Abstract
Polyhydroxyalkanoate (PHA) production by Bacillus thuringiensis EGU45 and defined mixed culture of Bacillus spp. were studied by using crude glycerol (CG) and hydrolyzed biowastes as feed material. Hydrolysates from onion peels (OP), potato peels, pea-shells (PS), apple pomace 2% total solids obtained with defined mixed hydrolytic cultures (MHC2) were inoculated with B. thuringiensis EGU45 and defined mixed bacterial cultures (5MC1), which produced PHA at the rate of 40–350 and 65–450 mg/L, respectively. Addition of CG (1%, v/v) to these hydrolysates resulted in 1.8-fold and 4.5-fold enhancement in PHA production from OP by B. thuringiensis EGU45 and 5MC1, respectively. Co-utilization of OP and PS (in 2:1 ratio) supplemented with CG (1%, v/v) by B. thuringiensis EGU45 resulted in 2-fold increase in PHA production in comparison to OP + CG. This co-metabolism of OP and PS also enabled PHA co-polymer production (1300 mg/L), having an enhanced HV content of 21.2% (w/w).
Keywords: Bacillus, Crude glycerol, Biowastes, Vegetable wastes, Mixed hydrolytic culture
Introduction
Petroleum based synthetic plastics are destroying fragile ecosystems. These plastics cause serious threat to human health and the environment as they are non-biodegradable in nature. Replacement of these polymers with biodegradable polymers has gained major attention in recent years [1, 2]. Among the biopolymers, polyhydroxyalkanoates (PHAs) have gained importance due to their biodegradable nature and the fact that these can be produced from natural and renewable resources. They have physical and chemical properties similar to petroleum based synthetic plastics [3]. PHAs have attracted the attention of many researchers due to their non-polluting properties. PHAs are produced by bacteria under stress conditions. Depending upon the type of substrates most bacteria produce homo- and co-polymers of PHAs [3]. Homopolymers (PHB) being brittle are restricted in their applications. Thus, there is the need for producing ductile co-polymers, which have the potential for medical applications and also have high commercial value [1, 2]. However, there are many challenges in customizing PHA compositions such as varying in monomeric compositions, molecular weight, tensile strength and elongation [4]. These modifications for improving PHA strength can be achieved through synthetic biochemical approaches [5].
PHA co-polymers production can be achieved by using biowastes as feed. As bacteria have an ability to exploit a variety of complex wastes, these turn out to be an obvious choice as feed material. Hydrolysis of these complex biowastes is essential for the release of intermediates, which will help in PHA co-polymer production [3, 6–9]. Although many biowastes have been used as feed for PHA production, however, in recent years effluent from biodiesel industry has gained attention as it is produced in large quantities. The effluent released from biodiesel industry is composed of 70% as crude glycerol (CG) among other impurities [10, 11]. CG has been shown as feed for PHA production by diverse bacterial species [1, 10]. On the other hand, biowastes originating from municipal markets such as apple pomace (AP), potato peels (PP), pea-shells (PS) and onion peels (OP) have been also shown to act as feed material for PHA producing bacteria [3, 8, 9]. Although co-metabolism of: (1) market wastes and glucose, and (2) CG, glucose and propionic acid combinations have been helpful in producing PHA co-polymers [1–3, 10]. However, PHA copolymer production by co-utilization of municipal market wastes and biodiesel industry wastes have not been demonstrated so far. In this study, we have shown that PHA co-polymers can be produced from municipal market wastes and CG in different combinations by Bacillus thuringiensis and defined mixed microbial cultures. It also resulted in enhanced 3-hydroxyvalerate (3HV) content in the co-polymer of PHA.
Materials and Methods
Organism and Its Growth Parameters
Bacterial strains used in this study were obtained from our laboratory stock. Different bacteria were used for preparing: (a) mixed hydrolytic bacterial culture (designated as MHC-2) constituted of Bacillus sphaericus strain EGU542; B. thuringiensis strain EGU378; Bacillus sp. strains EGU85, EGU367 and EGU447; and Proteus mirabilis strain EGU30, and (b) defined mixed microbial culture (designated as 5MC1) for producing PHA was constituted of Bacillus cereus strains EGU3, EGU43, EGU44, and EGU520, and B. thuringiensis strain EGU45 [9]. These bacterial strains were grown on nutrient broth (NB) (13 g/L) and incubated at 37 °C at 200 rev/min for a period of 16–20 h. The cultures of bacterial strains so prepared were used to inoculate media at the rate of 10 µg cellular protein/mL [3].
Preparation of Biowaste Hydrolysate
Biowastes—PS, OP, AP and PP were collected from municipal market of Delhi. These wastes were cut into 1–3 mm pieces, and mixed with 250 mL of distilled water in 300 mL BOD bottles to make a slurry (total solid, TS—2%) as described earlier [3]. Each slurry was inoculated individually with MHC2, to a concentration of 100 µg cell protein/mL. Digestion of biowaste slurries was performed at 37 °C for 48 h [3].
PHA Production on Waste Hydrolysate
200 mL of PS, PP, AP or OP slurry (2%, TS) hydrolyzed with MHC2 was filtered through 0.45 µm Whattman filter and used for PHA production. The pH of the hydrolysates was set at 7.2 and inoculated with B. thuringiensis EGU45 and 5MC1 at the concentration of 10 µg cell protein/mL of feed [3]. These hydrolysates were supplemented with crude glycerol (CG), (1%, v/v). The hydrolysates of PS, PP, AP, OP not supplemented with CG were used as controls. Subsequently, MHC2 hydrolysates of OP were mixed with AP, PP or PS in three different ratios of 1:2, 1:1 and 2:1. 100 mL of each of these mixed hydrolysates supplemented with or without CG (1%, v/v) was used as feed for B. thuringiensis EGU45 and 5MC1. The PHA production was monitored for 48 h of incubation at 37 °C and 200 rev/min.
Analytical Methods
PHA Analysis
100 mL aliquots of bacterial cultures were used to estimate the dry cell mass (DCM) and PHA production as described previously [1, 3]. The polymers were also analyzed for their monomeric composition using GC fitted with DB-5 (fused silica with 5% phenylpolydimethylsiloxane) column (30 m × 0.32 mm × 0.25 µm) [1].
Results
Utilization of biowastes such as AP, PP, PS and OP has been shown for producing PHA and its co-polymers. Supplementation of the biowaste with glucose proved helpful in further improving PHA production and its composition [3]. As CG is now available as waste from biodiesel industry hence, we explored the possibilities of its co-metabolism with these biowastes.
Influence of CG on PHA Production from Biowastes
B. thuringiensis EGU45 grows well on slurries of different biowastes such that the DCM of 720 mg/L on AP to 3590 mg/L on OP was recorded (Table 1). B. thuringiensis EGU45 could produce 40–350 mg PHA/L on slurries containing 2%, TS of 4 different biowastes: AP, PS, PP, OP. PHA constituted 2–17% of the total DCM. OP as feed proved to be the best in terms of DCM (3590 mg/L), and PHA (350 mg/L) production by B. thuringiensis EGU45. Addition of CG at the rate of 1%, v/v to these biowastes proved effective in enhancing DCM and PHA production. The influence of CG on PHA production by B. thuringiensis EGU45 was: 1.08-fold on AP, 1.28-fold on PS, 1.8-fold on OP and 3.75-fold on PP. B. thuringiensis EGU45 didn’t produce HV on AP as feed, whereas it produced, co-polymers of PHA with the rest of the feeds. The addition of CG greatly influenced PHA co-polymers composition, where HV content varied from 15 to 72 mol%. With AP as feed HV content improved from nil in AP alone to 45 mol% in AP + CG as feed. Here, OP turned out to be the best feed either alone or along with CG, producing 5400 mg/L of DCM and 630 mg/L of PHA having 72 mol% HV (Table 1).
Table 1.
Influence of crude glycerol on bacterial polyhydroxyalkanoate production from biowastes
| Feed | Bacterial culture | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Bacillus thuringiensis EGU45 | Mixed culture | |||||||||
| DCM (mg/L) | PHA (mg/L) | % | Mol% | DCM (mg/L) | PHA (mg/L) | % | Mol% | |||
| HB | HV | HB | HV | |||||||
| Apple pomace (AP) | 720 | 120 | 17 | 100 | 0 | 600 | 120 | 18 | 93 | 7 |
| AP + 1% CG | 820 | 130 | 16 | 55 | 45 | 1850 | 200 | 10 | 65 | 35 |
| Potato peels (PP) | 1040 | 40 | 2 | 92 | 8 | 1500 | 120 | 11 | 97 | 3 |
| PP + 1%CG | 2340 | 150 | 6 | 85 | 15 | 1700 | 105 | 5 | 75 | 25 |
| Pea-shells (PS) | 1200 | 70 | 6 | 98 | 2 | 850 | 65 | 8 | 95 | 5 |
| PS + 1% crude glycerol (CG) (v/v) | 1230 | 90 | 6 | 81 | 19 | 1700 | 455 | 26 | 72 | 28 |
| Onion peels (OP) | 3590 | 350 | 8 | 83 | 17 | 1900 | 450 | 23 | 91 | 9 |
| OP + 1% CG | 5400 | 630 | 11 | 28 | 72 | 3400 | 2040 | 60 | 70 | 30 |
PS pea-shells, PP potato peels, AP apple pomace, OP onion peels, CG crude glycerol, DCM dry cell mass, PHA polyhydroxyalkanoate, HB hydroxybutyrate, HV hydroxyvalerate
On the other hand, defined mixed bacterial culture (5MC1) was found to produce PHA in a significant quantity. Hydrolysates of AP, PP, PS and OP with MHC2 resulted in a PHA content of 65–450 mg/L of the total DCM recorded. PHA constituted 8–23% of the total yield (Table 1). Addition of CG to the biowastes slurries resulted in higher DCM (1700–3400 mg/L) and PHA (200–2040 mg/L). On individual basis, PHA yield improved 1.67-fold on AP, 7-fold on PS, 4.53-fold on OP. There was a decline in PHA yield on addition of CG to PP. In comparison to B. thuringiensis EGU45, PHA co-polymer production was observed with all the biowastes treated with mixed bacterial culture: 5-fold on AP + CG, 8.3-fold on PP + CG, 5.6-fold on PS and 3.3-fold on OP + CG compared to their respective individual feeds (Table 1). In this case also OP was found to be the suitable feed material for PHA and its co-polymer production, which was observed to vary up to 450 mg/L on OP with a DCM of 1900 mg/L. Supplementation of CG (1%, v/v) to the biowastes (OP) feed resulted in an enhanced DCM of 3400 mg/L. PHA production also increased up to 2040 mg/L, which is equivalent to a yield of 60%. The supplementation of CG to other biowastes also resulted in enhanced bacterial growth in the range of 1700–1850 mg/L. PHA content varied from 105 to 455 mg/L. Although addition of CG (1%, v/v) proved helpful in improving DCM and PHA production, however, it negatively influenced the PHA co-polymer composition. The HV mol% was found to decrease from 72 mol% with B. thuringiensis EGU45–30 mol% with mixed bacterial culture (5MC1) (Table 1).
Influence of CG on PHA Production from Co-utilization of Biowastes
In view of the fact that OP turned out to be the best performer in terms of PHA production either individually or along with CG, we checked the influence of AP, PS and PP on this combination. Hence, OP was mixed with these wastes in different ratios.
Apple and Onion Wastes
The combination of OP: AP in different ratios produced PHA in the range of 100–250 mg/L B. thuringiensis EGU45. Addition of CG (1%, v/v) to OP + AP helped in enhancing PHA content i.e. up to 400 mg/L, which was 4-fold higher than the control (Table 2). Although, there was an increase in PHA production however, HV content did not improve significantly and continued to be around 75–82 mol% (Table 2). In conclusion, combination—OP: AP:: 2:1 +1% CG proved to be the most effective for PHA production by B. thuringiensis EGU45. Similarly, co-utilization of these wastes along with CG also proved to be effective for improving PHA yield and composition with mixed bacterial cultures (Table 2).
Table 2.
Bacterial polyhydroxyalkanoate production by co-utilisation of biowastes and crude glycerol
| Feed | Bacterial culture | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Bacillus thuringiensis EGU45 | Mixed culture | |||||||||
| DCM (mg/L) | PHA (mg/L) | % | Mol% | DCM (mg/L) | PHA (mg/L) | % | Mol% | |||
| HB | HV | HB | HV | |||||||
| OP:AP | ||||||||||
| OP:AP::1:1 | 2000 | 245 | 12 | 20 | 80 | 1260 | 290 | 23 | 41 | 58 |
| OP:AP::1:2 | 2010 | 250 | 12 | 35 | 65 | 570 | 28 | 5 | 64 | 36 |
| OP:AP::2:1 | 2200 | 100 | 4 | 20 | 80 | 1450 | 260 | 18 | 25 | 75 |
| OP:AP::1:1 + 1% CG | 2800 | 385 | 14 | 25 | 75 | 1400 | 280 | 20 | 23 | 77 |
| OP:AP::1:2 + 1% CG | 2800 | 350 | 13 | 25 | 75 | 1590 | 216 | 14 | 35 | 65 |
| OP:AP::2:1 + 1% CG | 3030 | 400 | 13 | 18 | 82 | 2400 | 275 | 11 | 20 | 80 |
| OP:PS | ||||||||||
| OP:PS::1:1 | 2000 | 185 | 10 | 35 | 68 | 1800 | 90 | 5 | 50 | 50 |
| OP::PS::1:2 | 1550 | 125 | 15 | 80 | 20 | 2600 | 65 | 3 | 95 | 5 |
| OP:PS::2:1 | 1900 | 80 | 5 | 65 | 35 | 2000 | 300 | 15 | 90 | 10 |
| OP:PS::1:1 + 1% CG | 2800 | 325 | 12 | 20 | 80 | 2300 | 35 | 12 | 10 | 90 |
| OP:PS::1:2 + 1% CG | 2700 | 1230 | 45 | 20 | 80 | 1600 | 280 | 10 | 35 | 65 |
| OP:PS::2:1 + 1% CG | 3700 | 1300 | 25 | 15 | 85 | 2600 | 265 | 5 | 35 | 65 |
| OP:PP | ||||||||||
| OP:PP::1:1 | 1200 | 40 | 4 | 75 | 25 | 950 | 115 | 12 | 95 | 5 |
| OP:PP::1:2 | 1500 | 50 | 3 | 60 | 40 | 650 | 71 | 11 | 98 | 2 |
| OP:PP::2:1 | 600 | 30 | 3 | 66 | 34 | 570 | 35 | 6 | 0 | 0 |
| OP:PP::1:1 + 1% CG | 900 | 75 | 8 | 60 | 40 | 900 | 87 | 9 | 97 | 3 |
| OP:PP::1:2 + 1% CG | 1000 | 20 | 3 | 75 | 25 | 975 | 66 | 7 | 98 | 2 |
| OP:PP::2:1 + 1% CG | 900 | 55 | 9 | 63 | 37 | 450 | 45 | 10 | 0 | 0 |
PS pea-shells, PP potato peels, AP apple pomace, OP onion peels, CG crude glycerol, DCM dry cell mass, PHA polyhydroxyalkanoate, HB hydroxybutyrate, HV hydroxyvalerate
Pea-Shells and Onion Waste
Mixtures of OP and PS without CG in diverse combinations resulted in drastic reduction in PHA yield to 80–185 mg/L in comparison to those recorded with either OP or AP. However, addition of CG (1%, v/v) resulted in improving PHA production by B. thuringiensis EGU45, which was 16.2-fold at 2:1, 9.8-fold at 1:2 and 1.7-fold at 1:1 in comparison to respective controls (Table 2). Although HV content in the OP and PS combinations was lower than that recorded with AP and OP, however, addition of CG didn’t result in any significant improvement. Although, with mixed microbial cultures PHA yields and their compositions from mixed biowastes (OP and PS) were lower than those recorded with B. thuringiensis EGU45, however, they were better than their respective controls (Table 2).
Potato and Onion Wastes
When OP and PP hydrolysates were mixed in different ratios, PHA production by B. thuringiensis EGU45 was quite low. In these cases PHA co-polymer content was also negligible. However, with CG (1%, v/v) as supplementation, the PHA content increased up to 75 mg/L at 1:1 of OP: PP. Addition of CG also led to reduction in HV content. Similar observations were made with mixed microbial cultures (Table 2).
In conclusion, OP turned out to be the best feed for PHA co-polymers production. Addition of CG proved effective in improving PHA and its co-polymer production with B. thuringiensis EGU45 and mixed microbial cultures. OP and PS mixed in 1:2 or 2:1 ratio along with CG proved effective in enhancing PHA yields.
Discussion
Different reports have shown the PHA producing ability of Bacillus species by co-utilizing different biowastes as feed substrates [1, 2, 6, 9, 12, 13]. Production of PHA from biowastes such as PP, OP, AP and PS has been reported with B. thuringiensis EGU45 and B. cereus EGU43 [3, 9, 14]. Subsequently, these wastes were studied in different combinations as feed for PHA production. It was realized that PS and OP hydrolysates along with glucose resulted in enhanced PHA yield and a 3HV content upto 7.5% (w/w) [3]. On the other hand, these strains have been also observed to metabolize CG, and produce PHA with a HV content of 13.4% (w/w) [10]. The potential of B. thuringiensis EGU45 to produce co-polymers from CG was also shown by adding propionic acid. In addition it was also observed that B. thuringiensis EGU45 can also tolerate a combination of CG and glucose [1]. Hence, in the present study, we have combined biowastes and CG to explore the possibility of extending the metabolic limits of B. thuringiensis EGU45 to produce PHA and its co-polymers. It was observed that PS and OP hydrolysates along with CG were the best combinations for PHA production by B. thuringiensis EGU45, as was the case where glucose was used as supplement [3]. The unique feature of the present study is that OP:AP::2:1 along with 1% v/v, CG could produce 1300 mg/L of PHA with a enhanced HV content of 21.2% (w/w). In addition, here the need for supplementation with precursors [1] for PHA co-polymers was replaced with naturally occurring biowastes. These precursors are generally volatile fatty acids (VFAs) such as propionic acid (PA), butyric acid (BA), acetic acid (AA) and iso-valeric acid (IVA), which act as intermediates in PHA co-polymer production [3, 4, 8, 9]. Here, this enhancement in PHA content was seen with change in metabolism i.e. addition of CG to biowastes. This led to changes in PHA co-polymer compositions (3HB:3HV mol%)—55:45, 85:15, 81:19, 28:72 by B. thuringiensis EGU45 and 5MC1 (Tables 1, 2).
Copolymers of PHA have gained attention due to their various biomedical applications. Due to their lower crystallinity, P (3HB-4HB) were employed for drug delivery and making medical devices such as: rivets, tacks, pins, sutured fastener etc. [15]. PHA co-polymers produced by Pseudomonas fluoroscenes act as immobilizing agent in clinical diagnostics [16]. Aeromonas hydrophila produces PHA copolymers poly-3-hydroxybutyrate-hydoxyhexanoate (PHB-HHx), which are useful in preparing vessel stent [15]. Poly-3-hydroxy-actylthioalkanoate-co-3-hydroxyalkanoate used as antibacterial agent against Staphylococcus aureus [17, 18]. PHB-HHx has been exploited for osteoblast attachment, proliferation and differentiation [19, 20]. PHA-copolymers can be used as scaffolds for liver tissue engineering, cartilage repair and promoting cell growth [21–23].
In general, we expect different wastes to get mixed while transportation hence, we need to treat wastes in combinations. The need is to have a versatile organism or a combination of organisms, which can withstand physiological stress arising out of variation in feed material [9, 10]. Since, Bacillus has a unique ability to produce hydrogen and PHA from different wastes even in non-limiting nitrogen conditions [10, 24], hence, this study will help in further exploiting them on different feed material in diverse combinations. Bacillus is an organism which is generally regarded as safe (GRAS). Hence, it can be used in an unrestricted manner without harming environment [4, 25, 26].
Acknowledgements
We are thankful to the Director of CSIR-Institute of Genomics and Integrative Biology (IGIB), and CSIR project CSIR-HRD (ES Scheme No. 21(1022)/16/EMR-II) for providing the necessary funds, facilities and moral support. SR is thankful to CSIR for granting Senior Research Fellowship. Authors are also thankful to Academy of Scientific and Innovative Research (AcSIR), New Delhi.
Compliance with ethical standards
Conflict of interest
None.
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