Introduction

Synthetic plastic takes years to degrade completely and at the same time produces toxins during its degradation process in nature. This is the major cause which led researchers to search for an alternative polymer that can be degraded easily by microorganisms in an eco-friendly manner and at the same time supporting the properties of commercial petroleum based plastics. Polyhydroxyalkanoates (PHAs) produced by bacteria are suggested as a potential alternative polymer for petroleum-derived synthetic plastics because of their good biodegradability and biocompatibility properties. More than 150 constituents of PHAs have been identified [1] and characterized and among them polyhydroxybutyrate (PHB) is one of the best characterized derivative of PHA. Though PHB could replace the present commercial plastics; its high cost of production is the major drawback that pulls down the possibility of commercialization of PHB. Therefore, production strategies can be improved using suitable carbon and nitrogen sources at suitable concentrations [2] which can lead to reduction in production cost of the final product implying wider use of PHAs in daily life [3]. Moreover, environment-friendly and cost-effective methods for large scale PHB production using novel microbial strains are also important for commercial applications of PHB. Most of the PHB producing bacteria was isolated from soil and activated sludge [4]. Therefore, municipal waste areas may serve as a stress prone environment favouring to explore a novel strain capable to produce PHB.

There are three important enzymatic steps involved in PHB production and for this the starting material is acetyl-coenzyme A (acetyl-CoA). Two acetyl-CoA molecules undergo reversible condensation reaction into acetoacetyl-CoA which is catalyzed by β-ketothiolase (encoded by phbA). NADPH-dependent acetoacetyl-CoA reductase (encoded by phbB) reduces acetoacetyl-CoA into 3-hydroxybutyryl-CoA and finally, polymerization of 3-hydroxybutyryl-CoA monomers by PHB synthase (encoded by phbC) produces the polymer PHB [5].

In PHB production, 40–48% of the total production cost goes to the raw materials where carbon source accounts for 70–80% of the total production cost [6]. Therefore, search for a carbon source which can be easily available and effectively used to produce PHB, is the need of the hour. Cow dung substrate was effectively used as a medium for the growth of bacteria [7]. Cow dung samples collected from different places provided with different feeds like grass, corn and palm tree waste; produces different composition of total carbon, total nitrogen, crude protein and organic matter. It was found that cow dung obtained from grass feed cows have more nutrient characteristics while compared to the others [8]. Therefore, cow dung collected from different feed sources may have significant effect on growth of bacteria also. So, bacterial growth may either increase or decrease depending on the sources of cow dung samples used in the media. The chemical composition of cow dung consists of 1.6% N, 0.70% P, 0.53% K, 0.91% Mg, 2.71% Ca, 0.50% Na, 56.8% organic matter and C:N of 7.9 [9]. Formulating a proper medium for maximum production of PHB is of critically important from an industrial point of view as the medium components significantly affect the product yield. An ideal substrate should be available throughout the year [10]. Considering its easy availability, an attempt was made to use cow dung as a cheap substrate for the production of PHB from Bacillus pumilus H9. Reports on the use of cow dung substrate as carbon source for the production of PHB are limited or, perhaps, not available.

In a fermentation process, the physical and chemical parameters have to be optimized owing to their impact on economy and practicability of the process. Conventional “one-factor-at a time” method was followed previously which is time consuming and painstaking for a large number of variables, and does not analyse the interactions among variables. So, statistical design of experiments is used in many studies in order to reduce the number of experiments despite large number of variables. In fermentation process seeking optimized conditions for improved product yield, response surface methodology (RSM) is an efficient tool used to study the interactive effects of the process parameters [11]. Therefore, for medium optimization and to study the interactions among various physicochemical parameters involved in biopolymer production, application of statistical methods and RSM has been extensively applied in microbial fields in the recent years [12,13,14].

The aim of the present study is to isolate a potential PHB accumulating bacterium and to evaluate the use of cowdung as a cheap carbon source through statistical media optimization. Also, PHB genes were amplified to confirm the production of PHB genotypically. To our knowledge, this is the first report claiming that cowdung can be utilized as a low cost substrate for PHB production thereby converting bio waste material into a value- added product biopolymer.

Materials and Methods

Isolation of PHAs Producing Bacteria

Soil samples were collected from the municipal waste areas of Hailakandi district of Southern Assam in sterile falcon tubes and transferred to lab under aseptic conditions. For screening and isolating PHB producing capability of the isolates, the pure cultures were grown in basally-defined M9 medium (DM9 medium) containing (g/L of distilled water): glucose, 4; NH4Cl, 1; Na2HPO4, 7; NaH2PO4, 3; 10 ml 0.01 M CaCl2 and 10 ml 0.1 M MgSO4·7H2O. The medium containing flask was autoclaved at 15 psi for 30 min, cooled and then inoculated with 1% (v/v) of 18 h old culture and incubated at 30 °C for 48 h. The contents of the flask were then centrifuged at 8000 g for 10 min at 4 °C, and the cell pellets were stained with Sudan Black B stain and observed under oil immersion microscope (Nikon Eclipse E200).

Characterization of the Isolated Bacteria

The morphological and physiological properties of the strain H9 were investigated according to Bergy’s manual of determinative bacteriology [15].

16S rRNA Gene Sequence Analysis

The genomic DNA was extracted using HiPurA™ Bacterial genomic DNA Extraction Kit and nearly full-length 16S rRNA sequences were amplified by PCR using primers 27F and 1492R [16]. PCR amplifications were carried out with the following temperature profile: 5 min at 95 °C, 30 cycles of denaturation (60 s at 94 °C), annealing (60 s at 55 °C), extension (2 min at 72 °C) and a final extension for 7 min at 72 °C. Amplified products were separated on 1% agarose gel and observed with a UV transilluminator and documented with GelDocXR software (Biorad). The amplification product was purified using Genejet Gel Extraction PCR purification kit according to the manufacturer’s instruction. The purified PCR product was sequenced by ABI 3500 Genetic Analyser. The 16S rDNA gene sequence analysis was carried out by Bio-Edit software and identification of bacteria was done using NCBI-BLAST (National Centre for Biotechnology Information http://www.ncbi.nml.nih.gov) program. The 16S rDNA sequence of the bacterium was compared with the available database using blastn program through NCBI. Search for the database nucleotide collection was done using Megablast (Optimize for highly similar sequences). The blast parameters used are as follows: Maximum target sequences of 100, Expect threshold of 10 and the scoring parameters involves 1, -2 for Match/Mismatch scores and a linear gap costs was used.

Media and Growth Conditions

For seed culture preparation, a loop full of the stock culture was inoculated in 100 ml shake-flask containing 20 ml of nutrient broth and the flask was incubated in rotary shaker for 24 h at 37 °C and 220 rpm. The seed was then inoculated into a 200 ml of nutrient broth and again incubated for 24 h under the same conditions. Growth was monitored spectrophotometrically by measuring the culture’s absorbance at 600 nm. The cells were harvested by centrifugation, washed twice with phosphate-buffer (pH 7.0) and the biomass was air-dried. Weight of the dried mass was considered as the dry weight of the sample.

Analytical Procedure

Cell concentration was determined by measuring the dry cell weight (DCW). For this, 5 ml broth culture was centrifuged, washed with distilled water and dried at 60 °C until the weight does not decrease further. The residual mass was defined as total DCW minus PHB weight and PHB (%) as the percentage of the ratio of PHB to DCW.

Cow Dung Substrate Treatment

Cow dung was collected locally from Irongmara village in Barjalenga, Cachar District, Assam, India and dried for 1 week by sun drying. Using a mixer grinder, it was powdered, sieved and stored at room temperature for further use. The powdered cow dung was mixed properly with distilled water and kept on shaker for 12 h at 120 rpm at 28 °C and filtered through double layer of muslin cloth. The filtrate obtained through muslin cloth was processed by autoclaving for 20 min at 121 °C and 15 psi. This cow dung filtrate was used as a cheap substrate carbon source in the production medium for PHB production by the bacterium B. pumilus H9.

Production of PHB

PHB from B. pumilus H9 was extracted following Hypochlorite method with slight modification [17]. The bacterium was grown in 50 ml of modified production medium (composing cowdung, 4 g/l; sucrose, 4 g/l; peptone, 1 g/l; Na2HPO4, 7 g/l; NaH2PO4, 3 g/l; 10 ml 0.01 M CaCl2 and 10 ml 0.1 M MgSO4·7H2O) and incubated at 30 °C for 48 h on rotary shaker at 160 rpm. 10 ml of the cell suspension was centrifuged at 6000×g for 10 min. The cell pellet was washed with 10 ml saline and was recentrifuged to get the pellet. Cell pellet was then suspended in 5 ml sodium hypochlorite (4% active chlorine) and incubated at 37 °C for 10 min with stirring. This extract was centrifuged at 8000×g for 20 min and the pellet of PHA was washed with 10 ml cold diethyl ether. The pellet was again centrifuged at 8000×g to get the purified PHA and was dried to constant weight at 60 °C. Amount of PHB produced was estimated by measuring its dry weight.

Effect of Substrates on PHB Production

Effect of various substrates (cow dung, sucrose, glucose, fructose, mannitol, maltose, xylose, trehalose, starch and lactose among carbon sources; peptone, urea, ammonium chloride, tryptone, yeast extract, beef extract, gelatin, casein, NH4NO3 and (NH4)2SO4 among nitrogen sources) on the production of PHB was determined. The concentration of the selected substrates was taken from low to high in the range of 2–6 g/l for cowdung, 3–7 g/l for sucrose and 0.5–2.5 g/l for peptone. The concentration of the substrates contributes for maximum production of PHB and was considered for central composite rotary design (CCRD).

Effect of Time and pH on PHB Production

Effect of time on the production of PHB was studied by inoculating B. pumilus H9 into the modified production medium containing cowdung, sucrose and peptone. The samples were then collected for every 12 h. The range of initial pH of the medium under consideration was 5–9. Time and pH of maximum production of PHB was determined by considering the dry weight of the PHB simultaneously.

Medium Optimization for Production of PHB

Experimental Design

CCRD obtained by using the software Design-Expert 6 (Stat-Ease, Inc., Minneapolis, USA) was applied to optimize and to study the interactive effects of four variables, viz. concentrations of cowdung, sucrose, peptone and initial pH of the medium. Central composite design at the given range of the above parameters in terms of coded and actual terms is presented in Table 1.

Table 1 The coding levels of four variables involved in optimizing the medium components for maximizing the PHB production
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Optimization of Medium for PHB Production

A two level four factorial CCRD was used to optimize the medium composition. The four variables used were cowdung, sucrose, peptone and pH. For CCRD experiments, the actual levels of variables were selected as a central point of experiments. An experimental design of 30 experiments with six trials for the central point was formulated using the Design-Expert 6 software (Stat-Ease, Inc., Minneapolis, USA). Experiments were conducted in 500 ml Erlenmeyer flask containing 90 ml of media prepared according to the design inoculated with 10 ml seed culture. The flasks were kept in incubator shaker maintained at 30 °C and 160 rpm. Response studied was PHB dry weights (g/l) at the end of 48 h. 3D graphs were created to understand the interaction of various factors. And the graphs were used to analyse the optimized components of the medium which influences the responses. The point prediction is a special feature of this software which was used to confirm the obtained optimum value.

Statistical Analyses

The software Design-Expert (Stat-Ease, Inc., Minneapolis, USA) was used for regression analysis of experimental data and to plot response surface. ANOVA was used to estimate the statistical parameters.

Amplification of phbA, phbB and phbC Genes

To amplify phbA, phbB and phbC genes in B. pumilus H9, the genomic DNA was extracted using HiPurA™ Bacterial genomic DNA Extraction Kit and the genes were amplified by polymerase chain reaction (PCR) using the specific primers (Table 2). PCR amplifications was carried out with the following temperature profiles separately: phbA: initial denaturation (5 min at 94 °C), 35 cycles of denaturation (30 s at 94 °C), annealing (40 s at 55 °C), extension (40 s at 72 °C) and a final extension for 5 min at 72 °C; phbB: initial denaturation (3 min at 94 °C), 35 cycles of denaturation (30 s at 94 °C), annealing (30 s at 55 °C), extension (30 s at 72 °C) and a final extension for 7 min at 72 °C and phbC: initial denaturation (5 min at 94 °C), 35 cycles of denaturation (30 s at 94 °C), annealing (40 s at 55 °C), extension (1 min at 72 °C) and a final extension for 7 min at 72 °C.

Table 2 Primers used in this study
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Results and Discussion

Screening of PHB Accumulating Bacteria

Different bacterial strains that effectively accumulated PHB were isolated from the soil samples of municipal waste areas of Hailakandi district, Assam, India. Screening was done using Sudan Black B staining method, in which the positive isolates showed blue–black coloured cytoplasmic granules inside the cytosol of the bacteria. An effective PHB producer was chosen based on the dry weight of the extracted PHB. Strain H9 was selected for further study as it shows highest PHB content among other bacteria.

Identification and Characterization of Strain H9

The isolate H9 was identified using a series of biochemical tests and morphological characteristics. According to the methods described in “Bergey’s manual of systematic bacteriology” [19], microbiological properties were investigated and the organism was identified as a member of the genus Bacillus. Further characterization was confirmed with 16S rRNA gene sequence, in which almost-complete 16S rRNA gene sequence of the strain H9 (1395 bp) put in pair-wise alignment exhibited 99% similarity at the DNA gene level. Further, following BLAST analysis of the 16S rRNA gene sequence of the strain H9 it is observed that B. pumilus B6 (KJ870186.1), Bacillus safensis AL-A32 (KC844810.1) and Bacillus safensis MUGA218 (KJ672379.1) are the nearest phylogenetic neighbors of the strain H9 with a similarity of 99, 99, 99% respectively. Therefore, the bacterium was identified as B. pumilus H9 and its nucleotide sequence has been deposited in NCBI database and the sequence was assigned the accession number: KT907044. Municipal waste sites have high BOD and COD values and also have high carbon content with less nitrogen and phosphorus. This variant nutrient condition (especially carbon to nitrogen) produces a stress prone environment which activates bacteria to accumulate PHB inside the cytosol of the bacteria. Different Bacillus species isolated from different environments like wastewater, sewage and sludge were found to accumulate PHB [20,21,22,23,24]. Bacillus is also a prominent genus found in soil and water, which can grow easily using cheap raw materials [25].

Production of PHB by B. Pumilus H9

Maximum cell density was reached at 48 h of cultivation by B. pumilus H9 and pH of the production medium was decreased from 7.0 to 5.6 during the growth of this bacterium. The biopolymer PHB synthesis was confirmed by Sudan black B staining. At the log phase of its growth, PHB accumulation was started and continued up to 48 h of incubation of B. pumilus H9. The maximum PHB synthesis was reached at stationary phase of growth and decreased slightly after the stationary phase which may be due to the intracellular utilization of PHB as energy and carbon reserves. The biomass (DCW) of 2.87 g/l of medium was achieved with 1.90 g/l of PHB (66.2% of DCW) from B. pumilus H9 (Fig. 1). Literature reveals that PHA production from Bacillus sp. varied from 13.06% by Bacillus sp. PHA 013 to 66.80% of dry cell weight by B. licheniformis strain PHA 007 [23]. This is somewhat similar with the findings of the present study of B. pumilus H9. Optimum PHB production by another strain Bacillus cereus FA11 was 48.43% obtained at pH 7, 30 °C, with glucose as carbon source after 48 h of incubation [26] which is very much less when compared to our B. pumilus H9. For PHB production, the modified production medium was found to be better and the productivity was higher than the yield of PHB obtained from other Bacillus species using commercial carbon sources [27].

Fig. 1
figure 1

Growth and accumulation of PHB by B. pumilus H9 with respect to time in hours. The culture was grown at 30 °C in the modified production medium at 160 rpm

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Effect of Substrates, pH and Time on PHB Production

The raw materials used in earlier studies are costlier, though PHB production was higher. Therefore, the cost of the carbon source is supposed to be cheap and yield should be the maximum. Cheap carbon sources derived from wastes like whey, cane, molasses and sugar beet molasses were used for PHB production and a mixture of different salts were used as mineral source [28]. Further, biowastes rich in organic matter is one of the targets for cheap carbon sources. Many biowastes have been reported as substrates for PHB production like whey, food scraps, agro-industrial by-products [29], palm kernel oil [30], vegetable oils [31, 32], pea-shells [33], etc. Cow dung is considered as a cheap bio resource almost available in every country. Though cow dung is produced as waste excreta, it has essential nutrients including carbon, nitrogen, phosphorus, potassium, calcium, magnesium, sulphur, manganese, copper, zinc, chloride, boron, iron and molybdenum [34]. Therefore, it is also regarded a valuable resource as fertilizer and also could be used as a good candidate for fuel. Moreover, owing to its essential nutrients, cow dung has been utilized as a novel and inexpensive substrate in enzyme bioprocesses by bacteria [34]. So, attempts have been made to use cow dung as a bio waste substrate for PHB production due to its cheap, easy availability and nutrient components.

To investigate the effect of substrates on PHB production, B. pumilus H9 was inoculated into the modified production medium containing different carbon and nitrogen sources (Fig. 2a, b). Consequently, cowdung, sucrose and peptone were selected as the best nutrient sources. Considering the concentration of the selected substrates optimized through conventional methods, 4 g/l of cowdung, 5 g/l of sucrose and 1.5 g/l of peptone produced maximum amount of PHB. Maximum PHB synthesis occurred at 48 h of incubation with pH of the medium being 7. All these data were used to design the experiments for CCRD [35].

Fig. 2
figure 2

a Effect of different carbon sources on cell growth and accumulation of PHB by B. pumilus H9 grown at 30 °C in the modified production medium at 160 rpm for 48 h. b Effect of different nitrogen sources on cell growth and accumulation of PHB by B. pumilus H9 grown at 30 °C in the modified production medium at 160 rpm for 48 h

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Statistical Optimization of PHB Production with RSM

RSM is an efficient tool applied in the optimization of medium constituents and other critical variables responsible for the production of biomolecules [36, 37]. The experimental results of PHB production by a complete four-factor-two-level factorial experiment designed with six replications of the central point and six axial points are shown in Table 3.

Table 3 Experimental design and results of CCD for response surface methodology
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$$\begin{aligned} PHB\left( Y \right) & = + 2.47 + 0.13 \times A - 0.032 \times B + 0.21 \times C + 0.030 \times D \\ & - 0.097 \times A^{2} - 0.095 \times B^{2} - 0.15 \times C^{2} - 0.28 \times D^{2} \\ & - 0.12 \times A \times B + 0.069 \times A \times C + 0.40 \times A \times D \\ & + 0.17 \times B \times C - 0.33 \times B \times D + 0.083 \times C \times D \\ \end{aligned}$$
(1)

The overall second-order polynomial equation for PHB production was given in Eq. (1) where, Y is PHB dry weight (w/v); A is cowdung (w/v); B is sucrose (w/v); C is peptone (w/v) and D is the initial pH of the medium. The statistical significance of the model equation was evaluated by the F-test for analysis of variance (ANOVA), which showed that the regression is statistically significant at 99% (p < 0.05) confidence level. Values < 0.1000 indicate the model terms are significant (Table 4). In this case A, C, A2, B2, C2, D2, AB, AD, BC and BD are found to be significant model terms. The P-values are used as a tool to check the significance of each of the coefficients which, in turn, are necessary to understand the pattern of the mutual interactions between the best variables. The smaller the magnitude of the P, the more significant is the corresponding coefficient. ANOVA for PHB production showed that the model F-value of 20.83 for PHB production is statistically significant at Prob > F-value < 0.0001. There is only 0.01% chance that a “Model F-value” this large could occur due to noise. “Adeq Precision” measures the signal (response) to noise (deviation) ratio. A ratio > 4 is desirable. The ratio of 18.813 indicates an adequate signal in the case of medium optimization for PHB production. The coefficient of determination (R2) was calculated and found to be 0.9511, indicating that the model could explain 95% of the variability in the production of PHB. The R2 value was always between 0 and 1 and the closer the R2 value to 1.0, the stronger the model and the better it predicts the response [38].

Table 4 Analysis of variance (ANOVA) for the regression model of PHB production obtained from the experimental results
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The effect of interaction of various nutrients on the PHB production (z axis) was studied by plotting three dimensional response surface curves against any two independent variables while keeping the other independent variable at their “0” levels. Therefore, three response surfaces were obtained by considering all three possible combinations. From the response surface (Fig. 3) it has been concluded that all the nutrients has significant effect on PHB production. With the optimized medium the production of PHB was found to be 2.47 g/l.

Fig. 3
figure 3

Three-dimensional curve showing the interactive effects of a peptone and cow dung, b sucrose and cow dung, c peptone and sucrose, d pH and peptone, e pH and sucrose, f pH and cow dung, on production of PHB by B. pumilus H9 grown in the modified production medium at 30 °C, 160 rpm for 48 h. These interactions were analysed by keeping the other two independent variables at their “0” level

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Amplification of phbA, phbB and phbC Genes

The PCR products of phbA, phbB and phbC genes of B. pumilus H9 were amplified and separated on 1% agarose gel, observed in UV transilluminator and documented with GelDocXR software (Biorad) (Fig. 4). Three fragments with the length 262 bp for phbA gene, 174 bp for phbB gene and 503 bp for phbC gene were obtained by PCR using forward and reverse primers. Amplification of these genes confirms the production of PHB by B. pumilus H9. This may be useful in studying the pathway and regulation of PHB biosynthesis in B. pumilus H9. It may also help in detecting PHB accumulating bacteria genotypically using various PCR protocols. Thus, the polyphasic approach including both phenotypic and genotypic detection of PHB producing bacteria may satisfy the minimal industrial standards for screening potentially useful PHB producer [39]. Consequently, these amplified PHB biosynthetic genes of B. pumilus H9 may help in producing a recombinant biodegradable polymer by cloning, to be used in medical and environmental biotechnology.

Fig. 4
figure 4

Amplified PCR product of PHB genes of B. pumilus H9 separated on 1% agarose gel. Lane A, 262 bp for phbA gene; Lane B, 174 bp for phbB gene and Lane C, 503 bp for phbC gene

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Conclusion

In this study, a medium was optimized using RSM to enhance PHB production by a potent bacterium B. pumilus H9 utilizing cow dung, sucrose and peptone as the main nutrient sources. This is the first report highlighting production of PHB using cow dung as one of the nutrient source. Therefore, this may help in reducing the production cost as cow dung can be used as a cheap carbon source and easily available and may provide a benefit of converting bio waste material into a value added product biopolymer. Also, amplification of biosynthetic genes in B. pumilus H9 provide a reference for further study.