In a previous study (Rodríguez-Contreras et al. 2013), B. megaterium uyuni S29 showed the formation of intracellular PHA granules. The granules were processed for analysis and the proton displacements and chemical shifts observed in the 1H NMR spectrum confirmed the chemical structure of PHB homopolymer. The polymer biosynthesis was carry out utilizing a conventional medium with low salt content as typically used for industrial production of PHAs (Atlić et al. 2011; Küng 1982). Considering the involved advantages of using conditions used industrially (Rodríguez-Contreras et al. 2013), the same medium was used in both fed-batch experiments.
The results of both fed-batch fermentations show that better growth conditions were achieved in the bioreactor experiment, while higher polymer content was achieved in fed-batch experiments in shaking flasks (Table 1). This is possible because the bioreactor allows the total control of fermentation parameters, while the shaking flasks experiment indicates the tendency of the behaviour (Büchs 2001).
In the first fed-batch experiment carried out in a 3-L bioreactor, no limitation of nitrogen was achieved in the entire fermentation. Because all other nutrients were under control, the system was likely to be in suboptimal conditions for biopolymer accumulation, and no high amount of polymer content could be attained. Thus, there was a margin for further improvement on the PHB production. Consequently, a second fed-batch experiment was carried out to totally restrict the nitrogen source. This time, nitrogen limitation was achieved after 12 h of fermentation, attaining the maximal polymer content at this point (69·20 ± 4·10% PHB). In this fermentation, the RB increased without any addition of nitrogen after 12 h, indicating that cell autolysis could occur (Wang and Yu 2007).
In the literature, studies with B. megaterium for PHB production were initially reported to show a maximal polymer content of 40% of CDM in a glucose medium containing acetate (Macrae and Wilkinson 1958). Later, a polymer yield of 48·13% with B. megaterium Y6 was reached (Yilmaz et al. 2005), and 42% in a fed-batch experiment using sugar cane molasses and urea as cheap carbon sources under nitrogen limiting conditions (Kulpreecha et al. 2009). Most recently, it was found that B. megaterium strain OU303A yielded a maximum of 62·43% DCM polymer in the medium containing glycerol as a carbon source (Reddy et al. 2009). Table 3 shows a summary of the main values related to PHB production by different strains of B. megaterium reported in the literature. As mentioned before, sporulation is the cause of low PHB productivity in Bacillus genus. B. megaterium produces spores under similar conditions to PHA formation, mainly as a consequence of the depletion of essential nutrients, leading to a decrease in the accumulated PHB (Omar et al. 2001). This could be the reason why such a low PHB content was reached by this genus before. To our knowledge and compared with the percentages found in the literature, the 70% of PHB (69·20 ± 4·10% PHB) reached by B. megaterium uyuni S29 is up to now the highest polymer content obtained by a Bacillus genus. Moreover, the results from the experiments carried out in this study with B. megaterium uyuni S29 indicate that PHB production and accumulation takes place when the nitrogen source is limited, reaching a high polymer concentration with no visible spore formation.
Table 3. Comparative values of Poly-3-hydroxybutyrate (PHB) production by different Bacillus megaterium strains
|B. megaterium uyuni S29||Glucose||Fed-batch||30||0·45||600–125||161||−11||This work|
|B. megaterium uyuni S29||Glucose||Fed-batch||70||0·25||600–125||136·8||−16||This work|
|B. megaterium KM||Glucose||Batch||40||–||–||–||–||Macrae and Wilkinson 1958;|
|B. megaterium P7||Yeast extract peptone||Batch||14·04||–||–||–||–||Yilmaz et al. 2005;|
|B. megaterium BA-019||Molasses||pH-stat fed-batch||42·1||1·27||3900||174||3·9||Kulpreecha et al. 2009;|
|B. megaterium OU303A||Glycerol||Batch||62·43%||–||510||175·23||8·85||Reddy et al. 2009;|
|B. megaterium OU303A||Glucose||Batch||58·63%||–||519||178||8·50||Reddy et al. 2009|
In addition and considering an already industrial scaled strain such as Azahydromonas lata (formerly known as Alcaligenes latus) which reaches its high PHB content (88% of CDM) with ammonium limitation (Quillaguamán et al. 2008), B. megaterium uyuni S29 is not far from this result. It has to be considered that A. lata has been studied for a long time, and its optimal fermentation conditions are being constantly improved. In comparison, the percentage reached with strain uyuni S29 (70% of CDM) was obtained from a shaking flask experiment with feed-batch fermentation, where the conditions were not completely optimal. Therefore, it is very important to continue the research with bacterial strain B. megaterium uyuni S29 to further improve its PHB content.
Furthermore, it is possible to consider B. megaterium strain uyuni S29 for polymer production on an industrial scale: first, because the 70% of PHB obtained in this fermentation is higher compared with the 60% of polymer that is necessary for considering a strain suitable to be used in an industrial process (Macrae and Wilkinson 1958; Reddy et al. 2003); and second, because industrial fermentation conditions (the used conventional medium and moderate salt content) were already taken into account for the bacterium selection in the previous study (Rodríguez-Contreras et al. 2013), those conditions were maintain in this work. Thus, no additional changes in the culture conditions are needed to adapt B. megaterium strain uyuni S29 to the required industrial conditions for biopolymer synthesis.
Polymer extraction and characterization
Two extraction methods have been carried out in this work in order to study the possibility of the strain to produce other qualities of PHA besides PHB. Extraction with acetone by Soxhlet was used in previous works to extract medium-chain-length PHA (Jiang et al. 2006). These are PHA biopolymers that contain 6 to 14 carbons in their subunits and possess attractive properties such as low melting points, high elasticity and biodegradability (Jiang et al. 2006). However, the main bands and peaks of the FTIR and 1H NMR spectra from the polymers extracted using both extraction procedures (via chloroform and via acetone) correspond to the characteristic ones of PHB homopolymer, a short-chain-length (scl) PHA, according to the literature (Oliveira et al. 2007). These results together with the GC analysis indicate that B. megaterium uyuni S29 produces and accumulates not other PHA but PHB homopolymer from glucose as sole carbon substrate, matching with the results from the preliminary study of polymer characterization with strain B. megaterium strain uyuni S29 (Rodríguez-Contreras et al. 2013).
The molecular masses of PHB produced from wild-type bacteria are usually in the range of 10 and 3000 kDa with a polydispersity around 2 (Sudesh et al. 2000). The results of the GPC analysis showed that the values for the biopolymer production by B. megaterium strain uyuni S29 are within this range. The values of the two molar masses concur with the results obtained in previous works with this bacterium (Rodríguez-Contreras et al. 2013).
Usually, scl-PHAs, especially PHB, constitute highly crystalline materials, although they are amorphous within the bacterial cell. The crystallization rapidly occurs after disruption of cells when the polymer is extracted. A common Xc is typically found between 60 and 80%, the Tg about 4°C and the Tm about 160–180°C (Sudesh et al. 2000; Khanna and Srivastava 2005; Valappil et al. 2007). In this study, the three PHB samples extracted from B. megaterium uyuni S29 showed very different thermal properties compared with the usual PHAs. The biopolyesters extracted from both fed-batch experiments (bioreactor and shaking flasks) showed lower thermal parameters compared with the common ones from PHBs. These results coincide with the thermal properties of the biopolymer extracted in initial studies with this strain (Rodríguez-Contreras et al. 2013). Particularly, the extracts from the bioreactor experiment showed even lower Xc and Tg (Table 2). The obtained thermal values confirmed that different fermentation processes and extraction techniques can influence the thermal properties, corresponding to the findings reported by Valappil et al. (2007). Low thermal properties shown by the chloroform extraction technique could be explained by the presence of possible impurities. The method may allow lipids, fatty acids and other hydrophobic cellular materials to be extracted along with PHB. However, the fact that the polymer extracted via Soxhlet-acetone also showed low thermal properties confirms that this is a property of the biopolymer synthesized by B. megaterium uyuni S29.
On the other hand, these uncommon thermal properties could be the result of the blend of the different PHBs fractions, as the two different molar masses resulted from the GPC analyses show. Therefore, the results obtained from DSC and GPC for these extracted PHBs could be connected to each other. The thermal behaviour of some polymers is influenced by the chain length and molecular mass of the polymer used (Rojas de Gáscue et al. 2002). As already reported by Lundgren et al. (1965), lower molar masses tend to melt at lower temperatures, because the end groups act as impurities. The different melting point values are related to polymer fractions which have undergone different degrees of degradation, yielding polyester with a relatively large fraction of chain ends (low degree of polymerization). The different peaks of the melting points from DSC results could be a consequence of these different chain lengths. Thus, there is evidence that the synthesized PHB features a blend of different PHB fractions with different molar masses (different degrees of polymerization). This matches with the DSC results obtained from previous studies with this strain (Rodríguez-Contreras et al. 2013).
The crystallinity of a polymer is known to play a major role in the degradation of a polymer: the amorphous regions in polymers degrade at a much faster rate compared with crystalline regions (Iannace et al. 2001). The relatively lower crystallinity of the PHB isolated from B. megaterium uyuni S29 can be an advantage, because it will depend on the application needs. Otherwise, the thermal properties of the polymers influence their mechanical properties (Odian 2004).Thus, the relatively low thermal properties of the PHBs isolated from B. megaterium uyuni S29 can be reflected on a decrease in the strength and on an increase in the extensibility of the material, enlarging its possible applications.