SBB is non-specific dye to screen PHA-producing isolates [11]. In this study, it was shown that there was a good indication of SBB positivity when the isolate was collected from sediment and then stained with SBB dyes. In this respect, Bacillus sp. LPPI-18 showed strong SBB positivity after staining with SBB dye.

PHA and non-PHA-producing species of bacteria can be differentiated from each other using various methods [20]. They used to stain viable colony. These methods are quite fast and sensitive and then result in dark blue which is similar to the current study (Steinbuchel and Schlegel, 1991; Bhuwal, et.al., 2013). However, it is time-consuming [11]. When properly stained, distinct black granules with red background due to counterstaining (0.5% safranin (w/v)) were detected. The longer the incubation period, the more granules developed when these isolates were supplied with 1% glucose as a carbon source.

NBA used to discriminate between PHA-producing and non-PHA isolates. Staining dyes are the oldest and fastest technique available to screen for PHA producers from their natural environments. It is the cheapest method of screening potential isolates used for PHA production. The staining dyes used for screening PHA-producing isolate from their natural environments were NBA and Nile Red. In accordance with previous reports, these dyes are the most broadly used dyes for selective staining of PHA granules within bacterial cells [4, 30].

SBB dye has a high sensitivity for PHA screening isolates from their natural habitat (Burdon, 1946). From a diversity of isolates of bacteria, a total of about 42 lipophilic isolates have been screened when stained properly with SBB dye. It is used to discriminate PHA granules with distinct structure. However, SBB positivity was lastly confirmed by using NBA which is a specific dye for PHA granule binding ability [31].

After SBB staining, PHA-positive isolates showed blue-black granules. It was indicated that the PHA-positive isolates have a tendency to store PHA. Following the results of SBB staining methods, the B3-D indicated a considerable amount of PHA granules after heat-fixed and staining had been performed using SBB. The same isolates of bacteria were further checked for PHA production by using NBA dye, a more selective and specific dye for PHA granules. Pure culture of isolates was inoculated and grown on minimal media and incubated at 30 °C for 90 h. Then, the cells were heat-fixed for smearing. Confocal fluorescence microscopic images have shown accumulation of PHA granules within a cell of bacteria [32]. When the cells had been stained with SBB, the granules were observed in the central region and occupying the maximum space within the cell. Moreover, the PHA granules were observed as hollow circles in the typical PHA producers within a microbial cytoplasm when it had been observed under a phase contrast microscope [33].

NBA is also a lipophilic dye most commonly used as a fluorescent probe for staining cells of bacteria that harbor PHA granules. It can be dissolved in water which is polar solvent and has positive charges and hence important for biotechnological application. It was used in the medium with DMSO as an organic solvent. NBA was first spread out to cytoplasm and later into the PHA granules. PHA-positive colonies will fluoresce under ultraviolet irradiation. It was used to discriminate between PHA-producing isolate and PHA-negative strains Therefore, it is the viable colony staining methods [11]. It is a satisfactory stain for PHA granules in bacterial cells. It is also specific and superior to SBB for PHA granule staining. NBA appears to be more selective and specific to stain PHB granules than SBB does. It is not as easily washed from the cell during decolorization procedures [4].

During plate staining methods, the presence of NBA and Nile red dyes have no effect on the growth of cells. This viable colony method of staining is used to apply for both gram-positive (e.g., Bacillus megaterium or Rhodococcus ruber) and gram-negative bacteria (e.g., Escherichia coli, Pseudomonas putida, Azotobacter vinelandii, and Ralstonia eutropha). Both Nile blue A and Nile red have an insignificant role for distinguishing between PHA-negative and PHA-positive isolate such as gram-positive Bacillus megaterium or Rhodococcus ruber, respectively. Normally, it was also used to differentiate between triacylglycerol-negative and triacylglycerol-positive and wax-ester strains of Acinetobacter calcoaceticus or Rhodococcus opacus [11]. Nile blue A (NBA) is a selective dye used to stain polyhydroxyalkanoic acids producing bacterial isolates. It has been shown that Escherichia coli cells are unable to produce polyhydroxyalkanoic acids when they have been stained with NBA and detected with flow cytometry. It is a simple, low-cost, and easy to stain cell using flow cytometry [12].

It was observed that a PHB accumulation ability of B. cereus SE-1 and Bacillus sp. CS-605 has been determined at 24, 48, and 72 h of interval [34]. The cells of B. cereus SE-1 and Bacillus sp. CS-605 are detected as bivariate distributions when these isolates were examined with a flow cytometer. It has been observed that more PHA accumulation is detected for isolate B. cereus SE-1 than Bacillus sp. CS-605 under the same condition after 72 h incubation. For instance, Bacillus sp. CS-605 is able to accumulate 5, 18.1, and 33% of PHB at an interval of 24, 48, and 72 h respectively. B. cereus SE-1 able to store 3.5, 22.1, and 40% PHB under the same condition [34].

A recent study revealed that the 16SrRNA of Bacillus sp. LPPI-18 had shown more affiliation with Bacillus cereus ATCC 14579T (AE016877). Our result indicates that this PHA-producing isolate has also shown more sequence similar to that of Bacillus wiedmannii FSL W8-0169T LOBC01000053 and Bacillus paramycoides NH24A2T MAOI01000012 strains. However, the GC composition and sequence between Bacillus sp. LPPI-18 and other closely related have shown variation. This indicates that the recent isolate shows to be various when it was compared to the closest strain, Bacillus cereus ATCC 14579T (AE016877). In contrary to this study, it is revealed that [10] the 16SrDNA sequences of E13 and C18 Mat obtained from Polluted Marine Microbial showed a 100% similarity with Bacillus thuringiensis DiSz8 sequence which is screened from a polluted sample of soil. The same authors reported that weathered granite have been recognized to be able to use as a source and habitat to harbor for PHA-producing C19 isolates which have shown a sequence similarity (99%) with endolithic Bacillus megaterium WN603. When Paracoccus homiensis DD-R11 grow on sandy beach sample, they are able to develop PHA granules and their sequence similarity (100%) were affiliated with E33, E45, and E46 strains. The sequence of strain C20R and E63 have been also shown 100% sequence similarity with gram-positive Staphylococcus cohnii (GTC) 728 strains. A 100% sequence similarity have been also observed between E4 and Staphylococcus arlettae (ATCC) (43957) sequences [10].

Certain isolates are able to produce pectinase enzyme and degrade pectin. In this study, Bacillus sp. LPPI-18 produce pectinase and produce a zone of utilization on a minimal salt medium that was supplemented with 1% pectin and incubated at 37 °C for 48 h (pH 7). In an agreement with the present study, it was [35] reported heat tolerance and acidic pectinase-producing Bacillus sp. ZJ1407 from soil sample after cultivated for 48 h at 37 °C. Bacillus sp. DT7 and Bacillus sp. TMF-1 were also other newly isolated bacterial species that are able to produce a thermotolerant pectinase using solid-state fermentation [36, 37].

It was [38] reported that out of 38 yeasts isolates, only Cyteromyces matritensis showed a small zone of hydrolysis. The same author further stated that extracellular proteases secreted by yeasts have been investigated for industrial application since they are fast growing and have the ability to grow in diverse substrates. A Bacillus marmarensis sp. nov from mushroom compost that was able to produce an alkaliphilic protease enzyme was reported when it had been incubated for 72 h at higher pH than the present study [39]. The present protease enzymes may be neutral protease enzyme. Those authors isolated this novel Bacillus marmarensis sp. at lower (30 °C) than the current temperature which is 37 °C. In this study, it shows that this isolate is unable to grow and produce more PCs when increasing the pH value as high as 11. Because of high pH, alkali region will be created and this might suppress these bacterial growths and hence affect PHA intracellular granule formation. In line with this result, it was [40] confirmed that more PHA yield can be obtained at pH12 to reduce the production cost of PHA polymers within a short time span.

The amount of P. conc. obtained from a newly isolated Bacillus sp. LPPI-18 is less than PHA contents obtained from Ps. fluorescens S48 (55% PHA content) [41] using the same methods of extraction. This could be due to different types of strains, carbon source, and the medium used for PHA productions. The highest amounts of P. conc., PP, and PC are obtained by CHCl3-NaOCl dispersion method of extraction when compared to SDS-NaOCl and NaCl-NaOCl methods of extraction. While pretreated with 3% SDS (w/v) and 6.25 M NaCl, PHA polymers may be degraded. This may further result in less amount of molecular weight of PHA polymers which is a similar finding with [5].

In agreement with our estimation, Ramsay et al. [29] obtained less molecular weight (730,000 kDa) and protein (0.7%) percentage with maximum purity of PHB (97%) using 1% SDS-NaClO solution. However, the molecular weight of PHB is 1,200,000 kDa for an untreated sample which is higher than 3% SDS pretreated sample with a less purity of PHB and more percentage of protein (50%) [29]. Sodium hypochlorite solution (4%) was used to degrade non-PHA cellular materials while extracting. The highest amount of PHA was obtained from R. eutropha (86%) and recombinant E. coli (93%) using NaOCl solution (4%) methods of treatment SDS which is a more PC than the present PC obtained from Bacillus sp. LPPI-18. In line with our recent study, a high level of purity (68%) and recovery yield (94%) for PHA polymer were reported from recombinant C. necator when a halogen-free method of extraction was used at 30 °C [42].

It was confirmed that there was less purity with the lower recovery of PHA polymer for NaCl-NaOCl methods of extraction when it was compared to 3% SDS and NaOCl-CHl3 methods of extraction. It could be due to high osmotic pressure developed against bacterial cell wall by NaCl solution which is 275.106 atm/mol (Π = єMRT) a similar result to [43]. The bacterial cell contents and PHA granules, as a result, were released outside and exposed directly to NaCl solution.

The highest PHA yield (52%) has been recovered from aerobic granules for Bacillus sp. using saturated sodium chloride solution (6.84 M) from a 24-h-old culture [20] which has higher yield than our current results for Bacillus sp. LPPI-18 (i.e., 27.13±3.26% purity PC). It should be noted that these isolates are able to produce more PC when extracted with SDS-NaOCl and CHCl3-NaOCl methods of extraction. Bacillus sp. LPPI-18 obtained from Loktak Lake sediment sample gave fewer PCs than those obtained from landfill sites (data not shown). This isolate obtained from sediment may contain a broad range of metabolic diversity for carbon source utilization for PHA polymerization. It was also true that sediment isolates are accessible to various carbon sources in the form of wastes that joined from dry-fill sites.

It was reported that different types of PHA were obtained from varieties of Pseudomonas putida strains (Wang et al. 2011). The maximum PHA yield obtained from a strain of P. putida KTOY06 (72.4±0.9) [44] is higher than our current results (27.13±3.26% pure PC with a 68.54±3.08% level of PHA recovery). However, still, certain P. putida strains are able to produce less PHA polymers under normal condition. This could be a due limitation of a broad range of metabolic diversity (Wang et al. 2011).

Π = єMRT (1 equation) is used to estimate osmotic pressure created against the cell wall of bacteria during extraction time while using 6.25M NaCl solution [43], where π is the osmotic pressure (atm), є is the van Hoff’s constant for NaCl (1.8), M is molarity of NaCl solution, R is the gas constant (0.08206 L atm /K mol), and T is the room temperature in K (298 K) [20].

Certain Bacillus species are also able produce PHA granules. They are also reported to be useful for various applications. The most commonly used Bacillus sp. for different applications beside PHA productions were B. amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. laterosporus, B. mycoides, B. licheniformis, B. macerans, B. cereus, B. firmus, B. subtilis, B. sphaericus, B. megaterium, and B. thuringiensis. The Bacillus species are also more potent than others since they produce homopolymer and copolymer PHAs that increase the diverse nature of the synthesized PHAs [45, 46].

All these carbon sources have not given the same amount of PHA contents. Glucose (1%) was able to produce the maximum PHA contents followed by molasses (1%). The least PC content has been obtained when Minimal Davis broth was supplied with 1% glycerol. These sugars are likely metabolized by this PHA-producing isolate at a very low rate and gradually produce different amounts of PCs. In agreement with the current study, Gouda et al. [47] reported the highest PHB yield from Bacillus megaterium using glucose as a sole carbon source. The same authors reported accumulation of PHB in Bacillus megaterium using fructose, glucose, and maltose carbon sources. Although the highest PHB yield was obtained from glucose, more cell dry mass was, however, obtained from maltose when it was used as a carbon source for Bacillus megaterium [47].

It was stated that [48] between a carbon source used for PHA synthesis, fructose is one of the most important substrate used in polymer (65.37%, 2.18 g/L) production which has more PHA contents than our present results. The same authors further stated that maltose was also able to produce a fewer amount of polymers. Palm olein (PO) (5 g/L) is the other substrate used for PHA production. About 67% PHA copolymers with 3HHx (27 mole %) were obtained from a 5.13 g/L bacterial biomass or cell dry weight (CDW) when Cupriavidus necator Re2058/pCB113 pure cultures grow on the PO [49].

It was revealed that these isolates were able to accumulate intracellular PHA granules under suitable condition. Since the fructose is a simple sugar, Bacillus sp LPPI-18 easily break down and produce intracellular PHA granules. It was also shown that isolates collected from Loktak Lake gave less PC on minimal medium supplemented with 10 g/L fructose concentration at 37 °C. It was [49] reported poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HHx) from palm olein and fructose using recombinant negative Cupriavidus necator Re2058/pCB113 strains. Fructose is naturally a homopolymer that is able to produce a P (3HB) when Cupriavidus necator Re2058/pCB113 strains grew on fructose (5 g/L fructose) as a sole carbon source). A 2.32 g/L CDW and 11% PHA/CDW homopolymer P (3HB) was produced from fructose, which is a higher CDW (g/L) and with less PC than the present study [49].

It has been observed that LPPI-18 is able to give a good PHA yield which is the closest yield to B. cereus SPV strain (38.0% ± 0.03%) [50]. It was also reported that Cupriavidus sp. KKU38 strains are capable to produce PHA when growing on cheap carbon sources like glucose, fructose, maltose, and xylose. Above all, glucose is the best carbon source for PHA production and has abilities to produce a 73.88% yield of PHA [8]. It is a higher PHA yield than our recent finding for newly screened Bacillus sp. LPPI-18 from Loktak Lake sediment suggesting that it could be due to the type of isolate used or other culture condition.

Fewer PCs were obtained from molasses when compared to glucose and fructose. Despite the fact that molasses are the potential substrate for PHA production, its level of PHA production is less than the same amount of glucose and fructose concentration which is probably due to the presence of sucrose concentration which is a double sugar broken by bacteria. Potential isolates of Bacillus sp. are able to produce PHA once grown on a minimal salt medium which is supplied with a carbon source. The estimated PHA contents are falling between 11.5 and 36.8% [51]. Different bacterial species are able to develop various amounts of PHA granules at 37 °C. For instance, the highest PHA contents were obtained at 24 h of fermentation for Bacillus sp. After 24 h of incubation, the PHA content was dropped which is inconsistent with our current results [51]. It was also reported a 42.10% PHA content for Bacillus megaterium BA-019 when grown on molasses [52] which is a higher yield than the current result.

Certain Bacillus species are able to produce PHA during fermentation. However, their DNA amplification for PHA gene did not occur when it was screened by using a primer. This might be a variation of gene type for PHA production. For instance, B. megaterium is able to produce PHA even though this isolate may not contain a gene of interest for PHA synthesis [51]. For the last 10 years, the polymers were produced from low-cost carbon source by using some potential industrially candidate pure cultures [53]. Molasses were used as a carbon source for PHA production when mixed with other organic acids. When sugar and organic acid are used, the conversion rate of PHA yield was not based on pH value. However, a slightly higher PHA yield is obtained when there are more acidic conditions. It has been reported that the highest yield of PHA was obtained from molasses which include about 0.22 g dry mass of PHA per 1 g of molasses. The PHA productivity was also 0.43 g/1 h in which it was in the range of reported PHA yield from other low-cost carbon sources from mixed cultures [53].

D-ribose can be used as a carbon source for microbial growth. Their role can be variously based on the type of microbial cells and culture condition. For instance, it was reported that new Bacillus sp. N-2 strains are also able to oxidize D-ribose when used as a carbon source. But the PHA production was not reported for this isolate [54]. In contrast to our recent finding, Diard et al. [55] employed D-ribose, and other carbon sources such as D-gluconate, D-galacturonate, itaconate, L-arabinose, L-tartrate, meso-tartrate, and tricarballylate for differentiation of Pseudomonas sp. GPo1 from P. monteilii and P. putida biotype A. However, PHA accumulating LPPI-18 isolate obtained from Loktak Lake is unable to utilize sucrose as a carbon source. Since the sucrose is a double sugar, it is hard for this isolate to break and be used as a carbon source. In agreement with the current study, it was [8] stated that some double sugars such as lactose, maltose, and sucrose were not utilized for A. eutrophus growth and PHA production.

All bacteria cannot grow on sucrose. For instance, it was recognized that Ralstonia eutropha, one of the most potential PHA-producing bacteria, is unable to grow on sucrose. However, when genetically engineered by expression of the M. succiniciproducens sacC gene encoding b-fructofuranosidase, R. eutropha is able to produce P(3HB) and P(3HB-co-LA) using sucrose as a carbon source (Park, et.al., 2015). Some bacterial spp. such as Azotobbacter vinelandii, Alcaligens latus, and Hydrogenophage pseudoflawa have been identified as PHA-producing bacterial species [56, 57]. PHA-producing isolate has shown higher PHA producers. This may suggest that the LPPI-18 isolate is able to harbor genes and enzyme associated with respective enzyme for PHA polymerization. This bacterial isolate has been found to utilize carbon source such as glucose, ribose, and other cost-effective and cheap carbon source for other metabolic products.

PHA film and its pore can be seen with SEM when the film has a coralloid surface. The pores can be generated and observed within the PHA film (PHB), and the crystallization degree and/or rapid crystallization rate of these biodegradable plastics were occurring. The more the contents of PHA like PHBHHx which is considered to be films, the lesser the size of the protrusions and the pores of the film surfaces. A PHA polymer that is only made up of 3-hydroxybutyric acid (98.7%) is able to form films [22].

Its structure was typically polyester, one of ester functional groups. Bacillus sp.LPPI-18 has shown similar fingerprinting parts which matched with PHB standard, natural polyester (sigma). In agreement with this results, Oliveira et al. [58] obtained absorption band at 1725 and 1277cm−1 (Fig. 9) that corresponds to a stretch of C=O bond, which is a functional group. The same authors stated that a series of intense bands located at 1000–1300 cm−1 corresponds to the stretching of the C–O bond which is an ester group. A biopolymer sample was extracted from Cupriavidus sp. KKU showed an intense band at 1731.61cm−1 wave number that corresponds to a stretch of C=O functional group when it was characterized with FTIR [8].

The intense band at 1280 to 1050 cm−1 indicated the presence of C-O-C atoms, vibrations of the aliphatic ester group. Similarly, intense absorption band was obtained at 1280, 1227, and 1181 cm−1, spectra attributed to the vibrations of the aliphatic ester group (C-O-C) which is a related finding with our current results [59]. The collective structure and functional groups of this FTIR spectrum indicate the type of polyester. This polymer might be PHB, a carbon four polyester which is a class of PHA that is produced by certain cyanobacteria and heterotrophic bacteria [60].

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