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Keywords:

  • Bacillus sp;
  • mahua flowers;
  • natural substrate;
  • PHA accumulation;
  • poly(hydroxybutyrate- co-hydroxyvalerate)

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Aims:  The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256.

Methods and Results:  In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l−1 sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) – P(HB-co-HV) – of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l−1) in a 3·0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2·7 g l−1, respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2·5 g l−1. The polymer was characterized by proton NMR.

Conclusions:  Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer.

Significance and Impact of the Study:  Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Polyhydroxyalkanoate (PHA) is produced by a number of bacteria under nutrient depletion conditions as intracellular storage material of carbon and energy. Their potential use as biodegradable and biocompatible thermoplastics has attracted worldwide attention. Polyhydroxybutyrate (PHB), which occurs most commonly in bacterial cells, does not posses material properties for practical application because it is crystalline and brittle. The copolymer of poly(hydroxybutyrate-co-hydroxyvalerate) – P(HB-co-HV) – is more elastic and flexible compared with PHB. Sugars such as sucrose and glucose are the most common main carbon sources used for PHB production and propionate or pentanoate is usually added to the medium for copolymer production. Composition of the carbon substrate used for fermentation and utilization of appropriate bacterial strain, controls the copolymer production where substrate cost represents nearly 40% of total cost. Thus, it is essential to explore an alternate substrate for bacterial growth and copolymer production. Cheaper raw materials, such as whey, wastewater from olive mills, molasses, corn steep liquor, starchy wastewater, palm oil mill effluent, have been used as nutrient supplements for bacterial PHA production (Page 1989; Hassan et al. 1997; Gouda et al. 2001; Yu 2001; Lapointe et al. 2002; Marangoni et al. 2002; Pozo et al. 2002).

Mahua (Madhuca longifolia and Madhuca indica) trees are distributed from India to Australia. Annual production of dry mahua flowers in India is about 2 million tonnes. The flowers, which are succulent, are eaten raw or cooked. They are used in the preparation of distilled liquors and vinegar. They are also used as feed for livestock. But so far they have not been used for bacterial fermentation for the production of other metabolites. The aim of the present work was to produce PHA copolymer using the succulent corolla of mahua flowers that drop down after maturation, which are rich in sugars and other organic carbon compounds.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Micro-organisms

Growth medium amended with mahua flower extract as a carbon source was used for the screening of the bacterial cultures for PHA production. The cultures used in the experiments were isolated from local soil samples, and maintained at the culture collection center of the Food Microbiology department, CFTRI, Mysore, India, which included: Bacillus sp-256, Rhizobium meliloti and Sphingomonas sp. Bacillus SPS-256 and Sphingomonas sp. were maintained on nutrient agar slants and Rh. meliloti on yeast mannitol agar slant (Media procured from Himedia, Mumbai, India), at 4°C.

Inoculum

A loop-full of cells from agar slant cultures of Bacillus sp-256 and Sphingomonas sp. were transferred into 10 ml of sterile nutrient broth contained in the test tubes. The test tubes were incubated for 18–20 h at 30°C, 250 rev min−1 and the contents were transferred to 100 ml nutrient broth (Himedia, Mumbai, India) sterilized in 500 ml capacity Erlenmeyer flasks. The inoculated flasks were further incubated up to 18 h and used as inoculum. Viable cell counts on nutrient agar indicated that the inocula contained 2 × 103 and 2·5 × 103 CFU ml−1, respectively. Rhizobium meliloti inoculum was developed similarly using yeast mannitol broth (Himedia, Mumbai, India) and contained 1·8 × 103 CFU ml−1.

Extraction of mahua flowers and characterization of the extract

Sun dried mahua (Madhuca sp.) flowers (succulent corollas) obtained from the forest area of Gujarat, India, contained: 17·1% moisture, 50% reducing sugar, 7·3% nonreducing sugar, 4% protein, 3·5% ash and 1·3% acidity. 50 g portion of the dried flower was suspended directly without shredding in 100 ml of water and boiled for 5 min. The suspension was passed through filter cloth and the residue was resuspended in 100 ml of boiling water twice for 5 min and filtered. Total volume of the supernatant collected was 200 ml. Dinitrosalicylic acid method (Miller 1959) was used to estimate reducing sugar in the extract before and after inversion with HCl. Succinic and malic acid in the extract were estimated using gas chromatographic (GC) method. For this, 5 ml of the extract was acidified with phosphoric acid to pH 2, the mixture was allowed to stand at room temperature for 30 min and centrifuged for 15 min at 1900 g. Column used for analysis was Chromosorb 101 (1·8 × 2 mm internal diameter). Column and detector temperature were maintained at 200°C and 225°C, respectively. Carrier gas used was nitrogen (40 ml min−1, at 2·8 Kg cm−2 pressure). One microliter of the sample was used for injection. Organic acids, such as succinic and malic, were used as standards.

Polyhydroxyalkanoate production in shake flasks

PHA production was carried out in triplicate in 500 ml Erlenmeyer flasks in sterile liquid medium (100 ml) containing (g l−1): Na2HPO4 2 H2O, 4·4; KH2PO4, 1·5; (NH4)2SO4, 1·5; MgSO4 7 H2O, 0·2 and (i) with sucrose 20 g l−1 (ii) 140 ml of mahua flower extract (filter sterilized) to impart 20 g l−1 of sugar (iii) whole mahua flower (35 g l−1) was used in Bacillus sp. fermentation to impart 20 g l −1 of sugar (iv) to ascertain the effect of malic acid (2 g l−1) or succinic acid (2 g l−1) on PHA copolymer production, these were supplemented individually (after neutralization and sterilization in 10 ml water) along with 5 g l−1 sucrose to the fermenting broth (after 24 h) which already contained sucrose (15 g l−1) as a main carbon source. The flasks were inoculated with 10% inoculum (v/v) and incubated at 30°C, 250 rev min−1 up to 72 h. Statistical analysis of the results was carried out using computer-based Microsoft Excel programme with nonbiased or n−1 method.

Polyhydroxyalkanoate production in fermentor

Fermentation was carried out in a jar fermentor (Bioflow 110; New Brunswick Scientific Co., Edison, NJ, USA) of 3·0 l capacity, containing 1·8 l of mineral medium, the composition of which is given under shake flask experiment. Sucrose (20 g l−1) or mahua flower extract (filter sterilized, 140 ml l−1 equivalent to 20 g l−1 of carbon source) were used as carbon sources. Medium was inoculated with 200 ml of Bacillus sp-256 inoculum that was prepared as mentioned above. Fermentation was carried out at 30°C and the culture pH was controlled at 7 automatically, by the addition of 1 mol l−1 NaOH. The dissolved oxygen was maintained at 40% of air saturation level. The airflow rate was kept constant at 1 v/v and stirrer speed was varied to achieve the required air saturation level automatically by operating the fermentor on cascading mode. The experiments were carried out for a period of 30 h.

Estimation of biomass

The biomass, which contained intracellular PHA, was collected by centrifugation of the culture broth at 2900 g for 15 min and the pellet was washed with distilled water and dried at 70°C in airflow drier to a constant weight.

Estimation of polyhydroxyalkanoate and identification of the polymer

PHA content of bacterial cells was initially determined gravimetrically by sodium hypochlorite hydrolysis (Williamson and Wilkinson 1958; Law and Slepecky 1961). GC analysis was also carried out using freeze-dried biomass (Akiyama and Doi 1993). Known weight of lyophilized cells (10 mg) was subjected to methanolysis in sealed ampoules using 1 ml chloroform, 0·85 ml methanol and 0·15 ml conc. H2SO4 at 100°C for 140 min. After cooling, the mixture was homogenized with deionized water and the bottom phase containing the methyl esters was used for GC analysis (Brandl et al. 1988). The analysis was carried out with a flame ionization detector, in a 30 m DB-1 (fused silica gel – polymethyl siloxane) capillary column (internal diameter 0·25 mm and film thickness 0·25 μm). Nitrogen was used as a carrier gas (1 ml min−1, at 0·07 Kg cm−2 pressure). Temperature of injector and detector were maintained at 220°C and 230°C, respectively. The temperature programme used was: 55°C for 7 min; ramp of 4°C min−1 up to 100°C; 10°C min−1 rise up to 200°C and hold at 200°C for 10 min. Benzoic acid was used as internal standard. PHB and P(HB-co-HV) containing 5 mol% of hydroxyvalerate (Sigma Aldrich, St Louis, MO, USA) were used as standards.

NMR

Lyophilized cells (100 mg) were suspended in 10 ml of chloroform and extracted overnight at 40°C. Cell sediment was separated by centrifugation at 1900 g for 20 min and PHA was isolated from clear chloroform layer by the addition of two volumes of hexane. Precipitated polymer was air-dried at 40°C. 1H NMR of the polymer was carried out in deuterated chloroform at 400 MHz on an AMX 400 spectrophotometer. PHB, P(HB-co-HV) (Sigma Aldrich) were used as standards.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Data in Table 1 indicate that the growth and PHA production by bacteria vary depending on the capacity of the bacteria to utilize sugars and organic acids present in the medium. Cultivation of three strains of bacteria on mahua flower extract as a substrate resulted in 22–51% of PHA in the biomass. Bacillus sp. grew well compared with others in the presence of mahua flower extract. In the whole flower medium, Bacillus sp. produced 54% PHA in its biomass (Table 1). Further, GC analysis indicated that only PHB was synthesized in the presence of sucrose, whereas P(HB-co-HV) in the molar ratio of 90 : 10 was synthesized in the presence of mahua flower in the medium. In addition to high concentration of sugar (57%, w/w), the flower is rich in succinic and malic acids (1·3 g%, w/w), which are found in the ratio of 1 : 2. To ascertain the effect of succinic and malic acids for copolymer production, these organic acids were used individually in pure form in the medium. Data in Table 2 indicate that amongst the organic acids tested, only malic acid was utilized for copolymer synthesis.

Table 1.   PHA production by bacteria in media containing sucrose and mahua flower extract as carbon sources
BacteriaBiomass (g l−1, w/v)PHA (% of biomass)PHB : PHV (Mol%)*
ABABAB
  1. A: mahua flower extract was used as a carbon source at 20 g l−1 sugar concentration; B: sucrose used as a carbon source at 20 g l−1 level.

  2. *By GC of lyophilized cells.

  3. †Mahua whole flower was used in the medium at 20 g l−1 sugar concentration.

Rhizobium meliloti2·32·8315894 : 697 : 3
Bacillus sp-2563·52·5514894 : 6100 : 0
3·7† 54 90 : 10 
Sphingomonas sp.2·02·2224595 : 598 : 2
Table 2.   Effect of succinic acid and malic acid on copolymer production by Bacillus sp. 256
Carbon sourceBiomass (g l−1)PHA (% of biomass)PHB : PHV (mol%)
  1. *Averages of three experiments and SD.

Sucrose2·9 ± 0·05*46·8 ± 0·7100 : 0
Sucrose + succinic acid2·6 ± 0·0553·3 ± 0·57100 : 0
Sucrose + malic acid3·8 ± 0·0567·8 ± 0·7294 : 6

Fermentor cultivation of Bacillus sp. in the presence of sucrose as carbon source yielded 4·8 g l−1 of biomass and 2·5 g l−1 of PHA. Marginal enhancement in growth (5·1 g l−1 biomass) and PHA production (2·7 g l−1) were observed in the mahua flower extract medium (Fig. 1). Sugar concentration at the end of fermentation was 0·8% (w/v, in sucrose medium) and 0·3% (w/v, in flower extract medium), respectively.

image

Figure 1.  Fermentor cultivation of Bacillus sp.: (a) in sucrose (20 g l−1) and (b) in mahua flower extract (140 ml l−1 equivalent to 20 g l−1 of carbon source) containing media. Fermentation was carried out at 30°C and at pH 7 and 40% of air saturation level. ♦, Biomass + PHA; bsl00001, PHA; bsl00066, biomass − PHA.

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1H NMR spectrum (Fig. 2) indicated that signal characteristic of PHB was observed in the polymer isolated from different carbon sources containing media, as a doublet at 1·29 ppm which is attributed to the methyl group, a doublet of quadruplet at 2·5 ppm which is attributed to methylene group and a multiplet at 5·28 ppm characteristic of methyne group. In addition to this, PHA obtained from malic acid and mahua flower extract media also showed a triplet at 0·9 ppm and a methylene resonance at 1·6 and methyne resonance at 5·1, which indicated the presence of valerate in the polymer.

image

Figure 2. 1H NMR spectrum of PHA extracted from Bacillus sp. cultivated on: (a) sucrose, (b) succinic acid and sucrose, (c) malic acid and sucrose and (d) mahua flower extract, as carbon sources.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

One of the limiting factors for the commercialization of PHA is the high cost of the carbon substrates used for fermentation. PHA copolymers posses better mechanical properties compared with homopolymer of PHB but costlier co-carbon substrates such as fatty acids should be supplemented in the medium for its biosynthesis (Choi and Lee 2000). Hence, the production of the polymer from unrefined and cost effective substrates rich in different carbon components appeared feasible for PHA production. Dry mahua flower is available in India at about 0·3$per kg. The carbohydrates identified in the flowers are mainly sucrose, glucose, fructose, and traces of maltose, arabinose and rhamnose. Other components present are amino acids, proteins, various vitamins and organic acids (The Wealth of India 1962). Because of richness of carbohydrates and organic acids, this substrate was examined for the purpose of PHA production. Cultivation of bacteria on this substrate indicated variability in yields of biomass and PHA concentration in the cells (Table 1). This may be because of varied metabolic activity and substrate utilization rates. Bacillus spp. is known to produce optimum PHA concentration in sucrose medium (Shamala et al. 2003). In the present work, the observed increase of biomass and qualitative and quantitative yields of PHA by Bacillus sp. in shake flask and fermentor cultivations with flower extract medium compared with sucrose may be due to the presence of various types of utilizable carbon compounds and growth promoting nutrients such as amino acids and proteins present in this natural substrate. GC and NMR data support the fact that the bacterium synthesized P(HB-co-HV) copolymer from carbon substrates present in mahua flower (Tables 1 and 2; Fig. 2). Although malic acid was found to enhance copolymer production, higher concentration of hydroxyvalerate may be synthesized in medium containing mahua flower extract. It is known that the flower contains various amino acids and essential oils (The Wealth of India 1962) and these can also be utilized for growth and polymer synthesis by bacteria.

It is known that PHB is synthesized from acetyl coenzyme A wherein β-ketothiolase catalyses condensation of two acetyl coenzyme A molecules to acetoacetyl CoA that is subsequently reduced to hydroxybutyryl CoA by acetoacetyl CoA reductase. PHB is then produced by the polymerization of hydroxybutyryl CoA by the action of PHB synthase (Madison and Huisman 1999; Tsuge 2002). Further, these authors have reported that the hydroxyvaleric (HV) unit, which is found as one of the components of copolymer, is produced either from propionic acid or valeric acid through β oxidation and deacetylation. In the absence of these typical HV precursors, HV most likely would be produced by some of the bacterial strains by the methylmalonyl-COA pathway where other carbon substrates other than propionic acid and valeric acid can be utilized. The co-carbon sources are usually provided in pure form, which makes the process more costly. Hence, only those bacteria, which utilize co-carbon source such as organic acids, would be able to synthesize copolymers from natural, economic substrate such as mahua flowers.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Mahua flower is a natural substrate, which contains nearly 60% of sugar, and it additionally contains organic acids, which are essential for copolymer synthesis. In PHA production medium, nearly 50% of the cost is due to carbon sources such as sugar and organic acids. This can be economized by using industrial by products or natural substrates. Next to molasses, mahua flower can be considered as a cheaper source of carbon for the synthesis of PHA copolymers.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The authors thank The Department of Biotechnology, Ministry of Science and Technology, Government of India for the financial support to the project and the Director, Central Food Technological Research Institute, Mysore, India, for his constant encouragement to carry out the research programme. P.K. Anil Kumar, L. Kshama, K.S. Latha Kumari and M.S. Divyashree wish to thank Council of Scientific and Industrial Research, India, for providing Research Fellowships.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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