Production and characterization of poly 3-hydroxybutyrate-co-3-hydroxyvalerate in wheat starch wastewater and its potential for nanoparticle synthesis

Abstract

Polyhydroxyalkanoates (PHAs) are polymers with biodegradable and biocompatible properties accumulated in a wide variety of bacterial strains. In the present study, active sludge, wheat starch wastewater (WSW), and oil wastewater were used for the isolation and screening of PHA-accumulating bacteria. WSW was then implemented as a cheap and economical culture medium for the production of PHAs by the selected isolate. The extracted PHA was characterized, and the capability of produced biopolymer for preparing nanoparticles was evaluated. Based on the results, 96 different bacterial isolates were obtained, of which the strains isolated from WSW demonstrated the highest PHA-accumulation capability. The maximum PHA content of 3.07 g/l (59.50% of dry cell weight) was obtained by strain N6 in 21 h. The selected strain was identified by molecular approaches as Bacillus cereus. Afterward, the physicochemical characterization of an accumulated biopolymer was specified as a PHBV copolymer. Finally, spherical homogenous PHBV nanoparticles with a size of 137 nm were achieved. The PHBV nanoparticles showed a suitable small size and good zeta potential for medical applications. Hence, it can be concluded that isolated wild strain (B. cereus) has the potential exploitation capability for cost-effective PHBV production using the WSW.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00430-5.

Introduction

The consumption of high volumes of petrochemical plastics (around 140 million tons/year) reflects the drastic threat of using fossil fuels for human and earth life. Polyhydroxyalkanoates (PHAs) are a group of polymers that can be considered good substitutes for traditional plastics. They are biodegradable plastics that are produced through cultivating a variety of microorganisms on natural resources. So, they can mitigate the current problems of decreasing fossil resources and the environmental impact caused by petrochemical plastics. Polyhydroxyalkanoates (PHAs) are produced in response to stressful conditions in the presence of enough carbon resources [1]. Among the bacteria, Ralstonia eutropha, Alcaligenes latus, Pseudomonas putida, Azotobacter vinelandii, Cupriavidus necator, and several strains of Methylobacteria and Bacillus spp. can accumulate various types of PHAs [2–4].

The main advantages of bioplastics in comparison with traditional plastics are biodegradability, reproducibility, and biocompatibility [5]. Biopolymers have a wide range of potential applications [6]. Despite their host of benefits, they suffer from a variety of weaknesses that limit their commercial production and applications. Polyhydroxybutyrate (PHB) as the most common type of bioplastics is a polymer with stiffness, brittleness, and thermal instability properties that greatly restrict its wide applications [7]. However, depending on the microbial species and the type of carbon source, a new combination of PHAs with different physicochemical properties can be generated. The incorporation of 3-hydroxyvalerate (3HV) into PHB improves its properties, such as decreasing stiffness and increasing more flexibility of the product [8, 9]. In this regard, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has more potential for biomedical and industrial applications than PHB. Its biocompatibility, non-toxicity, and cell adhesion properties have enabled PHBV to produce innovative medical products such as biodegradable carriers for drug delivery, bone tissue substitutes, and sutures [7, 10]. Enhancement strategies have been developed in order to improve the physical and biological properties of PHBV to increase even more its potent industrial applications [11].

Despite several attempts made for the isolation and screening of more efficient microorganisms in PHA production, finding an economical and accessible substrate for commercial production has been always a major research concern. The price of bioplastics is currently much higher than traditional plastics due to the cost of carbon sources [12]. Despite many efforts to optimize the production of PHAs using pure cultures, commercial production is limited because of the high costs involved in the manufacturing processes. Over 50% of the total production expenses are allocated to the raw materials, with the carbon source being a major part of this cost. Thus, different economical carbon sources have been tried to produce high content of bioplastics using microorganisms [2, 13].

Presently, the use of industrial and agricultural wastes has been proposed as inexpensive substrates for reducing the costs of PHA production. Agro-industrial residues such as molasses [14], vinasse [15], dairy, pulp industry waste [16], waste potato starch [17], wheat straw [18], and several others have been scrutinized for economical PHA production. However, high-quality types of PHAs (PHBVs) are produced in the presence of organic acids such as valerate, propionate, and levulinic acid. PHBV production has been studied by the addition of organic oils as a co-carbon source, as well [19]. However, the addition of organic acids or organic oils to the culture medium would lead to an increase in PHBV production cost. Thus, finding new strains of microorganisms with high efficiency of PHBV production along with cost-effective and abundant industrial culture media is of high interest among the researchers.

In the present study, different bacterial strains from active sludge, oil industrial wastewater, and wheat starch wastewater (WSW) are isolated and screened for finding high-efficiency PHA-producing bacteria. Furthermore, raw WSW is used as a cost-effective medium for producing a high content of PHBV. WSW is suitable wastewater with high biological oxygen demand (BOD) and chemical oxygen demand (COD) content that separation of its mass as a by-product is not economically justified for small- and medium-sized plants. This paper also proposes a possible method via the emulsification/solvent evaporation technique for preparing the most widely used PHBV nanoparticles. Biopolymeric nanoparticles play an important role in the development of a variety of fields including drug delivery, tissue engineering, biosensors, and environmental applications [7, 20].

Materials and methods

Isolation of bacteria from WSW, active sludge, and oil industrial wastewater

WSW samples from Ardineh Company (Esfahan, Iran), active sludge from Water and Wastewater Company (Ahvaz, Iran), and oil industrial wastewater from Bandar Imam Petrochemical Company (Mahshahr, Iran) were collected for the isolation of PHA-producing bacteria. To isolate the bacteria, a serial dilution of each sample was prepared in distilled water. Then, 500 μl of diluted samples (10⁻² and 10⁻³) was spread on nutrient agar (Merck, Darmstadt, Germany) plates and incubated at 37 ± 1 °C for 48 h. All isolates were then stained with Gram stain procedure kit (Becton Dickinson Microbiology Systems, Cockeysville, Md.), controlled for purity, and stored at 3 ± 1 °C on nutrient agar (Merck) slant.

Preparation of seed culture medium

Seed culture medium was composed of 1 g/l of meat extract, 5 g/l of peptone, 5 g/l of sodium chloride, and 2 g/l of yeast extract. The medium was prepared in 250-ml Erlenmeyer flasks at a pH of 7.0 and autoclaved for 15 min at 121 °C. Each isolated strain was inoculated in the separate flask and incubated at 37 ± 1 °C for 36 h with an agitation rate of 150 rpm. The optical density (OD) of the medium was measured using a WPA Lightwave II UV-visible spectrophotometer (Biochrom Ltd.) at 625 nm every 3-h interval. When the cell concentration was equal to 1 McFarland, 5% (v/v) of seed media including 3 × 10⁸ cells/ml was aseptically transferred to the production media.

Primary screening of PHA-producing strains in synthetic medium

To screen the capability of PHA production by the isolates, 50 ml of basally defined M9 (DM9) medium was prepared in 250-ml Erlenmeyer flasks and sterilized at 121 °C. The medium consisted of 4 g/l of glucose, 1 g/l of NH₄Cl, 7 g/l of Na₂HPO₄, 3 g/l of NaH₂PO₄, 10 ml of 0.01 M CaCl₂, and 10 ml of 0.1 M MgSO₄·7H₂O [21]. Each production medium was inoculated by a 5% v/v seed culture with 3 × 10⁸ cell/ml. All cultures were incubated at 37 ± 1 °C for 72 h with an agitation rate of 150 rpm.

Growth curve studies of isolates

To find the most efficient time of biomass and PHA production for each isolated strain, bacterial biomass production was evaluated by measuring the OD of the culture medium using a UV-visible spectrophotometer (Biochrom WPA) at 625 nm for 72 h with 3-h intervals. PHA production was also evaluated as explained in the next sections.

Pretreatment of WSW

WSW was collected from a starch industry, Ardineh Company (Esfahan, Iran), and stored at 3 ± 1 °C until use. According to the accomplished analyses, the chemical oxygen demand (COD) and biological oxygen demand (BOD) of the starch were 17,050 ± 413 mg/l and 4180 ± 167 mg/l, respectively. The wastewater contained 21.3 ± 0.6 g/l of reduced sugar and 19.1 ± 0.3 g/l of total nitrogen. For removing the suspended solids and decreasing the nitrogen content, the samples were centrifuged (Beckman Sorvall RC-5B Refrigerated Superspeed Floor Centrifuge., USA) at 5500g for 15 min. The supernatant was then separated, and the centrifuged wastewater containing 18.1 g/l of reduced sugar and 2.9 g/l of total nitrogen was used as a stock medium for further screening of selected bacterial isolates. To make this waste similar to the DM9 medium, it was diluted with distilled water four times. Subsequently, the pH was adjusted to 7.0. The diluted treated starch wastewater contained 4.49 ± 0.3 g/l of reduced sugars and 0.71 ± 0.2 g/l of total nitrogen and had a COD of 4160 ± 216 mg/l and BOD of 920 ± 145 mg/l.

Final screening to find the best PHA-producing bacteria

Three isolates from each wastewater source with the highest productivity of PHAs in the synthetic medium were selected for further screening of the WSW medium. Briefly, 50 ml of diluted treated starch wastewater, as culture medium, was placed in a 250-ml Erlenmeyer flask at an adjusted pH of 7.0 and was autoclaved. Culture media were then inoculated with 5% v/v of the seed culture of each isolated strain and incubated as described earlier. The bacterial strain giving the highest productivity of PHAs was then selected for further studies.

Estimation of total dry cell weight, yield, and productivity

Fermented broth (10 ml) was centrifuged at 7000 rpm for 15 min and washed three times with distilled water. The bacterial pellet was dried in the oven at 60 °C until a constant weight was achieved.

The product-biomass yield was estimated based on the percentage of the PHAs existing in the dry cell weight and calculated by Eq. (1):

$$\left[\mathrm{Y}\frac{\mathrm{p}}{\mathrm{x}}\left(\%\right)=\text{extracted PHAs}\left(\frac{\mathrm{g}}{\mathrm{l}}\right)\times \frac{100}{}\mathrm{dry}\text{cell weight}\left(\mathrm{g}/\mathrm{l}\right)\right]$$ 1

The product-substrate yield was calculated as the percentage of the extracted PHAs per consumed sugar measured by the dinitrosalicylic acid (DNS) method [22], which is expressed by Eq. (2):

$$\left[\text{Yield}\mathrm{p}/\mathrm{s}\left(\%\right)=\text{extracted PHAs}\left(\mathrm{g}/\mathrm{l}\right)\times 100/\text{consumed sugar}\left(\mathrm{g}/\mathrm{l}\right)\right]$$ 2

The productivity was also estimated by PHA accumulation per hour as indicated by Eq. (3):

$$\left[\text{Productivity}\left(\mathrm{g}/\mathrm{l}\mathrm{h}\right)=\text{extracted PHAs}\left(\mathrm{g}/\mathrm{l}\right)/\text{time}\left(\mathrm{h}\right)\right]$$ 3

Morphological, biochemical, and molecular characterization of isolated bacterial strain

The bacterial isolates were morphologically identified, and biochemical properties were determined as prescribed by Bergey’s Manual of Determinative Bacteriology. For molecular characterization of the best strain, the 16S rRNA sequencing method with universal primer 16sF-5′ AGA GTT TGA TCC TGG CTC AG 3′ and 16sR-5′ ACG GCT ACC TTG TTA CGA CTT 3′ was used [23]. The genomic DNA was extracted using a DNA extraction kit (Bioneer, Korea). The sequenced DNA was evaluated with sequences in the public nucleotide database of the National Center for Biotechnology Information (NCBI) through the employment of basic local alignment search tool for nucleotides (BLASTN).

PHA extraction

A modified chemical extraction was utilized to extract PHAs from bacteria. Briefly, samples were centrifuged at 7000 rpm for 15 min. The pellets were digested with 30% sodium hypochlorite solution at 37 ± 1 °C for 60 min and then centrifuged at 7000 rpm for 15 min. This was followed by the process of washing residue by implementing boiling water, acetone, and absolute ethanol, in the order of their appearance, and centrifuged at each step [24]. The pellets were then dissolved in chloroform, methanol, and water with a ratio of 7:3:1 and incubated at 37 ± 1 °C for 2 h [25]. Finally, the suspension was centrifuged at 4500 rpm for 10 min. After centrifugation, the bottom layer of the chloroform solution was separated and kept in the oven at 55 °C. After the evaporation of chloroform, a white film of PHAs was obtained and assayed through the crotonic acid method. After the screening of bacteria and during PHA production using WSW by selected strain, the method of estimation was changed to weight/volume as high yields of PHAs were obtained. It is of note that the extracted PHAs were completely pure because of the serial washing of the samples during the extraction procedures as shown in online resource 1. This was confirmed by measuring the produced PHAs using the crotonic acid method and comparing it with the weight of the extracted biopolymers.

Estimation of PHAs by crotonic acid assay

The small amount of PHAs in samples can be estimated by spectrophotometric crotonic acid assay [24]. Briefly, 10 ml of concentrated H₂SO₄ was added to the extracted PHAs and heated for 10 min at 100 °C in a water bath to convert PHA crystals into crotonic acid. The absorption of the solution was then measured at 235 nm in a UV/Vis spectrophotometer (Biochrom WPA) against a boiled sulfuric acid blank. For making the standard curve, different concentrations (0 to 100 μg) of commercial standard PHB (Sigma-Aldrich, USA) in chloroform were prepared and treated with sulfuric acid to obtain crotonic acid. Finally, the absorption was plotted against concentrations to obtain a standard curve as shown in online resource 2 [24].

Characterization of the polymer PHAs

Nuclear magnetic resonance

¹H-NMR spectra were obtained by dissolving the biopolymer in deuterochloroform (CDCl3) at a concentration of 10 mg/ml on a Bruker Avance II 500 spectrometer at 20 °C with a pulse width of 7.4 ms (30° pulse angle), 1 s pulse repetition, and 10,330 Hz spectral width. Overall, 65,536 data points were analyzed. The internal standard was tetramethylsilane [26].

Fourier-transform infrared spectroscopy

Important functional groups presented in the extracted biopolymer were identified using FTIR spectroscopy. The biopolymer samples, mixed with 2% KBr, were compressed into transparent sample disks and placed in the FTIR spectrometer (Bruker IFS 120 HR). IR spectra were recorded by scanning with the spectral range of 4000–400 cm⁻¹ [27].

Differential scanning calorimetry

Differential scanning colorimetric test was performed to determine the thermal properties of the extracted biopolymer. The experiments were carried out using a Perkin-Elmer DSC 6000 instrument with sample and reference cells. After calibration, 3 mg of the extracted biopolymer was sealed in an aluminum plate and subjected to a temperature profile in the range of − 50 to 200 °C. The rate of heating was 10 °C min⁻¹ for the first heating scan and kept at isothermal temperature for 2 min. The sample was then cooled to 50 °C and reheated during a second heating scan to 200 °C at the same rate as the first step. The endothermic peaks were used for calculating both melting temperature (Tm) and glass transition temperature (Tg) [28].

The crystallinity (Xc) of polymer was determined according to Eq. (4):

[Xc (%) = ΔHf/(WPHBV ∗ ΔHfref) × 100] (4) [29].where ΔHf is melting enthalpy of the extracted PHBV, W_PHBV is the weight fraction of PHBV, and ΔHfref is theoretical melting enthalpy of the 100% crystallized PHBV, which was assumed to be 146 J g⁻¹ [29].

Preparation of P (3HB-co-3HV) nanoparticles via emulsification/solvent evaporation

PHBV nanoparticles were prepared by a modified emulsification/solvent evaporation technique [20, 30]. To obtain adequate and uniform nanoparticles, the effects of different concentrations of PHBV (1, 3, and 5 mg), solution phase (organic and aqueous phase), and ultrasonication time (20 and 40 min) were evaluated on the size of nanoparticles. Briefly, different amounts of PHBV (1, 3, and 5 mg) were dissolved separately in 100 ml of dichloromethane using both sonication and heating at 40 °C to obtain various concentrations of the organic phase. To study the effect of the aqueous phase, two different surfactants of ethanol 20% and PVA 20% were prepared in distilled water. For achieving the ethanol phase, the ethanol was added to water and the mixing was performed at room temperature for 3 min, whereas the heating at 90 °C with continuous stirring for 20 min was necessary for preparing the PVA solution. Afterward, 50 ml of each concentration of organic phases was injected into a 250-ml beaker containing 150 ml of each aqueous phase, separately. The variety of emulsions was then formed by ultrasonication of the various organic/aqueous compositions with an ultrasonic processor (Bandelin Sonoplus-HD3200; power density—200 watts; frequency—20 kHz) for 20 and 40 min. To evaporate the organic solvent, all the solutions were stirred separately at 1000 rpm overnight at 40 °C. The PHBV nanoparticles were collected in the aqueous solution by centrifugation at 19,000 rpm for 30 min and washed in triplicate with Milli-Q water. The nanoparticles were morphologically characterized using a scanning electron microscope (SEM). The particle size and surface charge of nanoparticles were identified by a dynamic light scattering machine (DLS).

Field emission scanning electron microscopy

To study the morphology of the PHBV nanoparticles, the field emission scanning electron microscopy (FESEM) analyses were performed using a TESCAN device (model MIRA 2). The nanoparticles were coated with a 10-nm gold layer and the FESEM analyses were carried out at 15 kV.

Dynamic light scattering

The particle size (diameter, nm) and surface charge (zeta potential, mV) of nanoparticles were evaluated by DLS using a Zetasizer 3000 (Malvern Instruments, UK). Each sample was suspended in 1 ml phosphate-buffered saline (PBS; pH 7.4), followed by calculating the size and zeta potential.

Statistical analysis

PHB production of isolated strains was statistically compared using one-way analysis of variances (ANOVA) and Tukey’s multiple range test at p value < 0.05. These tests were applied to distinguish significant differences between means of three replicates of each isolate using the Minitab© V.14 (Minitab Inc., USA) software.

Results

Screening of PHA-producing bacteria in synthetic media

A total of 96 different bacterial isolates were obtained. Among the isolated strains, 32 isolates from active sludge, 24 isolates from starch wastewater, and 15 isolates from oil wastewater were able to produce PHAs. The strains isolated from WSW showed the highest PHB accumulation capability. The average values of the top eight strains of each isolation source are given in Table 1. The strain N6 isolated from WSW accumulated the highest amount of PHAs (1.603 g/l) in the synthetic medium (DM9).

Table 1.

The top eight strains of each wastewater source with the highest PHA accumulation capability in synthetic media

Strain	Dry cell weight (g/l)	PHAs (g/l)	Time (h)	Productivity (g/l h)
N1	2.021 ± 0.005	0.121 ± 0.002	24	0.0050
N2	2.523 ± 0.004	0.613 ± 0.005	21	0.0291
N3	3.012 ± 0.006	0.062 ± 0.004	27	0.0023
N4	2.879 ± 0.003	0.031 ± 0.002	21	0.0015
N5	3.287 ± 0.006	1.070 ± 0.009	18	0.0511
N6	3.491 ± 0.005	1.486 ± 0.008	18	0.0825
N7	3.231 ± 0.004	0.075 ± 0.005	27	0.0028
N8	3.981 ± 0.004	0.327 ± 0.008	21	0.0156
S1	1.012 ± 0.006	0.013 ± 0.001	24	0.0005
S2	1.672 ± 0.005	0.454 ± 0.007	27	0.0168
S3	1.981 ± 0.004	0.007 ± 0.001	27	0.0003
S4	1.895 ± 0.005	0.012 ± 0.002	27	0.0004
S5	2.034 ± 0.005	0.031 ± 0.003	30	0.0010
S6	2.510 ± 0.004	0.044 ± 0.001	33	0.0013
S7	1.101 ± 0.003	0.294 ± 0.006	21	0.0140
S8	1.947 ± 0.004	0.088 ± 0.004	30	0.0029
O1	1.129 ± 0.002	0.064 ± 0.003	24	0.0027
O2	1.720 ± 0.003	0.041 ± 0.001	27	0.0015
O3	1.829 ± 0.004	0.027 ± 0.001	27	0.0010
O4	1.301 ± 0.002	0.101 ± 0.005	24	0.0042
O5	1.506 ± 0.003	0.019 ± 0.001	24	0.0008
O6	0.872 ± 0.002	0.070 ± 0.003	24	0.0029
O7	2.053 ± 0.003	0.033 ± 0.002	27	0.0012
08	1.962 ± 0.003	0.057 ± 0.003	27	0.0021

Based on Gram reactions, 49 isolates of PHB producing bacterial isolates were Gram-positive and 22 isolates were phenotypically Gram-negative. Although there are many reports of the ability of Gram-negative bacteria to produce PHAs, the presence of LPS in the cell wall is always a concern for medical applications [10].

Additional screening of the highest PHA-producing bacteria in treated WSW medium

Three Gram-positive isolates from each wastewater with the highest PHA production were further screened for their capability of producing PHAs in the WSW medium as a low-cost carbon source. To the best of our knowledge, there is no study available for PHA-producing bacteria using WSW. The results showed that the amount of PHB accumulation increased significantly (p < 0.05) compared to the synthetic medium. The maximum PHB content of 59.50% (3.07 g/l) was obtained by the same strain N6 in 21 h (Table 2). The selected PHA-producing bacterium was identified as a member of the genus Bacillus sp. Further characterization with 16S rRNA sequencing and its comparison with available data in the NCBI database showed the closest matching of strain N6 with the Bacillus cereus (99% homology). Thus, the isolated bacterium was identified as B. cereus.

Table 2.

The top three selected strains of each wastewater source with the highest PHA accumulation capability in WSW

Strain	Dry cell weight (g/l)	PHAs (g/l)	YP/X (%)	Consumed sugar (g/l)	YP/S (%)	Productivity (g/l h)
N2	2.71 ± 0.1	1.43 ± 0.02	52.77	3.74	38.23	0.0433
N5	4.88 ± 0.09	2.69 ± 0.03	55.12	4.18	64.35	0.1121
N6	5.16 ± 0.08	3.07 ± 0.04	59.50	4.31	71.23	0.1462
S2	1.89 ± 0.09	0.786 ± 0.02	41.59	2.39	32.89	0.0238
S7	1.34 ± 0.05	0.429 ± 0.009	32.01	2.65	16.19	0.0159
S8	0.98 ± 0.07	0.056 ± 0.07	5.71	3.25	1.72	0.0016
O1	0.89 ± 0.05	0.098 ± 0.008	11.01	1.48	6.62	0.0033
O4	1.78 ± 0.08	0.167 ± 0.01	9.38	2.73	6.11	0.0051
O6	1.29 ± 0.05	0.102 ± 0.006	7.91	3.92	2.60	0.0038

The comparison between growth pattern and PHA production of isolated Bacillus cereus in synthetic and WSW media

In the synthetic medium, Bacillus cereus accumulated the highest level of PHAs of 1.49 g/l after 18 h (Fig. 1a). When WSW was used as a culture medium, the maximum PHA concentration was 3.07 g/l, which was obtained after 21 h of fermentation (Fig. 1b). Although the screened strain reached the maximum PHA production in a shorter time in the synthetic medium, the higher production (more than twofold) in the effluent medium resulted in the higher yield and productivity (59.5% and 0.146 g/l h, respectively).

Fig. 1.

The growth pattern and PHA production of N6 strain in the synthetic (a) and wheat starch wastewater (b) medium. The circle represents the dry cell weight and the square represents the extracted PHA. All data are the means ± SD of experiments (n = 3)

Unlike PHAs, the highest content of CDW in synthetic and WSW medium was 4.138 g/l and 5.587 g/l, which was obtained at 30 and 33 h, respectively. However, the yield and productivity of PHAs increased from initial exponential growth to near the end of the logarithmic phase. The result suggested that cells accumulated PHAs while growing, but cell growth continued until the stationary growth phase (Fig. 1 a and b).

Characterization of extracted biopolymer

¹H-NMR spectroscopy

The structure of the extracted biopolymer was investigated by ¹H-NMR (Fig. 2). The peaks at 2.62, 5.27, and 7.28 ppm are indexed to protons of methylene side group, methyne group, and impurities of protons in the solvent (chloroform), respectively. The characteristic peaks at 0.86 and 1.28 ppm are attributed to the absorption of protons of methyl (CH3) from the hydroxyvalerate and methyl (CH3) from the hydroxybutyrate, respectively. To determine the mole percent of hydroxyvalerate in the poly (3HB-co-3HV) biopolymer, Eq. (5) was applied [31].

$$\mathrm{HV}\mathrm{mol}\%=\frac{\text{Area}\mathrm{CH}3\left(\mathrm{HV}\right)\ast 100}{\text{Area}\mathrm{CH}3\left(\mathrm{HV}\right)+\text{Area}\mathrm{CH}3\left(\mathrm{HB}\right)}$$ 5

Fig. 2.

¹H-NMR spectroscopy of extracted biopolymer

Thus, the percentage of the HV compound was calculated to be 16%.

FTIR

The FTIR spectra of extracted biopolymer indicated the specific peaks of PHBV (Fig. 3). The absorption peaks at approximately 1277 cm⁻¹ and 1735 cm⁻¹ are related to the saturated ester bond of C–O and carbonyl (C=O) groups, respectively. The high absorption at 1378 and 1452 cm⁻¹ is consistent with the tensile and flexural modes of bending vibration of the methyl (–CH3) groups, respectively. The methine (–CH) and hydroxyl (–OH) groups were characterized by absorption peaks at 2933 and 3437 cm⁻¹ as reported earlier [32].

Fig. 3.

FTIR spectrum of extracted biopolymer

Differential scanning calorimetry analysis

The DSC thermogram was applied to measure the characteristic calorimetric properties of biopolymer (Fig. 4). Crystallization temperature (Tc) was calculated by the cooling curve. The curve showed that the Tc value was 61.46 °C. An exothermic peak was observed during recrystallization in the same temperature range to Tc during the second heating (green curve). Moreover, a wide crystallization peak was observed (blue curve), which could be due to low crystallization speed and incomplete crystallization process during the cooling. Therefore, cooling should be performed at a slower rate to crystallize polymer completely. However, the high melting enthalpy of approximately 94.35 J g⁻¹ indicated that the extracted biopolymer was highly crystalline. Also, the crystallization percentage was calculated at around 64.6% using the melting enthalpy obtained during the scan [33] based on Eq. (6):

$$\left[\mathrm{Xc}\left(\%\right)=\left(\mathrm{\Delta Hf}/\text{ΔHfref}\right)\ast 100\right]$$ 6

where ΔHfref is the enthalpy of fusion of 100% crystallized PHBV, 146 J g⁻¹, ΔHf is 94.35 J g⁻¹, and Xc (%) is 64.6%.

Fig. 4.

Differential scanning calorimetry of extracted biopolymer

Glass transition temperature (Tg) and melting point (Tm) were measured based on the second heating curve. Tg was − 0.89 °C and Tm was calculated with the twin melting peak (60.05 °C and 149.36 °C, respectively). The results were similar to those of the DSC reported for PHBV and the twin melting peak confirmed the formation of the copolymer.

Particle size and zeta potential of nanoparticles

The result revealed that increasing the homogenization rate significantly (p < 0.05) reduced the size of nanoparticles. The increase in biopolymer concentration significantly increased particle size. The optimum nanoparticles were obtained by injecting a 1% PHBV solution in 20% PVA solution under 40 min ultrasonication. FESEM micrograph of PHBV nanoparticles indicated that the average particle size of produced nanoparticles was about 100–150 nm (Fig. 5). The size of nanoparticles calculated by dynamic light scattering measurement was also around 136 nm in diameter (Fig. 6). Furthermore, these PHBV nanoparticles indicated a negative surface charge of about 33 mV (Fig. 7).

Fig. 5.

Scanning electron microscopy image of the PHBV nanoparticles

Fig. 6.

The dynamic light scattering analysis of PHBV nanoparticles

Fig. 7.

The zeta potential of PHBV nanoparticles

Discussion

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polymers that are accumulated in a wide range of bacteria. In the present study, DM9 media has been employed for screening of PHA-producing bacteria from various wastewaters. Based on cell growth rate and PHAs content, three top Gram-positive isolates from each wastewater were selected for the screening of the most efficient isolate using treated WSW as the production medium.

The isolated bacterium was identified as B. cereus. Bacillus genus is widely used in PHA production due to its high growth rate and PHA accumulation [34, 35]. Aslim et al. investigated the capability of PHB production in 40 Bacillus sp. and found that the highest product-biomass yield of PHB (48%) by Bacillus megaterium [36]. Labuzek and Radecka found that the PHB content of dry cell weight of B. cereus UW85 was 25% when cultured on glucose media [37]. Screening of 29 isolates of the Bacillus genus from soil samples, consisting of three B. cereus strains with a PHB production yield of 28%, was also reported by Yilmaz et al. [38]. In the present study, the product-biomass yield of biopolymer for B. cereus was approximately 43% on glucose media, which was moderately higher than that obtained by Aslim et al., Labuzek et al., and Yilmaz et al. [36–38]. However, a few reports are available on PHB production by Bacillus strains using industrial media. Halami showed that biopolymer production yield in B. cereus CFR06 was 48% of dry cell weight with the PHB concentration of 0.9 g/l on starch medium [12], while Tufail et al. reported the production of 18.1% PHA using waste frying oil [39]. Comparatively the results of the present study showed a higher product yield and concentration (59.5% and 3.07 g/l, respectively). According to Sharma and Bajaj, biopolymer production efficiency in an industrial medium can be higher than the synthetic medium [13], which is completely in agreement with the present study (Fig. 1 a and b).

Furthermore, the C:N ratio of the treated medium was around 6:1, which was close to the C:N ratio suggested by many investigators for producing PHB in bacterial strains such as Bacillus sp. [34]. An explanation for this result is that the WSW is composed of a high amount of organic substrates that are biodegraded and converted to PHAs by bacteria. As previously described, starch effluent contains high levels of reduced sugars, making it a valuable medium for the fermentation processes. Also, the treated effluent had a suitable COD/BOD ratio (4.57) for microbial growth. Bhuwal et al. reported that Bacillus sp. NG220 accumulated 3.951 g/l PHB in a cardboard industry wastewater with a COD/BOD ratio of 3.9 to 5 in 72 h [35]. In the present study, the maximum PHB content of 59.50% (3.07 g/l) was obtained by the strain N6 in 21 h using treated WSW (Table 2). Thus, productivity was almost 3 times higher than that reported by Bhuwal et al.

Based on the results, the production of PHAs increased up to the highest level and decreased, thereafter. Because of the interesting fast reduction of PHAs during the stationary phase, the experiments were repeated in triplicate precisely and confirmed. This reduction indicates that a high concentration of PHAs inhibits the further production of a biopolymer. Also, it showed that bacteria have utilized PHAs as a nutrient source due to inadequate carbon source in the medium [4, 39, 40]. Moderately lower reduction of biopolymer in the effluent medium in comparison with synthetic medium might be due to the consumption of some complex compounds in the WSW medium after the expiration of simple carbon and nitrogen sources. In comparison, in the synthetic medium, due to the absence of complex compounds, the bacteria may consume PHAs as carbon and nitrogen sources. Therefore, the biopolymer content decreased rapidly.

According to the FTIR spectroscopy and thermal properties, the extracted biopolymer could be classified as a thermoplastic PHBV copolymer. There are a few reports on the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by B. cereus. Masood et al. showed that Bacillus cereus strain S10 could produce PHBV with a yield of 69.91% after 48 h in glucose medium [41]. Further studies have confirmed the effect of propionic acid as an expensive inducer of polyvalerate versus polybutyrate in PHBV [33]. Since propionic acid is used as a fungicidal active ingredient in grain marketing, the WSW probably has the required propionate content. Hence, finding a wild Gram-positive strain, which produced the polymer efficiently, and producing the PHBV without the addition of extra complements indicated that the WSW medium could be proposed as a cost-effective source for mass production of PHBV.

As mentioned, the production of PHBV is more attractive than PHB due to its biomedical and industrial applications such as drug delivery carriers [42]. The biological functions of biopolymer nanoparticles depend on several parameters such as size, surface charge, and cell type [43]. The small size of the nanoparticles increases their specific surface area and thus increases their contact surface with the epithelial surface. As a result, it has greater potential for nonspecific uptake into the cell or receptor-mediated endocytosis [44]. In the present study, the size of the nanoparticles was optimized using different homogenization rates and surfactants. The negative charge of PHBV nanoparticles increases their targeted efficiency and reduces the ability of immune proteins to bind to drug-carrying nanoparticles. Hence, nanoparticles will remain in the bloodstream for a long time without causing an immune response [30]. Furthermore, the produced nanoparticles could be considered suitable nanoparticles for medical applications because of their appropriate size and zeta potential and their valerate structure.

Conclusion

The wild type strain of B. cereus isolated from wheat starch wastewater was able to consume efficiently this wastewater as a nutrient source for growth and PHBV production. The utilization of wastewaters not only reduces the cost of PHBV production but also prevents environmental pollution due to petrochemical plastics use. WSW is a high efficient substrate for biopolymer production, especially 3HB-co-3HV copolymer, without the addition of any inducer such as propionic acid and valeric acid. The PHBV nanoparticles showed a suitable small size and good zeta potential. Hence, isolated wild strain (Bacillus cereus) because of using WSW can be exploited for cost-effective PHBV production.

Authors’ contributions

Design of study was accomplished by Davood Zare, Neda Sinaei, and Mehrdad Azin. Data and draft of manuscript were collected by Neda Sinaei and finalized by Davood Zare and Mehrdad Azin. All authors read and approved the final manuscript.

Data availability

All isolated strains are preserved in Persian Type Culture Collection (PTCC) and data are available by N. Sinaei.

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Conflict of interest

The authors declare that they have no conflict of interest.

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