Biosynthesis of Polyhydroxybutyrate from Glycerol
A large number of microorganisms can uptake glycerol from ambient environment and metabolize it into building blocks for microbial growth and development. In native PHA producing bacteria, when abundant carbon sources (e.g., glycerol, sugars, fatty acids) are present and at least one of other nutrient is (e.g., nitrogen, phosphate, oxygen) depleted, PHAs are produced as carbon and energy reserves. Under such conditions, glycerol is generally converted into P(3HB) by various native PHA producing bacteria.5, 83–87 As shown in Figure 3, glycerol is converted to glyceraldehyde-3-phosphate (GAP) by three enzymes, glycerol kinase (GlpK), glycerol-3-phosphate dehydrogenase (GlpD), and triosephosphate isomerase (TPI). GAP is an intermediate in the glycolysis pathway and eventually metabolized to pyruvate. The pyruvate dehydrogenase complex contributes to transforming pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Microbial biosynthesis of P(3HB) starts with condensation of two molecules of acetyl-CoA by β-ketothiolase (PhaA) into acetoacetyl-CoA, which is subsequently reduced to (R)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (PhaB). The last step of PHA biosynthesis is dependent on PHA synthase (PhaC) to polymerize 3-hydroxybutyryl-CoA moieties to P(3HB).73, 88, 89
Figure 3. Proposed pathway for metabolism of glycerol and short-chain-length PHA production. Glycerol enters the cell via the glycerol facilitator protein (GlpF) and is phosphorylated by GlpK to produce glycerol-3-phosphate. Glycerol-3-phosphate is reduced by GlpD to produce dihydroxyacetone phosphate which enters into the glycolytic pathway to be converted to pyruvate, and eventually acetyl-CoA. Two molecules of acetyl-CoA are condensed by the β-ketothiolase (PhaA) enzyme to produce acetoacetyl-CoA. This molecule is reduced by acetoacetyl-CoA reductase (PhaB) to produce the substrate (R)-3-hydroxybutyryl-CoA, which is polymerized by the PHA synthase (PhaC).
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The P(3HB) homopolymer is the most extensively studied PHA and has been produced by several species of bacteria using glycerol as a carbon source.5, 90–92 Teeka et al.92 reported that P(3HB) accumulated to 45% of the cell dry weight (CDW) with biomass yield at 3.5 g/L in 72 h. Shrivastav et al.93 isolated and identified two bacterial strains from soil and marine environments for producing P(3HB) from Jatropha biodiesel byproduct as a carbon source and found that P(3HB) was accumulated up to 76% of CDW in 4.0 g/L biomass in shake flasks. Compared to these shake flask experiments, fed-batch fermentation technology has been shown to dramatically increase cell density, up to 82.5 g/L CDW (Table III), using waste glycerol as a feedstock. In addition, P(3HB) production increased to 51 g/L using fed-batch reactors.84 Improved fermentation processes and optimized fermentation conditions have the potential to be scaled-up for PHA industry to commercialize these biodegradable plastics with comparable market price while using renewable and inexpensive feedstocks, e.g., crude glycerol.31
Table III. Survey on Bioconversion of Glycerol to P3HB Using Various Bacteria
|Strains||Glycerol purity||CDW (g/L)||PHB yield||References|
Impurities found in crude glycerol, such as methanol and salts, can dramatically effect bacterial growth and PHA yield.94 Mothes et al.83 found that NaCl at concentration of 5.5% in the crude glycerol resulted in a lower PHA yield due to osmoregulation. Ashby et al.87 reported that the usage of untreated crude glycerol with 40% methanol affected microbial growth and P(3HB) production. Therefore, optimization of biodiesel production process for efficient catalysis and the following methanol recycling will not only reduce the production cost for biodiesel manufacturing, but also help subsequent utilization of crude glycerol during fermentation processes.
However, some impurities, such as free fatty acids and alkyl esters, could act as complementary carbon sources and be utilized by bacteria to enhance their growth. Ashby et al.87 studied the compositions of crude glycerol and found that Pseudomonas oleovorans could use fatty acids and unrecovered alkyl esters as additional carbon sources for bacterial growth. Burkholderia sp. USM was identified to be capable of converting palm oil, fatty acids, and glycerol byproducts into P(3HB).95 Zhu88 used Burkholderia cepacia to grow on tall oil fatty acids, consisting of 52% oleic acid and 45% linoleic acid, for PHA production. The maximum CDW and P(3HB) yield reached 3.8 g/L and 50% of CDW, respectively, under these conditions. B. cepacia grown on crude glycerol generated by a small batch biodiesel processor at SUNY-ESF containing free fatty acids as a carbon source for comparable growth, yet much lower glycerol consumption, indicating that B. cepacia metabolized fatty acids, along with glycerol, for bacterial growth and PHA production.88 Teeka et al.96 isolated a previously unidentified strain, AIK7, which also demonstrated enhanced bacterial growth, even improved PHA yield, when using waste glycerol containing free fatty acid instead of pure glycerol.
Zhu et al.5 studied the effects of glycerol concentration on P(3HB) production. High concentrations of glycerol (≥3%) in fermentation broth exhibited obvious inhibitory effects on bacterial growth due to osmotic stress on the cells. Maintenance of proper concentration of glycerol in the medium (1–3 wt %) should be considered for high cell density and PHA yields during fermentation of crude glycerol.5, 10, 91
The efficiency of converting glycerol to PHA varies due to the substrate concentration in the medium. Ibrahim and Steinbüchel85 reported that glycerol concentration at 10 g/L gave the highest product yield at 0.31 g P(3HB)/g glycerol by Zobellella denitrificans MW1. Higher glycerol concentrations of 20, 30, and 50 g/L in the media resulted in the low product yield at 0.21, 0.12, and 0.03 g P(3HB)/g glycerol, respectively. The same group97 studied the fed-batch fermentation for P(3HB) production. The product yield could be increased from 0.10 g P(3HB)/g glycerol to 0.25 P(3HB)/g glycerol after optimization of the fed-batch process. Cavalheiro et al.84 used pure glycerol and waste glycerol as carbon sources in a two-stage fermentation by Cupriavidus necator (Ralstonia eutropha), and found that the product yield reached 0.36 and 0.34 g P(3HB)/g glycerol from pure and waste glycerol, respectively. Mixed microbial communities obtained from activated sludge in a municipal wastewater treatment plant could also efficiently utilize crude glycerol and the product yield reached 0.40 g PHA/g glycerol, which was comparable to the conversion efficiency of those using fatty acids as carbon sources.98
Characterization of Polyhydroxybutyrate from Glycerol
The P3HB homopolymer has been produced by many microorganisms utilizing carbon sources such as sugars and fatty acids. However, it is noteworthy that conversion of glycerol to P(3HB) results in polymers with relatively low molecular mass compared to P(3HB) polymers produced from sugars. Molecular mass analysis by gel permeation chromatography (GPC) showed significant decreases for PHA polymers isolated from strains utilizing glycerol as a carbon source compared to xylose.5 The number average molecular weights (Mn) of P(3HB) produced from xylose and glycerol were 468 kDa and 175 kDa, respectively, indicating that the size of P(3HB) polymers produced from xylose was approximately three-fold larger than the size of P(3HB) polymers produced from glycerol. Several other reports have also shown that P(3HB) polymers produced from glycerol exhibited lower molecular mass than P(3HB) polymers produced from sugars.90, 91, 99 1H-NMR detected that P(3HB) polymers produced from glycerol feedstocks were end capped with glycerol molecules through covalent esterification. Glycerol acts as a chain transfer agent resulting in early termination of P(3HB) polymerization, which led to low molecular mass of P(3HB).5, 87, 91 High concentrations of glycerol in bacterial growth media inhibited bacterial growth and also resulted in lower Mn and Mw (number-average molecular weight and weight-average molecular weight, respectively). When concentrations of glycerol were increased from 3% to 9%, both Mn and Mw decreased gradually from 173 kDa and 304 kDa to 87 kDa and 162 kDa, respectively.5 The polydispersity indices (PDIs) of all P(3HB) samples were between 1.9 and 2.1 in this research. However, P(3HB) produced from glycerol or xylose did not show any significant differences in thermal properties (Tm, Tg, and Tdecomp) compared to P(3HB) polymers produced from glycerol.5
It has been proposed that PHA synthesis occurs within the active site of PhaC polymerase where two thiol groups are responsible for locating the PHA monomer unit and the other holding onto the propagating chain. The type of polymerization itself was also assumed to be a chain transfer polymerization.100 Without any exogenous factors, high molecular weight PHAs are produced. With the addition of exogenous chain transfer agents with hydroxyl end groups such as glycerol and low molecular weight polyethylene glycol derivatives (PEG), PHA polymerization can be terminated early.101, 102 Since glycerol contains three hydroxyl groups per molecule and is much smaller than the PEG derivatives, it makes a good candidate as a chain transfer agent. By varying the concentration of glycerol in the starting media the molecular weight of PHAs can be controlled, due to the prevalence of glycerol terminating the PHA synthesis. Since glycerol is a small molecule it can find itself within the active site of the polymerase and covalently bond with the propagating chain.5, 87 A proposed mechanism is shown in Figure 4.
Figure 4. Proposed mechanism for chain transfer termination in PHA synthase enzymes by glycerol. A: If glycerol is present, it can enter the active site of the PHA synthase and prematurely terminate the extention of the PHA polymer, resulting in a glycerol end capped polymer. B: Under circumstances where chain transfer agents are not present, polymerization of PHA can proceed via transfer of the substrate (R-3-hydroxyacyl-CoA) and product between two active site cysteine residues.
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In addition to the effects of glycerol on molecular mass of P(3HB) polymers, residual methanol in the waste glycerol from the biodiesel process can also affect the size of P(3HB). Ashby et al.87 reported that Mn (31 kDa) of P(3HB) produced from waste glycerol (47% glycerol and 40% methanol) was 10-fold lower than the Mn (314 kDa) of P(3HB) produced from pure glycerol as a carbon source. One-dimensional 1H-NMR and two-dimensional DOSY (diffusion ordered spectroscopy) identified that the P(3HB) homopolymer was end-capped not only with glycerol, but also with methanol, which formed an ester linkage with P(3HB). The amount of methoxy group was found to be 100 times higher from the P(3HB) sample using waste glycerol than the P(3HB) sample using pure glycerol. Also, the PDI (Mw/Mn) of P(3HB) from pure glycerol and waste glycerol were 1.66 and 2.77, respectively. The higher PDI of P(3HB) produced from waste glycerol containing 40% methanol revealed that methanol in the crude glycerol may exacerbate premature chain termination by itself, which could serve as another chain termination agent. Previous research also showed poly(ethylene glycol) (PEG) could end cap P(3HB).103, 104 In a word, addition of chain termination agents, such as glycerol, PEG, and methanol, results in the production of P(3HB) polymers with lower molecular mass during biosynthesis, and thus, needs to be taken into account for PHA production when using waste glycerol as a carbon source in fermentation process.