Isolation and optimization of extracellular PHB depolymerase producer Aeromonas caviae Kuk1-(34) for sustainable solid waste management of biodegradable polymers.

Bioplastics, synthesized by several microbes, accumulates inside cells under stress conditions as a storage material. Several microbial enzymes play a crucial role in their degradation. This research was carried to test the biodegradability of poly-β-hydroxybutyrate (PHB) utilizing PHB depolymerase, produced by bacteria isolated from sewage waste soil samples. Potent PHB degrader was screened based on the highest zone of hydrolysis followed by PHB depolymerase activity. Soil burial method was employed to check their degradation ability at different incubation periods of 15, 30, and 45 days at 37±2°C, pH 7.0 at 60% moisture with 1% microbial inoculum of Aeromonas caviae Kuk1-(34) (MN414252). Without optimized conditions, 85.76% of the total weight of the PHB film was degraded after 45 days. This degradation was confirmed with Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscope (SEM) analysis. The presence of bacterial colonies on the surface of the degraded film, along with crest, holes, surface erosion, and roughness, were visible. Media optimization was carried out in statistical mode using Plackett Burman (PB) and Central Composite Design (CCD) of Response Surface Methodology (RSM) by considering ten different factors. Analysis of Variance (ANOVA), Pareto chart, response surface plots, and F-value of 3.82 implies that the above statistical model was significant. The best production of PHB depolymerase enzyme (14.98 U/mL) was observed when strain Kuk1-(34) was grown in a media containing 0.1% PHB, K2HPO4 (1.6 gm/L) at 27 ℃ for seven days. Exploiting these statistically optimized conditions, the culture was found to be a suitable candidate for the management of solid waste, where 94.4% of the total weight of the PHB film was degraded after 45 days of incubation.

1. Introduction

Plastics play an essential role in daily life; hence, their requirement increased tremendously from 1.5 million tonnes in 1950 [1] to ~300 million tons in 2015 [2]. These synthetic polymers are usually low-cost but have a major negative impact on our environment [3]. Due to their non-degradable nature, plastics are the main culprit of environmental nuisance. Despite many recycling efforts, the results of plastics disposal in municipal landfills still create significant problems. Consequently, many attempts have been undertaken to generate renewable, degradable, and recyclable materials, i.e., green materials, for sustainability [4, 5]. Poly(lactic acid) (PLA), poly(butylene succinate adipate) (PBSA), polycaprolactone (PCL), and poly(hydroxy alkanoates) (PHAs) are few biodegradable aliphatic polymers, which can replace polyethylene (PE) and polystyrene (PS) that took hundreds or thousands of years to degrade [4].

PHAs are produced by a diversity of microorganisms as carbon and energy storage material under stress conditions. Poly-β-hydroxybutyrate (PHB) is the most commonly occurring PHA, which comprises of packed monomers of (R)-3-hydroxybutyric acid (R3HB) [6, 7]. PHB depolymerase (EC 3.1.1.75) is an extracellular and intracellular hydrolyzing enzyme that degrades PHB effectively [8]. Partially structured (denatured) PHB is degraded by extracellular depolymerase [9], while intracellular depolymerase operates on unstructured (native) PHB [10]. A plethora of reports are available on the biodegradation of PHAs in a terrestrial, marine, soil environment [11-13]. As a result, several microbes have been characterized responsible for the degradation of these polymers [14]. In the present study, biodegradation of PHB polymer from a novel bacterial enzyme isolated from the sewage waste soil bacteria was observed. Their detailed statistical production optimizations were also studied, including soil burial applications for the sustainability of the environment.

2. Materials and methods

2.1. Polymer studied

PHB powder with a linear formula of [COCH2CH(CH3)O]n was purchased from Sigma-Aldrich, CAS Number: 29435-48-1.

2.2. Sample collection and isolation of PHB degrading microbes

Soil samples from sewage sludge in semi-solid form were taken from four different sources in Lucknow, Uttar Pradesh, India (Table 1) for isolation of potent PHB depolymerase-producing microorganisms. Samples were processed by serial dilution following spread plate method on Bushnell Hass medium and incubated for 48 h at 37 ºC for the proliferation of microbial growth and isolation of microorganisms.

Table 1

Soil samples taken from different locations of Lucknow, UP, India.

Sampling site	Latitude/Longitude	Total Isolates	Positive Isolates
Kukrail	26.91 N/ 80.98 E	73	15
Gomti Nagar	26.84 N/ 81.00 E	31	04
IIM Road	26.80 N/ 80.76 E	16	02
Molviganj	26.85 N/ 80.92 E	07	01

2.3. Screening of PHB depolymerase producing isolates

PHB depolymerase producers were screen out by clear zone assay on Bushnell Hass Medium (BHM) (g/L): Magnesium sulphate, 0.20 g; Calcium chloride anhydrous, 0.02 g; Potassium di-hydrogen phosphate, 1.0 g; Di-potassium hydrogen phosphate, 1.0 g; Ammonium nitrate, 1.0 g; Ferric chloride, 0.05 g with 0.15% PHB powder sonicated in an ultrasonic water bath (Labman-LMUC 3, 40 KHz and 100 W) at 40 ºC for 20 min followed by addition of 2% agar powder to generate solid media. After sonication, plates were incubated for seven days at 37 ºC, and then a clear hydrolysis zone was measured.

2.4. PHB depolymerase assay

PHB depolymerase assay was performed as per the modified method of Kobayashi et al. [15]. Tris-HCL buffer (50 mM, pH 7.0) with 0.15% of PHB powder was suspended and subjected to sonication immersed in an ultrasonic water bath (40 kHz and 100 W) for 30 min. In 0.9 mL of the substrate suspension, 0.1 mL of culture supernatant was added and incubated for 25 min at 37 ºC. The activity of the decrease in turbidity of the PHB suspension was measured at OD650 against blank (1 mL of Tris HCL buffer) [14].

2.5. Morphological, physiological, biochemical identification of isolates

The positive bacterial isolates that gave a clear zone of hydrolysis on PHB plates were identified in accordance with Bergey’s Manual [16]. Detailed analysis was done for morphological, physiological, and biochemical identification. The 16S rRNA gene sequence analysis was performed by Biokart, Bangalore, India Pvt. Ltd. using forward primer sequence 27F and backward primer sequence 149R [17]. The sequences were examined and compared to the nucleotide sequences stored in the NCBI (National Center for Biotechnology Information) database using Basic Local Alignment Search Tool (BLAST) search engine. The phylogenetic tree, as implemented in MEGA X from nucleotide sequences, was developed using the neighbouring ClustalW method [18].

2.6. Preparation of PHB film

PHB film was prepared by solvent casting method where 0.1 g of PHB powder (Sigma-Aldrich) was suspended in 30 mL of chloroform while kept on a magnetic stirrer for 20 min at 45 ºC. PHB-suspension was poured into clean autoclaved glass Petri plates, and chloroform was then vaporized. Petri plates were further incubated at 37 ºC for 24 h, resulting in the formation of PHB films about 10 cm in diameter and 2 mm in thickness [19].

2.7. Soil burial biodegradation analysis with PHB film

The change in the weight of PHB polymer film was calculated before and after the treatment with the positive bacterial strains. PHB films (0.024–0.026 g) were buried in autoclaved soil in pots (at 3 cm depth) at ambient temperature (35–37°C) with moisture content uphold to 60% in the presence of mineral salts and incubated for specified time period (i.e. 15, 30 and 45 days). The pre-weighed PHB polymer films were then taken off from the soil after different incubation times and washed several times by distilled water to remove soil particles and then dried at room temperature [17]. The polymer degradation was calculated by given formula: where, Wif = initial weight of films, Wff = final weight of buried films.

2.8. Scanning Electron Microscopy (SEM) analysis of soil buried PHB films

Soil fragmented PHB film surface was analyzed using SEM for detailed visualization [17]. Before SEM analysis, the PHB films were fixed in 4% (v/v) glutaraldehyde in sodium cacodylate buffer (100 mM, pH 7.2). After the glutaraldehyde fixation, PHB films were again fixed in 20 g/L of aqueous osmium tetroxide and, after drying to the critical point, examined under SEM [20].

2.9. Fourier-Transform Infra-Red (FTIR) spectroscopic analysis

FTIR spectra of PHB polymer film were obtained using a Perkin Elmer System 2000 Fourier transform infrared spectrometer. A small amount of PHB polymer film was immersed in an organic solvent, milled with KBr, and pressed into a transparent film for FTIR analysis. FTIR spectra were collected over the range of 4000 to 450 cm-1 [21]. FTIR determined functional groups in the degradation patterns were compared with control [22].

2.10. Statistical optimization of the media variables for PHB depolymerase enzyme production using Placket-Burman (PB) experimental design

PB experimental design was used in the initial phase of optimization using Minitab 19. In this experiment, ten independent variables were used, and each variable was checked at high (+1) and low (-1) values. All experiments were carried out in triplicates and repeated twice. PHB depolymerase activity was used as a response based on experimental design and polynomial model of the first order as follows: where, Y is response (enzyme production), β0 is model intercepts, β1 is a linear coefficient, and Xi is the level of the independent variable. This model was used to screen and assess the key factors influencing response. The p value ≤0.05 was a probability that defines the magnitude of a contrast coefficient arising from the variability of the random process and was calculated using Analysis of Variance (ANOVA). A Pareto chart analysis was drawn using standardized effects, and the results of F-value validate the importance of significant effects [23].

2.11. Statistical optimization of the PB variables for PHB depolymerase enzyme production using Response Surface Methodology (RSM)

Response surface central composite design (CCD) specifies optimal concentration of the significant variables and the interacting effect of media ingredients obtained in PB design. Out of these ten variables, five of them (namely time, temperature, PHB, KH2PO4, and K2HPO4) were further optimized with 32 runs of experiments, while the rest five variables remained constant. A high (+2) to low (-2) values have been checked for each variable. The model was validated by ANOVA and response surface plots to ensure efficiency.

2.12. Soil bioremediation and solid waste management under statistically optimized conditions

The biodegradation of PHB-based biofilm treated with bacterial strain by soil burial method under statistically optimized conditions for the application of solid waste management was performed as previously described in section 2.7 for 15, 30, and 45 days and the polymer degradation was calculated in the similar manner [22].

3. Results

3.1. Screening of PHB depolymerase isolates

In the present study, a total of four sewage waste soil samples were collected from different dumping zones of Lucknow. These samples produced maximum number of bacterial strains that were found to be potent producers of PHB depolymerase and were capable of degrading PHB-based bioplastics. After the serial dilution, the sewage waste soil samples were spread on BHM+PHB agar plates. Among 127 isolates, a total of 22 positive PHB degraders were selected based on their ability to form a zone of hydrolysis on PHB agar plate (Fig 1A and 1B). Out of these 22 bacterial strains, three were selected on the basis of large zone of diameter, and named as CB2-(20), Kuk1-(34) & CA6-(55). Zone diameters were observed in the range of 2.5 to 11.3 mm (Table 2). Zone of hydrolysis showing enzyme production is clearly visible in the Fig 1C–1E.

Fig 1

Images showing clear zone of diameter of microbial colonies in (A) and (B). Positive colonies showing PHB hydrolysis on PHB agar media (C) CA6-(55), (D) CB2-(20), and (E) Kuk1-(34).

Table 2

Enzyme activity and CFU count of positive bacterial isolates.

S. No	Positive isolates	Colony size in mm (a)	Zone of hydrolysis in mm (b)	Zone diameter in mm (b/a)	Enzyme activity (U/mL/min)	Decrease in turbidity measured at OD650	CFU/mL
1	Control	NA	NA	NA	NA	0.823	NA
2	CA6-(55)	4	10	2.5	1.55	0.379	1.29 x109
3	CB2-(20)	4	24	6	1.71	0.404	4.80 x109
4	Kuk1-(34)	3	34	11.3	2.06	0.320	4.10 x109

3.2. PHB depolymerase activity

The degradation ability of 22 positive isolates was also checked by PHB depolymerase assay. The isolates, CB2-(20), Kuk1-(34) & CA6-(55), exhibited potent activity in the range of 1.55–2.06 U/mL/min (Table 2). Among these three significant PHB degraders, Kuk1-(34) gave the best PHB depolymerase activity (2.06 U/mL/min) and maximum zone of hydrolysis (11.3 mm) and, therefore, was selected for further study.

3.3. Morphological, biochemical, physiological, and phylogenetic analysis

All the physical and physiological characteristics of bacterial strain Kuk1-(34) are represented in Table 3. Phylogenetic evaluation of the Kuk1-(34) strain was concluded with the help of alignment and cladistics analysis of a homologous sequence of known bacteria. 16S rRNA sequences were submitted in NCBI through GenBank(accession number MN414252), and their identity was performed through BLAST. The phylogenetic tree of the submitted strain in the NCBI is shown in Fig 2, and the strain was identified as Aeromonas caviae Kuk1-(34) sp (Table 4).

Table 3

Morphological, biochemical and physiological characteristics of the bacterial strain Kuk1-(34).

Characteristics	Bacterial strain Kuk1-(34)
Morphological tests
Grams staining	Negative
Pigmentation	Yellow
Form	Irregular
Elevation	Umbonate
Cell shape	Rod
Margin	Undulate
Biochemical tests
Cellulose	+
Casein	+
Indole	-
Methyl red	-
Voges-Proskauer	-
Citrate	+
Hydrogen sulphide	+
Catalase	+
Physiological tests
Optimum temperature for growth	17–37 ºC
Growth at NaCl (%)	0.5–5
Optimum pH for growth	6.0–8.0

Fig 2

Phylogenetic tree of Aeromonas caviae Kuk1-(34) sp. drawn using UPGMA tree (MEGA X software) with the evolutionary distances showing the relationship of PHB depolymerase producing bacteria with the known sequences of related genera.

Table 4

16S rRNA identification of positive isolates.

S.No.	Submitted microbial strain	Identity	Accession Number	Similarity Index %
1.	CA6-(55)	Enterobacter cloacae CA655	MN088848	97.88
2.	CB2-(20)	Stenotrophomonas sp. CB220	MN736124	96.60
3.	Kuk1-(34)	Aeromonas caviae Kuk1 sp.	MN414252	99.43

3.4. Weight loss analysis of PHB film by soil burial method

PHB films (0.024–0.026 gm) were buried in 200 g of sterile soil inside the pot with different strains, viz. Kuk1-(34), CA6-(55) and CB2-(20), separately. It was observed that the weight of PHB film was maximally reduced 45 days after pre-treatment with strain A. caviae Kuk1-(34) sp. as compared to the other isolates (Fig 3). Up to 85.76% degradation of PHB film was observed in comparison to the control (Table 5) under unoptimized conditions.

Fig 3

Degradation of PHB film treated with (A) control and Aeromonas caviae Kuk1-(34) sp. after (B) 15 days (C) 30 days and (D) 45 daysin soil burial method.

Table 5

Weight loss degradability of PHB film after different incubation time in un-optimised conditions.

Bacterial species	Weight of PHB film (g)	Weight of pre-treated soil buried PHB film (g)			Weight loss degradability
		15 days	30 days	45 days	After 45 days (%)
Control	0.0240	0.0240	0.0240	0.0240	0.00%
CA6-(55)	0.0250	0.0224	0.0190	0.0145	42.00%
CB2-(20)	0.0240	0.0229	0.0202	0.0170	29.16%
Kuk1-(34)	0.0260	0.0232	0.0187	0.0037	85.76%

3.5. SEM analysis of soil buried PHB films

In the present study, soil-buried PHB films of 15, 30, and 45 days were analyzed by SEM. Several holes, crests, surface erosion, and significant roughness were clearly observed on all PHB films as compared to the control. Multiple bacterial colonies of A.caviae Kuk1-(34) sp. were seen attached on the surface of the PHB films, which implies that this species actually secretes extracellular depolymerase enzyme that degrades PHB film (Fig 4).

Fig 4

Morphological changes in SEM micrographs of PHB film degraded in soil burial method after incubation with (A) Control PHB film without treatment, and Aeromonas caviae Kuk1-(34) sp after (B) 15 days (C) 30 days (D) 45 days.

3.6. FTIR Analysis of soil buried PHB films

FTIR analysis of soil buried PHB film treated with Kuk1-(34) sp. showed changes in the functional groups and significant shift of wave-numbers, indicating biodegradation of PHB film. It was observed that in control, the FTIR chromatogram peak for functional group O-H appearing at 3435.25 cm-1 shifted to 3392.51 cm-1 after 15 days incubation with Kuk1-(34) strain. This peak moved further to 3401.97 cm-1 and 3409.23 cm-1 with an intensity of 62.2 and 44.67, up on increasing the incubation time to 30 and 45 days, respectively. The functional group, ester bond, present in control showed a characteristic peak at 1729.63 cm-1; while in Kuk1-(34) treated samples, this peak shifted to 1623.16, 1724.7 and 1633.04 cm-1 respectively, after incubation for 15, 30, and 45 days. There is the formation of a new-fangled bond and an additional functional group after degradation at 45 days, emerging as a peak at 2923.08 cm-1 with an intensity of 47.84. This supplementary peak could be due to the -C-H functional group. Another peak at 1047.18 cm-1 with an intensity of 46.02 is indicative of the -C-O functional group. These changes in wavelength frequency of major functional groups in degrading samples as compared to control give preliminary proof of biodegradation of PHB film by A. caviae Kuk1-(34) sp. after incubation for 45 days (Fig 5).

Fig 5

FTIR chromatogram of soil buried PHB film after the degradation by Aeromonas caviae Kuk1-(34) sp.

The functional groups changes were compared with each film after incubation with (A) Control PHB film without treatment, Aeromonas caviae Kuk1-(34) sp after (B) 15 days (C) 30 days (D) 45 days.

3.7. Statistical optimization to determine the interactive impact of Kuk1-(34) sp. on enzyme production using PB design

For PB analysis, ten independent variables were selected (Table 6). The experiment was carried out with 12 runs in different combinations to identify significant parameters for extracellular PHB depolymerase production. Higher (+1) and lower (-1) values were screened for each variable (Table 7). Experimental data was statistically analyzed using F-test for ANOVA. The model F-value of 202.74 implies that the model was significant, and p-values were used as a tool to check the significance of each parameter (Table 8). Thus, p-values <0.05 denoted the importance of factors on enzyme production. The Pareto Chart illustrates the order of significance of the variables affecting the Kuk1-(34) production in PB design (Fig 6A). Among ten variables, temperature showed the highest positive effect, followed by time, PHB, and K2HPO4; while KH2PO4 comes near the positive and effective parameter. The negative impact was demonstrated by NH4NO3, pH, CaCl2, FeCl3, and MgSO4. Therefore, for the next optimization step, the optimum level of time, temperature, PHB, K2HPO4, and KH2PO4 were checked by RSM using CCD. The model equation for enzyme production (Y) can be written as:

Table 6

Component design of Plackett Burman for PHB depolymerase production with the bacterial strain Aeromonas caviae Kuk1-(34) sp.

Variable	Symbol	(+1) High value	(0) Central value	(-1) Low Value
Time (days)	A	7	5	3
Temp (ºC)	B	47	37	27
pH	C	9	7	5
PHB (%)	D	0.2	0.15	0.1
MgSO4 (g/L)	E	0.25	0.20	0.15
CaCl2 (g/L)	F	0.005	0.003	0.001
KH2PO4 (g/L)	G	1.3	1	0.7
K2HPO4 (g/L)	H	1.3	1	0.7
NH4NO3 (g/L)	I	1.3	1	0.7
FeCl3 (g/L)	J	0.07	0.05	0.03

Table 7

Placket Burman experimental design for PHB depolymerase production with the bacterial strain Aeromonas caviae Kuk1-(34) sp.

Run Order	(A)	(B)	(C)	(D)	(E)	(F)	(G)	(H)	(I)	(J)	Enzyme activity (U/mL)
1	-1	1	-1	-1	-1	1	1	1	-1	1	3.467
2	1	1	-1	1	1	-1	1	-1	-1	-1	0.967
3	-1	-1	-1	1	1	1	-1	1	1	-1	9.8
4	-1	1	1	1	-1	1	1	-1	1	-1	0.701
5	1	1	1	-1	1	1	-1	1	-1	-1	1.048
6	1	-1	-1	-1	1	1	1	-1	1	1	1.88
7	1	1	-1	1	-1	-1	-1	1	1	1	3.79
8	-1	-1	1	1	1	-1	1	1	-1	1	9.25
9	1	-1	1	-1	-1	-1	1	1	1	-1	2.9
10	1	-1	1	1	-1	1	-1	-1	-1	1	6.153
11	-1	1	1	-1	1	-1	-1	-1	1	1	0.887
12	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	8.38

Table 8

ANOVA analysis for Plackett Burman design for PHB depolymerase enzyme production.

Source	DF	Adj SS	Adj MS	F-Value	p-Value
Model	10	129.354	12.9354	202.74	0.055
Linear	10	129.354	12.9354	202.74	0.055
Time (Days)	1	20.664	20.6640	323.88	0.035
Temperature (ºC)	1	63.035	63.0346	987.97	0.020
pH	1	4.496	4.4958	70.46	0.075
PHB (%)	1	12.199	12.1988	191.20	0.046
MgSO4 (g/L)	1	0.203	0.2025	3.17	0.326
CaCl2(g/L)	1	0.814	0.8138	12.76	0.174
KH2PO4(g/L)	1	9.888	9.8881	154.98	0.051
K2HPO4(g/L)	1	10.616	10.6164	166.40	0.049
NH4NO3(g/L)	1	7.218	7.2184	113.14	0.060
FeCl3(g/L)	1	0.222	0.2217	3.47	0.313
Error	1	0.064	0.0638
Total	11	129.418

Fig 6

Pareto chart of the significant factor in the (A) Plackett Burman (PB) design, (B) Central composite design (CCD) for the enzyme production,(C) Surface plot of enzyme activity of the bacterial strain Aeromonas caviae Kuk1-(34) sp., (D) Contour plot showing the interaction effect of temperature and time on PHB depolymerase enzyme production by Aeromonas caviae Kuk1-(34) sp.

3.8. Statistical optimization of significant factors of PB plots by CCD

Significant factors of the PB experiment were then further tested by CCD to evaluate the optimized value for extracellular PHB depolymerase production. Time, temperature, PHB, K2HPO4, and KH2PO4 were assessed using Minitab 19 software (Table 9). Each variable was tested from high (+2) to low (–2) levels (Table 10). The ANOVA result showed that the regression is statistically significant for enzyme production (Table 11). The F-value of 3.82 implies that the model was significant. The Pareto Chart (Fig 6B) illustrates the order of significance of the variables affecting the production of an enzyme from Kuk1-(34) sp. The autonomous K2HPO4showed the most significant beneficial impact among all factors, followed by the independent time variable, autonomous KH2PO4 variable, and finally, interaction between time and PHB that also showed the beneficial impact. Each response surface for enzyme activity indicated a clear peak, which means that the optimum point was inside the design boundary level (Fig 6C). The effect of time and temperature on enzyme activity while keeping PHB, K2HPO4, and KH2PO4 at zero level was depicted. The response surface plot showed that the maximum production of an enzyme could be attained at optimum temperature (27 ºC) and maximum incubation time (3 days). As a result, enzyme production increased exponentially with a decrease in the incubation time and keeping the temperature at an optimum level. By applying multiple regression analysis on experimental data, the following second-order polynomial equation was obtained to describe the enzyme production efficiency (Y):

Table 9

Component design of CCD for PHB depolymerase production with the bacterial strain Aeromonas caviae Kuk1-(34) sp.

Variable	Symbol	(+2) value	(+1) value	(0)	(-1) Value	(-2) Value
Time (days)	A	9	7	5	3	1
Temp (ºC)	B	57	47	37	27	17
PHB (%)	D	0.25	0.2	0.15	0.1	0.05
KH2PO4 (g/L)	G	1.6	1.3	1	0.7	0.4
K2HPO4 (g/L)	H	1.6	1.3	1	0.7	0.4

Table 10

Central Composite Design for PHB depolymerase production with the bacterial strain Aeromonas caviae Kuk1-(34) sp.

Run Order	A	B	D	G	H	Enzyme activity (U/mL)
						Predicted value	Experimental value
1	0	0	0	2	0	13.87346	13.87
2	-1	1	-1	-1	-1	14.14433	14.09
3	0	-2	0	0	0	14.32713	14.71
4	0	0	0	0	0	14.30838	14.11
5	0	2	0	0	0	14.34463	13.98
6	1	1	1	-1	-1	14.29292	14.38
7	-1	-1	1	1	1	14.23675	14.17
8	1	1	-1	-1	1	14.60967	14.80
9	-2	0	0	0	0	14.05696	14.29
10	0	0	0	0	0	14.30838	14.1
11	1	1	-1	1	-1	14.11608	14.18
12	0	0	0	0	0	14.30838	14.47
13	1	-1	-1	-1	-1	14.60925	14.48
14	-1	-1	-1	1	-1	13.40492	13.17
15	0	0	0	0	-2	14.24346	14.53
16	-1	1	1	1	-1	13.99858	13.98
17	-1	-1	-1	-1	1	14.2325	14.13
18	2	0	0	0	0	14.65479	14.44
19	-1	-1	1	-1	-1	14.29175	14.08
20	-1	1	-1	1	1	13.95933	14.05
21	0	0	0	0	0	14.30838	14.41
22	0	0	0	0	0	14.30838	14.53
23	0	0	0	0	2	14.91829	14.65
24	0	0	0	-2	0	14.44829	14.47
25	1	-1	1	1	-1	14.0125	13.91
26	1	-1	1	-1	1	14.43308	14.47
27	1	1	1	1	1	14.37792	14.61
28	0	0	0	0	0	14.30838	14.2
29	1	-1	-1	1	1	14.96425	14.98
30	0	0	-2	0	0	13.93996	14.008
31	-1	1	1	-1	1	14.75617	14.87
32	0	0	2	0	0	14.02979	13.98

Table 11

ANOVA analysis of CCD for PHB depolymerase enzyme production.

Source	DF	Adj SS	Adj MS	F-Value	p-Value
Model	20	2.99525	0.149763	1.66	0.195
Linear	5	1.72742	0.345484	3.82	0.030
Temperature (ºC)	1	0.00046	0.000459	0.01	0.944
Time (Days)	1	0.53611	0.536107	5.93	0.033
PHB (%)	1	0.01211	0.012105	0.13	0.721
K2HPO4 (g/L)	1	0.68310	0.683100	7.56	0.019
KH2PO4 (g/L)	1	0.49565	0.495650	5.49	0.039
Square	5	0.40545	0.081090	0.90	0.516
Temperature*Temperature	1	0.00139	0.001386	0.02	0.904
Time*Time	1	0.00414	0.004136	0.05	0.834
PHB*PHB	1	0.19186	0.191862	2.12	0.173
K2HPO4*K2HPO4	1	0.13614	0.136136	1.51	0.245
KH2PO4*KH2PO4	1	0.03989	0.039886	0.44	0.520
2-Way Interaction	10	0.86238	0.086238	0.95	0.525
Temperature*Time	1	0.10808	0.108077	1.20	0.297
Temperature*PHB	1	0.04337	0.043368	0.48	0.503
Temperature*K2HPO4	1	0.00985	0.009851	0.11	0.747
Temperature*KH2PO4	1	0.01015	0.010151	0.11	0.744
Time*PHB	1	0.46410	0.464102	5.14	0.045
Time*K2HPO4	1	0.00001	0.000005	0.00	0.994
Time*KH2PO4	1	0.11408	0.114075	1.26	0.285
PHB*K2HPO4	1	0.00501	0.005006	0.06	0.818
PHB*KH2PO4	1	0.00000	0.000001	0.00	0.998
KH2PO4* KH2PO4	1	0.10775	0.107748	1.19	0.298
Error	11	0.99380	0.090345
Lack-of-Fit	6	0.81247	0.135412	3.73	0.085
Pure Error	5	0.18133	0.036265
Total	31	3.98905

The contour plot (Fig 6D) is used to predict optimal levels of components for different test variables. Two variables, time and temperature, were used to observe the enzyme activity of PHB depolymerase from A. caviae Kuk1-(34) sp. while keeping the hold values of PHB, KH2PO4, and K2HPO4 as zero. It was observed from the contour plot that PHB depolymerase from A. caviae Kuk1-(34) sp. gave maximum enzyme activity with an increase in the incubation time (9 days) and decrease in the temperature (17 ºC).

3.9. Soil bioremediation and solid waste management with A. caviae Kuk1-(34) sp. under optimized conditions

The optimized parameters obtained from CCD were further used for solid waste management by soil burial method. Autonomous factors like time, KH2PO4, K2HPO4 with (+1) values and combined interaction of time and PHB with (-1) values were used for the soil burial method treated with Kuk1-(34) strain. After the incubation for 15, 30, and 45 days, soil-buried PHB polymer film was taken from soil pots. It was observed that maximum degradation occurred when incubation was done for 45 days (Fig 7). The weight loss of PHB film after 45 days of incubation was 94.4% after the soil burial method (Table 12), which is 8.64% more under optimized conditions as compared to the weight loss in unoptimized conditions. This indicates that the CCD model validates the optimized values that can be used on the industrial level for the degradation of PHB based bioplastics.

Fig 7

Soil burial method for the degradation of PHB film as compared with (A) control after treatment with the Aeromonas caviae Kuk1-(34) sp. for (B) 15 days (C) 30 days and (D) 45 days.

Table 12

Weight loss degradability of PHB film after different incubation time in optimised conditions.

Bacterial species	Weight of PHB film (g)	Weight of pre-treated soil buried PHB film (g)			Weight loss degradability (%) after 45 days
		15 days	30 days	45 days
Control	0.0250	0.0250	0.0250	0.0250	0.00%
Aeromonas caviae Kuk1-(34) sp.	0.0250	0.0198	0.0089	0.0014	94.4%

4. Discussion

A rapid increase in the production and utilization of synthetic plastics on a daily basis and their non-degradability in nature leads to the introduction of biodegradable PHB-based bioplastics. A wide variety of micro-organisms accumulate PHB as intracellular granules in a highly reduced and insoluble polymer state [24]. In the present study, soil samples were collected from sewage waste from different locations and grown on BHM+PHB medium. BHM contain all nutrients except carbon source, necessary for the growth of bacteria. Only those bacteria that are able to decompose hydrocarbon will grow in this media. Specific carbon source i.e. hydrocarbon (in this case PHB) can be added to this medium and their utilization in terms of hydrolysis can be studied. Out of 127 isolates, a total of 22 were positive PHB degraders; among them only three displayed significant growth and exhibited maximum PHB hydrolysis. The largest zone (11.3 mm) was produced by the bacterial isolate A. caviae Kuk1-(34) sp. on BHM media containing 0.15% (w/v) of PHB when incubated for seven days at 37 ºC. This isolate was consequently selected as a prominent PHB depolymerase producer for the degradation PHB based bioplastic. A fungal isolate, Penicillium citrinum S2, produced PHB depolymerase when grown in BHM containing 0.2%, w/v PHB [25].

In literature, several articles are available regarding the isolation of different bacterial and fungal species that are potent degraders of PHB-based bioplastics. In one study, Mergaert et al. [26] isolated PHB and P(3HB-co-3HV) copolymer, while Elbanna et al. [27] described Pseudomonas indica K2 and Schlegelella thermo depolymerase as the PHA degrader. Similarly, Sayyed et al. [28] isolated PHB degrader strain from soil microbes on MSM containing PHB as sole carbon source. Typical enzyme assays for PHB depolymerase have also been described earlier by several researchers. In our study, isolated strain A. caviae Kuk1-(34) sp. was analyzed for the PHB depolymerase assay. Previous investigation conducted by Sayyed et al. [22] using Stenotrophomonas sp. RZS7 yielded 0.721 U/mL/min of PHB depolymerase after four days of incubation. Likewise, the yield of enzyme production was found to be 0.721 U/mL under unoptimized conditions [23]. However, in our case, the strain A. caviae Kuk1-(34) sp. yielded about 2.0623 U/mL/min of PHB depolymerase after incubation for seven days at 37 ºC [22]. Similar enzyme activities for the PHB depolymerase have been reported by many researchers [17, 24, 28, 29]. Up to 85.76% of total weight of PHB film was degraded after the pre-treatment with A. caviae Kuk1-(34) sp. after the incubation for 45 days in the soil under unoptimized conditions (Table 5), while no changes were observed in control PHB film (without pre-treatment). This data is quite significant than those of Bano et al. [17] regarding the degradation of PHB film using Paenibacillus alvei PHB28. As reported by Pati et al. [30], Bacillus sp. C1 (KF626477) showed significant PHB degradation within 7–21 days. Similarly, 87.74% biodegradation of PHB was obtained using isolate Stenotrophomonas sp. RZS7 under natural soil environment [22].

SEM analysis of the PHB polymer film was used to observe the progress of degradation. In the present study, surface analysis of treated PHB polymer film after incubation of 15, 30, and 45 days in soil burial method showed significant variation in the morphology of PHB polymer film. There was clear visualization of cracks and holes on the surface of the PHB polymer film. Morphological analysis of polyethylene surface comparing with control film was done by SEM and was reported by Gautam and Kaur [31]. Another study conducted by Calabia and Tokiwa [32] observed the growth of Streptomyces sp. SC-17 on the surface of the PHB film, which was responsible for the presence of crust and holes [22, 32]. Microbial degradation from soil burial method of the PHB film was also reported by Wen and Lu [33]. A similar study reported 15% of PHB film degradation in the soil after 45 days of incubation and observed surface morphology under SEM [17].

Treated PHB films were also analyzed by FTIR. The findings suggest that the chemical structure of PHB possesses molecules terminated by a hydroxyl and a carboxyl group. The hydroxyl and carboxyl end functional groups showed peaks at approximately 3435 cm-1 and 1729 cm-1, respectively. Previous characteristics of PHB vibrations were found to be around 1290 cm-1 and 980 cm-1. The peak at 1290 cm-1 determines the–C-O-C- group, while the peak at 980 cm-1 can be allocated to bending vibrations of olefinic -C-H [34]. As deprivation begins with time, it incorporates vinyl (crotonate) ester, and carboxyl groups end groups in PHB structure [35].

Consequently, a steady rise in crotonate ester groups with extrusions paths can be expected, as well as a decline in hydroxyl groups available in the original polymer. The absorption band assigned to stretching vibrations of double carbon/carbon bond, -C = C-, is to be shown at around between 1600 and 1700 cm-1 [36]. Band at 1729 cm-1, allocated originally to the carbonyl absorption band in infrared spectra, is shifted to 1633 cm-1 when combined with vinyl end groups after 45 days [37]. The availability of absorption bands associated with the formation of new chemical groups due to deprivation mechanisms of the polymer was noticed at 2923.08 cm-1 and 1047.18 cm-1, respectively.

Traditionally, improving one parameter at a time is exhaustive and expensive; therefore, statistical methods are utilized for optimization [38]. Plackett and Burman’s statistical method involves a two-level fractional factorial saturated strategy that uses only treatment combinations to estimate the main effects of factors independently, assuming that all interactions are insignificant [39]. Full factorials design the number of factors increases exponentially leading to an unmanageable number of experiments [39]. Hence, fractional factorial design like Plackett-Burman becomes a method of choice for initial screening of medium components [23].

To maximize enzyme output yield, PB design and RSM were implemented, which was demonstrated to be an effective screening method for significant medium components and their optimum amounts for total yield. In this study, to increase the enzyme production by A. caviae Kuk1-(34) sp., four factors were screened out from 10 factors by PB design. These four factors were further optimized for the RSM using CCD, where maximum enzyme activity of A. Caviae Kuk1-(34) sp. was obtained at 14.98 U/min/mL (p<0.05). Shivkumar [40] reported the production of depolymerase from Penicillium expansum using Placket Burman design. Bansal et al. [41] reported Aeromonas punctate sp. for the production and optimization of depolymerase enzyme using PB design and RSM [41]. Similarly, the production of PHB depolymerase from E. minima W2 (PhaZEmi) was studied [42]. The study revealed the importance of carbon sources in growth medium for the production of depolymerase enzymes, as the rate of polymer degradation was affected by the source of carbon [41, 43]. Findings from our CCD model suggest that autonomous factors like K2HPO4, time, KH2PO4, and interactive effect of time with PHB were the crucial variables in the PHB depolymerase enzyme production [23].

To validate the above study, a soil burial method with significant factors screened from RSM was applied on the PHB film to observe the degradation of PHB polymer film. In the present study, it is observed that the degradation rate of PHB polymer film increased from 85.76% to 94.4% after PHB film was treated with A. caviae Kuk1-(34) sp. in optimized conditions. This remarkable degrading characteristic of the strain was utilized for the soil bioremediation to deal with solid waste management. Since degradation proceeds at natural environmental conditions of temperature (35–37 ℃), pH 7.0, presence of nutrients in the soil, and oxygen availability allow faster microbial growth by utilizing PHB film as carbon source. So, the soil was the most promising ecosystem for PHB degradation in the presence of microbial activity that is enzymatic degradation is more relevant than composting or chemical treatment for solid waste.

5. Conclusion

The isolated strain A. caviae Kuk1-(34) sp. from sewage waste showed great potential to degrade PHB-based biodegradable polymer film almost completely in soil under optimized conditions within a limited time period. The growth and the maximum enzyme activity of the PHB depolymerase enzyme assay of the isolated strain were observed at ambient temperature (27°C), indicating that the isolate has the capability to survive well under natural environmental conditions. SEM and FTIR analysis of the PHB polymer film confirmed morphological and structural changes due to the biodegradation of polymer film. The statistical analysis for production optimization recognized significant factors of the medium for increased production of PHB depolymerase enzyme that saves time and currency as well. It can be concluded that A. caviae Kuk1-(34). isolated from sewage waste emerges as a potent producer of extracellular PHB depolymerase enzyme, having the potential to act as a bio-catalyst for biodegradation of PHB-based bioplastics for large scale bioremediation of soil.

(XLSX)

Click here for additional data file.

20 Sep 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Marcos Pileggi, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide in your methods section the geographical coordinates of the sites from which sewage samples were collected.

3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file)”

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

7. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. Authors did an excellent job of explaining the concept clearly and accurately.

2. Author should be congratulated for the novel endeavour, however language throughout the MS has to be improved, typographical errors, grammatical errors and syntax errors should be removed before submission.

3. Uniformity should be maintained, while writing the author's name.

4. There is a variation of statement between line no. 43 & 52. Author should check that properly.

5. In line no. 89, 'soil waste' should be replaced by 'sewage waste soil'.

6. In line no. 93, author should only mention the abbreviated form of 'PHB', as the full form is already mentioned in the introduction section.

7. Recast line no. 140.

8. The sub-section 2.11. should be deleted as this protocol is already mentioned in the sub-section 2.7. Author should properly check and modify that.

9. Sub-section 3.1. should be merged with 3.2. and in line no. 198 'soil sample' should be replaced by 'sewage waste soil sample'.

10. In line no. 201, before screening how did they get confirmed that all the 22 isolates were PHB degraders? Author should rearrange the result.

11. In Fig. 1, how the author measure zone of diameter by continuous streaking?

12. In line no. 141, it is already mentioned that 0.0245 g PHB film was used for soil burial, then why term 'different' is used in line no. 222.

13. If only A. caviae Kukl-(34) strain was examined for PHB film degradation, then why did author stated that different strains were examined in line no. 223?

14. Author should properly explain the requirement of section 4. This section includes the results, which were already stated in the individual section.

15. Result pertaining to percentage of PHB degradation should be mentioned in sub-section 3.5. instead of section 4, line no. 322.

16. The total weight of PHB degraded after pre-treatment with A. caviae Kukl-(34) varied in line no. 43, 52, 322 & 349. Author should check it properly.

17. In line no. 352, insert space between 'Bano' and 'et al'.

18. All the tables should be arranged as per the sequence of result throughout the MS. For eg. Table 2 should be captioned as Table 1 as it is the first table in the result section.

19. Reports are available (Pati et al., 2020, https://doi.org/10.1007/s00284-020-01922-7) for PHB degradation within 7-21 days by composting method. Thus, author should compare degradation of PHB in laboratory as well as environmental condition.

20. Throughout the MS, term 'the' is used repeatedly. Author should look into that and rectify wherever necessary.

The MS is accepted for publication; however, minor modification is required as per suggestion.

Reviewer #2: 1) Line 40- depolymerase enzyme activity – change it to depolymerase activity

2) Line 44 - FTIR, SEM- Expand

3) Line 48 - ANOVA

4) Line 51- This strain – this culture

5) Line 52 – was found to be- rephrase

6) 2.2. Sample collected- Provide coordinates/GPS location of the sampling site

7) Line 100- hours – h

8) Line 122- Cite the reference for Bergey's Manual of Determinative Bacteriology

9) Line 123- Table 3 ?

10) Line 191 – isolated bacterial isolate?

11) Line 227- SEM analysis – what is SEM?

12) In Methodology – details of SEM are not mentioned

13) Line 307-308 - 4. Application of A. caviae Kuk1-(34) sp. for soil bioremediation by soil burial method in aspects of solid waste management. What is author intend by in aspect of solid….?

14) Discussion can be improved

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Deviprasad Samantaray

Reviewer #2: Yes: R. Z. Sayyed

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Reviewers Report PONE-D-21-27980.docx

Click here for additional data file.

Submitted filename: PONE-D-21-27980.pdf

Click here for additional data file.

29 Jan 2022

Comment 1. Please provide in your methods section the geographical coordinates of the sites from which sewage samples were collected.

Compliance: The geographical coordinates of the sites are illustrated in the Table 1 and now mentioned in the section 2.2.

Comment 2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Compliance: As per the suggestion, we thoroughly edit the manuscript for language usage, spelling, and grammar.

Comment 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

Compliance: It is now corrected.

Comment 4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Compliance: We have submitted some raw data.

Comment 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing ID in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Compliance: ORCID iD of the corresponding author is mentioned in the revised MS and also will be mentioned while resubmission in the portal.

Comment 6. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

Compliance: All tables are now included in the revised MS after the references and figure caption.

Point-by-point responses to the Reviewer #1 comments

Manuscript Number: PONE-D-21-27980

Title: Isolation and optimization of extracellular PHB depolymerase producer Aeromonas caviae Kuk1-(34) for sustainable solid waste management of biodegradable polymers

We are grateful to the reviewer for his careful reading of our manuscript, and for providing useful feedback to clarify both of our results and conclusions. The concerns raised by the Reviewer have all been addressed and highlighted with Turquoise colour which is summarized below.

Comment 1. Authors did an excellent job of explaining the concept clearly and accurately.

Compliance: Authors are very thankful to the reviewer for his appreciation.

Comment 2. Author should be congratulated for the novel endeavour, however language throughout the MS has to be improved, typographical errors, grammatical errors and syntax errors should be removed before submission.

Compliance: Authors are very thankful to the reviewer for his appreciation. However as per the suggestion, typographical errors, grammatical errors and syntax errors have been checked and removed.

Comment 3. Uniformity should be maintained, while writing the author's name.

Compliance: Uniformity has been kept.

Comment 4. There is a variation of statement between line no. 43 & 52. Author should check that properly.

Compliance: Line 43 (now line no. 50) indicates result of soil burial method without optimized condition and line 52 (now line no. 61) indicates result of soil burial method using optimized condition from CCD.

Comment 5. In line no. 89, 'soil waste' should be replaced by 'sewage waste soil'.

Compliance: As per the suggestion of the reviewer, “soil waste” is replaced with “sewage waste soil”.

Comment 6. In line no. 93, author should only mention the abbreviated form of 'PHB', as the full form is already mentioned in the introduction section.

Compliance: As per the suggestion the abbreviated form of PHB has been added.

Comment 7. Recast line no. 140.

Compliance: As per the suggestion, line no. 140 has been recast.

Comment 8. The sub-section 2.11. should be deleted as this protocol is already mentioned in the sub-section 2.7. Author should properly check and modify that.

Compliance: As per the suggestions, section 2.11 was checked and modified. This section explains the methodology of soil burial method using statistically optimized parameters obtained from CCD and is clearly distinct form section 2.7 that deals with soil burial method without optimized conditions. Moreover, we have modified the title of this section to make it more understandable.

Comment 9. Sub-section 3.1. Should be merged with 3.2. and in line no. 198 'soil sample' should be replaced by 'sewage waste soil sample'

Compliance: As per the suggestion, sub-section 3.1 is merged with 3.2 and the word “soil sample” is replaced with “sewage waste soil samples”.

Comment 10. In line no. 201, before screening how did they get confirmed that all the 22 isolates were PHB degraders? Author should rearrange the result.

Compliance: After the serial dilution the sewage waste soil samples was spread on BHM+PHB agar plates. The positive colonies form zone of hydrolysis as shown in Fig 1 on BHM+PHB agar plates. These colonies were marked and selected for further experiments. Also mentioned in Results section.

Comment 11. In Fig. 1, how the author measure zone of diameter by continuous streaking?

Compliance: Fig 1A and 1B were used to measure the zone of diameter as the circular colonies were there and continuous streaking in Fig 1C, 1D and 1E is just to show the zone of hydrolysis.

Comment 12. In line no. 141, it is already mentioned that 0.0245 g PHB film was used for soil burial, then why term 'different' is used in line no. 222.

Compliance: Since the PHB films were prepared in the laboratory using PHB powder (Sigma), so there was definitely a variation in the weight of the PHB film used for the soil burial method. Therefore, in the manuscript and in the Table 2A, we have mentioned the range of the weight of the PHB film (0.024 – 0.026 g) that was used in the soil burial method. In order to maintain similarity and avoid confusion, we have used the same weight range in methodology and results section.

Comment 13. If only A. caviae Kukl-(34) strain was examined for PHB film degradation, then why did author stated that different strains were examined in line no. 223?

Compliance: It is clearly mentioned in Table 2A that along with the strain Aeromonas caviae Kuk1-(34), two different strain i.e., CA6-(55) and CB2-(20) were also examined for the PHB film degradation in soil. Therefore we stated different strains were examined.

Comment 14. Author should properly explain the requirement of section 4. This section includes the results, which were already stated in the individual section.

Compliance: In the section 4 (now section 3.9), it explains the application of the bacterial strain Aeromonas caviae Kuk1-(34) for soil burial method in totally optimized conditions. The new result section 3.4 explains the soil burial method for unoptimized conditions. It has been found that weight loss of PHB film is 8.64% more in optimized conditions as compared to the weight loss of PHB film in unoptimized conditions.

Comment 15. Result pertaining to percentage of PHB degradation should be mentioned in sub-section 3.5. instead of section 4, line no. 322.

Compliance: Section 3.5 (now section 3.4) indicates the result of weight loss analysis of PHB film by soil burial method under the un-optimized conditions whereas in section 4 (now section 3.9), line no. 306, the result 94.4% indicates the degradation of PHB film using the optimized conditions from CCD. An increment of 8.64% was found during the weight loss of PHB film under optimized conditions.

Comment 16. The total weight of PHB degraded after pre-treatment with A. caviae Kukl-(34) varied in line no. 43, 52, 322 & 349. Author should check it properly.

Compliance: Line no. 43 (now line no. 50) indicates the result of PHB film degradation without using the optimized condition whereas line no. 52 (now line no. 61) and line 322 (now line 400) indicates the result of PHB film degradation using the optimized conditions after the CCD. As per the suggestion, line no. 349 (Line 340) is checked and corrected to 85.76% as this result also indicates the PHB film degradation without using the optimized conditions.

Comment 17. In line no. 352, insert space between 'Bano' and 'et al'.

Compliance: As per the suggestion, space is inserted between Bano and et al.

Comment 18. All the tables should be arranged as per the sequence of result throughout the MS. For eg. Table 2 should be captioned as Table 1 as it is the first table in the result section.

Compliance: As per the suggestion, table are checked and rearranged.

Comment 19. Reports are available (Pati et al., 2020, https://doi.org/10.1007/s00284-020-01922-7) for PHB degradation within 7-21 days by composting method. Thus, author should compare degradation of PHB in laboratory as well as environmental condition.

Compliance: As per the suggestion, we have mentioned the reference for Pati et al. (2020) in the discussion section as a comparative study of our work.

Comment 20. Throughout the MS, term 'the' is used repeatedly. Author should look into that and rectify wherever necessary.

Compliance: As per the suggestion, unnecessary use of term “the” have been checked and rectified.

Point-by-point responses to the Reviewer #2 comments

We are grateful to the reviewer for his careful reading of our manuscript, and for providing useful feedback to clarify both of our results and conclusions. The concerns raised by the reviewer have all been addressed and highlighted with yellow color which is summarized below.

Comment 1. Line 40- depolymerase enzyme activity – change it to depolymerase activity

Compliance: As per the suggestion depolymerase enzyme activity has been replaced with depolymerase activity

Comment 2. Line 44 - FTIR, SEM- Expand

Compliance: As per the suggestion, FTIR and SEM have been expanded.

Comment 3. Line 48 – ANOVA

Compliance: As per the suggestion, ANOVA has been expanded.

Comment 4. Line 51- This strain – this culture

Compliance: As per the suggestion “this strain” word has been replaced with “this culture” word.

Comment 5. Line 52 – was found to be- rephrase

Compliance: As per the suggestion “was found to be” rephrased in the manuscript.

Comment 6. 2.2. Sample collected- Provide coordinates/GPS location of the sampling site

Compliance: As per the suggestion, the coordinates of the sampling site are mentioned in the Table 1, section 2.2.

Comment 7. Line 100- hours – h

Compliance: As per the suggestion, modification hours into “h” have been done (Line 108).

Comment 8. Line 122- Cite the reference for Bergey's Manual of Determinative Bacteriology

Compliance: As per the suggestion, the citation of Bergey`s Manual of Determinative Bacteriology has been added (Bergey, Krieg and Holt, 1984).

Comment 9. Line 123- Table 3?

Compliance: As per the suggestion, Tables have been checked and renumbered.

Comment 10. Line 191 – isolated bacterial isolate?

Compliance: As per the suggestion, this line has been checked and removed.

Comment 11. Line 227- SEM analysis – what is SEM?

Compliance: As per the suggestion, SEM has been expanded

Comment 12. In Methodology – details of SEM are not mentioned

Compliance: In methodology section 2.8, details of SEM are explained from line no. 153-57.

Comment 13. Line 307-308 - 4. Application of A. caviae Kuk1-(34) sp. for soil bioremediation by soil burial method in aspects of solid waste management. What is author intend by in aspect of solid….?

Compliance: In section 4 (Noe section 3.9), regarding application, we are actually focusing on the degradation of PHB based bioplastics which are dumped in the soil as a solid structure. Therefore, we have mentioned the term solid waste management. Nevertheless, we changed the subtitle to accommodate the reviewer’s comment.

Comment 14. Discussion can be improved

Compliance: As per the suggestion, discussion has been modified.

Submitted filename: Rebuttal letter_Kuk1.docx

Click here for additional data file.

7 Feb 2022

Isolation and optimization of extracellular PHB depolymerase producer Aeromonas caviae Kuk1-(34) for sustainable solid waste management of biodegradable polymers

PONE-D-21-27980R1

Dear Dr. NA,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Marcos Pileggi, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors should be congratulated for doing an excellent job to improve the quality of research article by making desirable modifications.

Reviewer #2: The authors have addressed all the concerns and made the corrections as per my suggestion. The manuscript is greatly improved

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Deviprasad Samantaray

Reviewer #2: Yes: RIYAZALI ZAFARALI SAYYED

6 Apr 2022

PONE-D-21-27980R1

Isolation and optimization of extracellular PHB depolymerase producer Aeromonas caviae Kuk1-(34) for sustainable solid waste management of biodegradable polymers

Dear Dr. Roohi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Marcos Pileggi

Academic Editor

PLOS ONE