prepared by dissolving 1g of PHA in 50 mL temperature of soil and water reaches up to 40 ◦ C. Hence, the temper­
of chloroform and pouring the polymer solution into glass plates used as atures selected for the study were 20 ◦ C, 25 ◦ C, 30 ◦ C, 35 ◦ C, and 40 ◦ C.
a mould. The films were obtained by evaporating the chloroform at PHA films were weighed and introduced in the setups prepared for the
room temperature and these films were dried in a hot air oven at 60 ◦ C experiments and each setup was then incubated at their respective
till constant weight was obtained (Pérez-Arauz et al., 2019). From every temperatures. Films were removed after a period of 30 days and washed
glass plate, a circular film was obtained, and further, it was cut into thoroughly with 70% ethanol and distilled water, dried in a hot air oven
rectangular pieces with dimensions of 4 cm × 4 cm and a thickness of at 60 ◦ C for 1 h, and weighed to observe the difference in weight.
0.2 mm (Fig. S1b). All the pieces were characterized for the loss in
weight, Fourier’s transform infrared spectrometry (FTIR), Nuclear 2.2.4. Effect of moisture content
Magnetic Resonance spectroscopy (NMR), Raman Spectroscopy, Dif­ The moisture content of soil varies with different geographical con­
ferential scanning calorimetry, and contact angle measurement before ditions and also with different seasons. Hence to study the degradation
and after degradation (Patil et al., 2022a). profile of the produced PHA in different seasons and at different loca­
tions around the globe, this study was performed. Garden soil was
2.2. Degradation of PHA film collected and placed in 5 different containers and the moisture content
of the soil was varied as 10%, 20%, 30%, 40%, and 50%. All setups were
The PHA films obtained were further studied for their degradation in incubated at room temperature and the moisture content of the soil was
different setups designed to mimic the different environmental condi­ maintained using sterile distilled water to ensure the negligible effect of
tions as mentioned below. This study was performed to test if the any external factor. Films were removed after a period of 1 month and

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washed with 70% ethanol and distilled water. Further, they were dried 3. Results
in a hot air oven and weighed to check the weight loss after respective
incubation days. 3.1. Degradation of PHA films

2.2.5. Effect of pH of the water 3.1.1. In soil

The pH of the water depends on different factors like weather pat­ The soil’s moisture content was studied and found to be between 35
terns, human activities, types of pollutants, types of salts present, and and 38%. It was observed that the PHA films in the soil were degraded
different natural processes. In different aqueous ecosystems, the pH completely, with almost 99% degradation, in terms of weight loss after
value changes with different environmental conditions. The pH of the incubation of 30 days (Fig. 1).
different types of water was varied as 5.0, 6.0, 7.0, 8.0, and 9.0 using 1N
sodium hydroxide (NaOH) and 1N hydrochloric acid (HCl). Preweighed 3.1.2. In different types of water
PHA films were then introduced in 500 mL of water and incubated at In the study of degradation in different types of water, the highest
room temperature under sunlight for a total of 30 days. Films were degradation was observed in the municipality wastewater with 89.75%
removed after incubation and washed with 70% ethanol and distilled degradation followed by seawater with 82.24% after 30 days of incu­
water. Further, films were dried in the oven and then weighed to observe bation. The percentage of degradation was low in the river water at
the loss in weight of the film after degradation. 78.60% followed by tap water at 75.11% compared to the former types
(Fig. 1). The rate of degradation in % weight loss per day was found to be
3.28%/d for soil, 2.74%/d for seawater, 2.99%/d for sewage water,
2.3. Model fitting and degradation prediction 2.62%/d for river water, and 2.50%/d for tap water.

The data obtained from the above studies were analyzed statistically 3.1.3. At different temperatures
to obtain the polynomial models. The data were fitted in polynomial A study for the effect of different temperatures on the degradation
models based on the obtained values for the coefficient of determination rate of produced PHA in different ecosystems was performed and it was
and the best-fitted models were selected. The equations for the respec­ revealed that the maximum degradation was obtained at 30–35 ◦ C in all
tive models were obtained and could be further used to predict the systems (Fig. 2a). The maximum degradation was obtained in soil with
percentage of degradation at different environmental conditions like 98.31% degradation at 35 ◦ C followed by 94.17% at 30 ◦ C. The mini­
temperature for all ecosystems, pH for aqueous ecosystems, and mois­ mum degradation in soil was observed at 20 ◦ C with about 70%
ture content for soil systems in 30 days (Hashemi et al., 2019). degradation. In the case of aqueous systems, the highest degradation
was observed in sewage water with 87.65% at 30 ◦ C followed by
seawater with 84.62% degradation. In the case of river water, maximum
2.4. Characterization of PHA film before and after degradation degradation was observed at 30 ◦ C with a 78.24% loss in weight, and for
tap water, the highest degradation was observed at 35 ◦ C with a 75.45%
PHA films were characterized after the degradation to study the loss in weight of the PHA film.
changes that occurred at the surface as well as structural levels.
Fourier Transform Infrared spectroscopy study was performed to 3.1.4. At different pH
study the different functional groups present on the surface of the film. It was observed that, with an increase in the pH value, the degra­
FTIR spectrum was studied from 500 cm− 1 to 4000 cm− 1 and different dation rate also increased to pH 8.0, and afterward it again decreased
peaks were studied for their respective functional groups. The obtained (Fig. 2b). The highest degradation was observed in sewage water with
spectrum was compared with the FTIR spectrum of the film before 89.32% loss in weight at pH 8.0. For the same system, the minimum
degradation (Patil et al., 2022a). degradation was observed at pH 5.0 with 68.32%. In the case of
Raman spectroscopy was performed in a Raman spectrometer seawater, the maximum and minimum percentages of degradation were
(XMB3000-3000 WItec GmBH) to study the changes in Raman intensity found to be 85.34% and 60.21% at pH 8.0 and pH 5.0 respectively.
of the film to find out the changes in structural properties before and Similarly for river water, the maximum (83.36%) degradation was ob­
after degradation. The Raman spectrum was studied from 500 to 4000 tained at pH 8.0 and the lowest (55.289) degradation was obtained at pH
cm− 1. The obtained spectrum was compared with the spectrum studied 5.0. In the case of tap water, similar results were obtained with
in the previous studies (Patil et al., 2022a). maximum (79.65) degradation at pH 8.0 and minimum (58.32) degra­
X-ray diffraction spectroscopy was performed to study the crystalline dation at pH 5.0.
characteristics as well as phase properties of the film before and after
degradation. The diffraction study was performed with Co-Kα radiation 3.1.5. In different moisture contents
and the scanning was performed for the 2θ values from 10 to 70◦ on an The effect of change in the moisture content of the soil on PHA
X-ray diffractometer (Make: Bruker, Model: AXS D8 advance with degradation was studied and it was observed that, with an increase in
Davinci Design) (Maity et al., 2022). the moisture content, the efficiency of degradation also increased. The
Drop shape analysis was performed to study the change in the hy­ maximum degradation (96.35%) was observed at 40% moisture content.
drophobic nature of the surface of the film after degradation. The It was also observed that the rate of degradation was significantly
experiment was performed in a drop shape analyzer (Model: DSA25B, affected by decreasing moisture content showing only 50.23% degra­
Kruss, Germany) using a drop of distilled water placed on the surface of dation at 10% moisture content (Fig. 2c).
the film (Patil et al., 2022a).
3.2. Degradation predication

2.5. Statistical analysis Based on the data and results obtained for the degradation of pro­
duced PHA under different environmental conditions, a numerical pre­
The experiments mentioned above were conducted in replicates and diction method was proposed based on the polynomial model fitting.
the results have been mentioned as the mean ± standard deviation The equations were obtained for respective models with their respective
which was calculated along with the analysis of variance (ANOVA). The environmental conditions, and are represented in Table S1. For the data
probability value i. e. (p-value) was considered less than 0.05 for the obtained in the study for the effect of change in the pH of seawater and
significant difference. the effect of moisture in the soil on the degradation, the 2nd-order

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Fig. 1. Degradation kinetics of produced PHA films in different ecosystems.

polynomial model was selected while for the rest of the data, 3rd-order colonize (Sevakumaran et al., 2019). This colonization of microbes on
polynomial models were selected based on the values obtained for the the surface of films and the enzymes secreted by them contribute to the
coefficient of determination. biotic factors involved in the efficient degradation of PHA (Meereboer
et al., 2020; Brodhagen et al., 2015; Kijchavengkul and Auras, 2008).
3.3. Characterization of PHA film before and after partial degradation The key enzymes involved in the biological degradation of PHAs are
PHA depolymerase and lipases and there are around 600 different PHA
The FTIR spectra were obtained for the films before and after depolymerase identified from different microbial species (Volova et al.,
degradation. A similar spectrum was obtained for both films with major 2010). During microbial degradation, the aerobic metabolism of PHAs
peaks at 2912, 1721, 1453, 1278, 1055, and 974 cm− 1. The major dif­ produces water (H2O) and carbon dioxide (CO2) as end products while
ference was the presence of peaks at 3279 cm− 1 and 1642 cm− 1 in the the anaerobic degradation produces methane (CH4) and water (La
spectrum of the degraded film. Also, the intensities of the peaks between Fuente et al., 2023). As the soil used in the study was collected from the
3000 and 2750 cm− 1 and 1000 - 1500 cm− 1 were reduced after the garden, the soil had a higher concentration of the different salts and
degradation (Fig. 3a). nutrients supporting higher microbial growth and improving the
In the XRD spectroscopy, major and sharp peaks were obtained in the degradation of PHA films (Sharma and Thakur, 2022). The moisture
spectra obtained for both films. The major peaks were found at the 2θ present in the soil reacts with the ester groups present in the long chains
values of 15.8, 20, 23.04, 26, 30, 32, 35, and 44◦ (Fig. 3b). Although a of PHA causing the hydrolysis of chains and it also aids in the growth of
similar spectrum was obtained for both films, the intensity of all peaks microbes. Also, it is involved in the dissolution of salts present in the
was significantly reduced for the film after degradation compared to the environment which again enhances microbial growth and in turn
film before degradation. degradation of PHAs (Siddiqui et al., 2021; Siracusa, 2019; Li and
Raman spectroscopy was obtained for the degraded film and Strathmann, 2019
compared with the film before degradation. A single broad peak was In the case of aqueous systems, the overall degradation was found to
observed for the degraded film compared to the sharp peaks at 846, be lower than the soil which could be due to the hydrophobic nature of
1460, 1733, 2878, and 2927 cm− 1 obtained for the native film (Fig. 3c). the PHA film used for the study which caused the PHA film to float on
The contact angle of distilled water with the PHA film after the the surface of the water for the first few days of incubation. The higher
degradation was studied and it was found to be 74.9◦ (Fig. 4b), which degradation was found in the sewage water which could be due to the
was significantly decreased from the contact angle of the film before presence of different types of reactive chemicals which interact with
degradation i. e. 129.8◦ , (Fig. 4a). different functional groups, mainly esters and alcohol, present on the
surface of the film and altering the surface properties (Chen, 2009).
4. Discussion These reactions change the surface properties of the films and induce the
colonization of microbes present in the water. Also, sewage water con­
4.1. Degradation of PHA films in different microenvironments tains a higher number of microbes compared to other considered water
samples which increases the rate of biological degradation. Also, envi­
The maximum degradation was observed in soil, as a larger surface ronmental parameters like biological oxygen demand (BOD) and
area was available for the microbes to colonize. Along with it, abiotic chemical oxygen demand (COD) significantly affect the degradation
stresses like pressure, abrasion, and weathering were present to a higher process. Higher BOD and COD values imply that the environment has an
extent in soil compared to other ecosystems which contributed to the abundance of organic pollutants. These organic pollutants will either
higher PHA degradation rate (Boyandin et al., 2013). Also, the microbial promote or inhibit microbial growth which in turn will affect the rate of
count is higher in the soil compared to other systems. The smaller par­ PHA degradation (Ruggero et al., 2020). After the sewage water,
ticles present in the soil could be responsible for inducing the abrasion maximum degradation was observed in seawater. The degradation of
and further weathering of PHA films as the films were continuously PHA films in the seawater was higher than in river water which could be
under the pressure of the soil present on top, which created cracks in the due to the higher salinity of seawater. The salts present in the water react
films and contributed to increasing the surface area for the microbes to with the functional groups present on the surface of the film inducing a

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Fig. 2. Effect of different parameters on the degradation of PHA film a) Temperature b) pH c) Moisture, in different ecosystems.

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Fig. 3. a) FTIR spectrum, b) XRD analysis, c) Raman spectrum of the PHA films before and after degradation.

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Fig. 4. Contact angle measurement of the PHA film a) before and b) after degradation.

chain of reactions responsible for further hydrolysis (Dilkes-Hoffman microbial growth will be affected which in turn affects the rate of
et al., 2019). The reactions also increase the hydrophilicity of the film degradation (Siracusa, 2019; Mergaert et al., 1994).
inducing colonization of bacteria on the surface and further the enzymes In the study of the effect of moisture content on the degradation of
secreted by microbes induce biodegradation. The lowest rate of degra­ PHA, it was observed that the percentage of degradation increased with
dation was found in tap water. As the supplied tap water undergoes an increase in the moisture content. As the moisture content decreases,
different treatment processes which decreases the concentration of the microbes present in the soil undergo stressed conditions which
reactive chemicals, salts, and microorganisms resulting in lower biotic decrease their growth rate (Iovieno and Bååth, 2008; Wei et al., 2022).
and abiotic factors compared to other considered systems. Also, the water molecules present in the soil interact with the ester
groups present in the carbon chain of the PHA (Riaz et al., 2021). Hence
at lower moisture contents, both biotic and abiotic factors were signif­
4.2. Degradation of PHA films in different environmental conditions icantly affected which in turn resulted in the lower degradation of films.
Similar results were obtained in the previously reported studies. For
The degradation profile of the in-vitro-produced PHA film was also example, a study was conducted by Volant et al. on the degradation of
studied in different environmental conditions like temperature, pH of PHBHV in the marine environment. It was observed that 90% degra­
the water, and moisture content of the soil to ensure the biodegrad­ dation was achieved after 250 days of immersion in the sea (Volant
ability of produced PHA at different geographical places. In the study of et al., 2022). In a study conducted on the blends of PHA and poly­
degradation at different temperatures, maximum degradation was caprolactone for the probable application as mulch film for agricultural
observed in the temperature range of 30–35 ◦ C in all considered eco­ applications. Maximum degradation of 48.34% was obtained in five
systems. Although the temperature of the collection sites varies from weeks (Othman et al. 2022). A study conducted on the degradation of a
3 ◦ C to 50 ◦ C throughout the year, the maximum number of microor­ 90:10 composite of PHA with distiller’s dried grains with solubles
ganisms present in the samples were mesophilic in nature. Hence, the (DDGS) showed that the composite had better mechanical strength, low
growth rate of most microbes was maximum in this temperature range. production cost, and better biodegradable properties than the neat PHA.
Along with the biodegradation of PHA, spontaneous chemical reactions Also, the composite was degraded at a faster rate compared to pure PHA
were favored in the obtained temperature (Turco et al., 2021; De Falco (Madbouly et al., 2014). A study was conducted on PHB/PHV copolymer
et al., 2021; Kai and Loh, 2014). Hence the combined maximum effect of for the degradation in different freshwater reservoirs. It was revealed
both abiotic and biotic factors increased the rate of degradation. At that the copolymer was degraded with a 21% loss in weight from the
temperatures below 25 ◦ C, the rate of undergoing chemical reactions initial weight (Voinova et al., 2008). In a study conducted by Bhatt et al.,
reduces along with the low viability of microbes (Madbouly et al., 2014; for the degradation of a blend of PHA with rubber, a soil isolated strain,
Jefferies et al., 2010), hence the degradation occurred at a very slow rate Pseudomonas sp. 202 was employed. It was observed that after 30 days of
which results in the lowest percentage of degradation in the 30 days. At incubation, a maximum of 16.12% degradation was observed (Bhatt
higher temperatures, spontaneous chemical reactions are favored but et al., 2008).
the majority of the microbes are inactive due to their mesophilic nature The rate of degradation of the PHA produced by Bacillus Sp. PhNs9
(Montano-Herrera et al., 2014). In the case of degradation in the soil at could be predicted using the models obtained by fitting the obtained
higher temperatures, the moisture content of the soil was reduced, data with the polynomial models. The obtained equations are mentioned
which affected the growth rate of native microbes which resulted in in Table S1. These equations could be used to measure the approximate
them being inactive at higher temperatures (Iovieno and Bååth, 2008). percentage of degradation under different environmental conditions in a
Hence only thermal degradation was responsible, which resulted in a period of 30 days. Although the rate of degradation will depend on
lower percentage of degradation. different factors like the diversity of microbes present in the selected
In the case of degradation at different pH values, it was observed that ecosystem, the presence of different salts, and chemicals, in the case of
the rate of degradation increased with an increase in the pH. This could aqueous systems, the BOD and COD values, an approximation of the rate
be because of the increasing concentration of OH− ions that react with of degradation could be established.
the ester groups present in the backbone of long chains of PHA. The
reaction results in the hydrolysis of the ester groups into alkyl alcohol
(-OH) and carboxylic acid (-COOH), which was confirmed in the FTIR 4.3. Biochemical characterization of PHA films before and after
analysis. This breakdown increases the hydrophilicity of the film and degradation
further induces the colonization of different microbes (Ngamdee et al.,
2022). At lower pH values, the growth of microbes was reduced due to During the characterization of PHA films before and after degrada­
the stressed environment (Obruca et al., 2021) and the chemical re­ tion, it was observed that the structural and chemical properties of the
actions were inhibited due to the absence of OH− ions which resulted in PHA films were significantly affected. The FTIR confirmed the presence
lower degradation (Lam et al., 2012). Hence if the pH is not optimum, of polar groups on the surface of the film which resulted in increased

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hydrophilicity. This was further confirmed by the contact angle mea­ observed after degradation (Weng et al., 2013). In a study conducted on
surement which established the decrease in the contact angle of distilled the homopolymer of PHB, an increase in the crystallinity was observed
water with film. The major peak at 3279 cm− 1 obtained in the spectrum after degradation with the rate of 0.08–0.33% per day for the films while
of the film after degradation represents the presence of an alcohol (-OH) 0.02–0.18% per day for pellets (Boyandin et al., 2013).
functional group (Shang et al., 2020; Manna et al., 2015). While the peak
at 1642 cm− 1 was due to the presence of an alkene (C– – C) functional
group (Ghosh and Chakraborty, 2020; Raza et al., 2016). The major 4.4. Probable mechanism of degradation
peaks obtained in the range of 3000 cm− 1- 2750 cm− 1 are representative
of the alkane chain (–CH2–CH2-), while the major peak obtained at 1379 Although the PHA films were degraded in different atmospheric
cm− 1 was due to the presence of an alkyl (-CH3) functional group. The conditions, the proposed underlying mechanism of degradation could be
peak at 1055 cm− 1 was attributed to the ether (C–O) group (Patil et al., similar (Fig. 5). It has been already established that both abiotic and
2022a, Saratale and Oh, 2015). Similar results were observed in a study biotic factors are responsible for the degradation of PHA films and
conducted by Phukon et al., the peaks representing the C–O–C and C– –O higher rates of degradation, both factors should perform synergistically.
groups in the range of 1127–1140 cm− 1 and 1675-1735 cm− 1 were There were many studies conducted on the degradation of PHA films
decreased in intensity (Phukon et al., 2012). Another study was con­ that have established the major steps during the degradation. The first
ducted on a composite of PHA and polylactic acid (PLA) and it was step of degradation was the disintegration of PHA film due to different
observed that the PHA fraction of the composite was degraded at a much abiotic factors like heat, pressure, abrasion, and sunlight. This deterio­
faster rate than the PLA. It was observed that there was an increase in the ration alters the surface properties, like hydrophobicity and surface
intensity of the bands representing –OH bending and stretching repre­ tension of the film, and further leads to the formation of cracks on the
senting their formation after degradation (Sintim et al., 2020). A study surface. These cracks help in initiating the colonization of microbes. The
was conducted on the degradation of PHA using a lipase enzyme isolated second step would be performed by the extracellular enzymes secreted
from Bacillus Subtilis and the band representing the –OH group was by colonized microbes which results in the cleavage of long chains of
witnessed in the spectrum of the degraded film (Kanmani et al., 2016). fatty acid esters into oligomers and monomers (Alshehrei, 2017). The
In Raman spectroscopy, it was observed that the peak at 1733 which primary class of enzymes catalyzing the reaction is PHA depolymerase
represents the crystalline characteristics of the film was absent, which like PHB depolymerase which acts on Polyhydroxybutyrate, Poly­
indicated that during the degradation of the film, the crystalline part hydroxyvalerate, and their co-polymer. Several microbes from diverse
was also degraded (Zheng et al., 2015). These results were further ecosystems have been identified for the production of PHA depolymer­
confirmed by the XRD analysis as the intensity of the crystalline peaks ase like Bacillus megaterium, Pseudomonas sp., and Comamonas sp. mainly
was decreased after degradation. Also, the peak at 2927 cm− 1 for the found in the soil (Bano et al., 2017; Dharmalingam et al., 2015), Bur­
presence of the –CH2 group, specific for the Polyhydroxybutyrate (PHB), kholderia sp., Desulfobotulus sapovorans, Halomonas hydrothermalis were
was absent showing the degradation of PHA due to different biotic and isolated from the marine environment (Suzuki et al., 2021), Alcaligenes
abiotic factors (Hermann et al., 2020). Also, the peak representing the faecalis and Pseudomonas were found in the activated sludge, Pseudo­
presence of the –CH3 group was absent, which was again confirmed with monas stutzeri was reported in the freshwater lake, while in anaerobic
FTIR analysis as the intensity of peaks representing the methyl groups sludge, Ilyobacter delafieldi was reported (Yean et al., 2017). The action
was decreased. These results confirmed that the process of degradation of these enzymes results in the formation of oligo and monomers which
resulted in the formation of an amorphous structure along with are then internalized by microbes as substrates. The breakdown of these
increased hydrophilicity which was similar to the reported studies. For substrates is performed by different proposed pathways shown in Fig. 5
example, in a study conducted by Weng et al., on the degradation of P (Altaee et al., 2016; Bano et al., 2017; Dong et al., 2022). Although
(3HB,4HB)/PLA blends, the appearance of alcohol (-OH) groups was different pathways are involved, the end products of the whole meta­
bolism are CO2 and H2O. The obtained results in this study had

Fig. 5. Probable mechanism and biochemical pathways for the degradation of PHA films.

8
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collinearity with this established theory of PHA degradation like Brodhagen, M., Peyron, M., Miles, C., Inglis, D.A., 2015. Biodegradable plastic
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Declaration of competing interest org/10.1016/j.ibiod.2020.105091.
Gu, J.D., 2003. Microbiological deterioration and degradation of synthetic polymeric
The authors declare no conflict of interest in the submission of this materials: recent research advances. Int. Biodeterior. Biodegrad. 52 (2), 69–91.
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Acknowledgment of Listeria monocytogenes, Shigella sonnei, Byssochlamys fulva and Saccharomyces
cerevisiae and ascorbic acid and β-carotene degradation kinetics in tangerine juice
The authors would like to acknowledge the Central Instrumentation by pulsed-thermosonication. Lwt 111, 612–621. https://doi.org/10.1016/j.
lwt.2019.05.060.
Facility of the National Institute of Technology Rourkela for providing Hermann, D.R., Lilek, D., Daffert, C., Fritz, I., Weinberger, S., Rumpler, V., Herbinger, B.,
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