Levulinic Acid: A Valuable Platform Chemical for the Fermentative

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Chapter 21

Levulinic Acid: A Valuable Platform Chemical for the Fermentative Synthesis of Poly(hydroxyalkanoate) Biopolymers Richard D. Ashby* and Daniel K. Y. Solaiman Eastern Regional Research Center, Agricultural Research Service, U.S. Departments of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, United States *E-mail: [email protected].

Levulinic acid (LVA) is a valuable platform chemical that can be produced from both 5- and 6-carbon sugars. As such, lignocellulosic biomass is a rich source of sugars for the large-scale production of LVA. Because LVA is relatively inexpensive, it may be viewed as a promising feedstock for biological syntheses. Many bacterial strains have been shown to utilize LVA for the synthesis of polyhydroxyalkanoate (PHA) biopolymers; however, only a few have been confirmed to use LVA in combination with other low-cost, renewable feedstocks (e.g., glycerine, whey permeate, and xylose) to produce copolymeric PHA polymers. This chapter focuses on our work as well as that of others in the synthesis of copolymers consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) (P-3HB-co-3HV) from separate mixtures of LVA with glycerine, whey permeate, and xylose. By utilizing LVA in conjunction with other low-cost feedstocks, the economics of PHA production can be improved, making these materials more attractive for large-scale use.

Introduction In a 2004 report from the National Renewable Energy Laboratory, levulinic acid (LVA; 4-oxovaleric acid) was included as one of the top bio-based platform molecules (1). It can be produced by acid-catalyzed dehydration from both 5- and Not subject to U.S. Copyright. Published 2018 by American Chemical Society Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

6-carbon sugars (Figure 1) (2). As LVA is inexpensive (with economic forecasts as low as $0.04–0.10/lb) (3), it has attracted interest from a number of large chemical companies as a starting material for the chemical or enzymatic conversion of many different compounds involved in the fuel, herbicide, polymer, and solvent arenas (Figure 2). It has been acknowledged that high concentrations of LVA are toxic to living systems. Recently, however, it was demonstrated that LVA could be tolerated by certain bacterial strains at concentrations of 20 g/L and higher. Specifically, members of the genus Rhodococcus, Cellulosimicrobium, Granulicatella, Brevibacterium, and Aeromonas were identified as capable of growth at high LVA concentrations with Brevibacterium epidermidis LA39-2 capable of growth at LVA concentrations as high as 80 g/L (4).

Figure 1. Biosynthetic pathways for the acid-catalyzed synthesis of LVA from 5and 6-carbon sugars.

Polyhydroxyalkanoate (PHA) biopolymers comprise a family of assorted bacterial polyesters that are produced as carbon and energy reserves by many bacterial species. The synthetic process is initiated when excess carbon is present but cellular growth is hindered by a deficiency of other essential nutrients. These biopolymers are biorenewable, biocompatible, and biodegradable to carbon dioxide and water in microbially active environments. Because of their structural variability, PHA biopolymers exhibit an array of material properties from rigid thermoplastics (short-chain PHA) to amorphous elastomers (medium-chain PHA) (5). Generally, it is accepted that short-chain PHA biopolymers typically contain 3-hydroxyalkanoic acids with monomer lengths ranging anywhere from 3 to 5 carbon units, whereas medium-chain PHA polymers are composed of monomers ranging from 6 to 14 carbon units and may contain alkenyl and/or functionally substituted chemical groups (6). 340 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Figure 2. Chemical derivatives from LVA. (BPA=Bisphenol A).

341 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Poly(3-hydroxybutyrate) (P-3HB) is the most well-known of the PHA biopolymers. It has been compared to polypropylene and polyethylene with respect to its material properties, but with the added benefit of improved environmental impact over both synthetic polymers. Unfortunately, because of its crystalline nature, P-3HB is considered too rigid and brittle for widespread industrial application. Material properties have been enriched by synthesizing copolymers with reduced crystallinity, increased flexibility, and decreased melting temperatures. The most commonly produced copolymer is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P-3HB-co-3HV) (for structure see Figure 3) (7). However, monomers such as 4-hydroxybutyric acid (8), 4-hydroxyvaleric acid (9), and 3-hydroxyhexanoic acid (10) have also been successfully copolymerized with 3-hydroxybutyric (3HB) acid to produce copolymers with manageable material properties. As LVA is structurally similar to pentanoic acid, it has been evaluated as a viable feedstock for fermentative synthesis of PHA biopolymers. Success in the production of P-3HB-co-3HV biopolymers has been realized when low concentrations of LVA were used in combination with other less harmful substrates. To this end, P-3HB-co-3HV copolymers have been produced from LVA when used in conjunction with typical monosaccharide cosubstrates (11–13). The metabolic pathway for 3HB and 3HV monomer synthesis from LVA in Cupriavidus necator was postulated in 2011 and is shown in Figure 3 (14). Subsequent work demonstrating the function of the LvaABCDE gene products in the catabolism of LVA to P-3HB-co-3HV copolymers through 4-hydroxyvaleryl-CoA and 4-phosphovaleryl-CoA intermediates has also been recently published (15). Although the use of LVA in the synthesis of copolymeric PHA biopolymers is not necessarily new, most of the work to date has been focused on the use of simple sugars as cosubstrates for polymer synthesis (11–13). In this chapter we describe efforts to use additional low-cost, nontraditional feedstocks in combination with LVA to produce unique PHA biopolymers.

Glycerine Glycerine is a renewable coproduct that has generated attention as a substrate for PHA biopolymer synthesis. It is derived from the transesterification of triacylglycerols (TAG) in the production of short-chain alkyl esters more commonly known as biodiesel (Figure 4). Biodiesel possesses many favorable qualities over petroleum-based diesel, including renewability and reduced emissions, and while the current cost to produce biodiesel is comparatively expensive, its benefits are driving increased production worldwide. This has affected the glycerine market by making glycerine more readily available, especially in its crude form, which has dropped the price and made it more attractive as a feedstock for value-added bio-based material synthesis.


Figure 3. Proposed metabolic pathway for the synthesis of P-3HB-co-3HV from LVA. Rxn 1: Enzyme – Acyl-CoA Synthetase, Cofactors – ATP, CoA; Rxn 2: Cofactors – ATP, CoA; Rxn 3: Enzyme – β-Ketothiolase A; Rxn 4: Enzyme – β-Ketothiolase B; Rxn 5: Enzyme – Acetoacetyl-CoA Reductase, Cofactors – NADPH; Rxn 6: Enzyme – Acetoacetyl-CoA Reductase, Cofactors – NADPH; Rxn 7: Enzyme – PHA Synthase.


Figure 4. Transesterification reaction in the synthesis of short-chain alkyl esters (biodiesel) and glycerine coproduct.

Unfortunately, biodiesel-derived crude glycerine is generated with many different levels of purity, depending on the efficiency of the transesterification reaction, the success of biodiesel recovery, and whether the alcohol involved in the reaction is recycled. Typically, the highest quality crude glycerine derived from transesterification contains approximately 85% glycerine, but the glycerine content can be much lower with the balance of the material made up of water, salts, free fatty acids (FFA), unrecovered alkyl esters, monoacylglycerols (MAG), diacylglycerols (DAG), TAG, etc. These differences in crude glycerine content can affect PHA biopolymer compositions in some circumstances, depending on the genetic capabilities of the producing bacterial strain. Those bacterial strains that are capable of producing short-chain PHA biopolymers are usually less susceptible to variability in their PHA compositions despite the quality of the crude glycerine (16). This is because acetyl-CoA is the metabolic precursor to P-3HB biosynthesis and whether that acetyl-CoA is derived from glycerine (as described in the next paragraph) or from the β-oxidation process, the effect is equal. The same cannot be said for the bacterial strains containing the genes involved in medium-chain PHA biosynthesis. These strains normally use shortened fatty acids (or fatty acids synthesized de novo from glycerine) as the metabolic precursors for PHA biosynthesis. As such, the amount and type of fatty acids (whether in the form of FFA, alkyl ester, MAG, DAG, and/or TAG) present in the crude glycerine can influence the final monomer content of medium-chain PHA biopolymers. For example, all things being equal, the crude glycerine derived from a soybean oil-based transesterification reaction would likely contain a higher linoleic acid content (depending on the efficiencies of the parameters described previously) than a palm oil-based reaction, which would 344 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

likely be higher in palmitic acid and/or oleic acid. Because the compositions of medium-chain PHA biopolymers reflect the fatty acids from which they are synthesized, compositionally distinct medium-chain PHA biopolymers would be produced with varying material properties. Glycerine easily passes through the bacterial cell membrane through facilitated diffusion by the action of a glycerol facilitator protein (17). Once inside the cell, it is easily assimilated into central metabolism through the sequential action of an ATP-dependent glycerol kinase, which phosphorylates glycerine to yield glycerol-3-phosphate and an NAD+-dependent glycerol-3-phosphate dehydrogenase enzyme. This catalyzes the conversion of glycerol-3-phosphate to dihydroxyacetone phosphate, which can be metabolized further to acetyl-CoA, a vital precursor to P-3HB synthesis. Because of this, many research groups have reported on the successful utilization of glycerine (both refined and crude) as a feedstock for the fermentative synthesis of PHA (particularly short-chain) biopolymers. For a thorough review of glycerine utilization in PHA biopolymer synthesis, see Zhu et al. 2013 (18). Unfortunately, using mixtures of glycerine and LVA to induce copolymer formation has been demonstrated by relatively few research groups (Table 1). Bera et al. (19) used Halomonas hydrothermalis MTCC 5445 to produce PHA biopolymers from crude glycerine (for composition of crude glycerine, see the footnote for Table 1) and two distinct LVA samples (20). A crude LVA sample was derived from the acid hydrolysis product of the seaweed Kappaphycus alvarezii, which was found to contain both LVA and formic acid in nearly equimolar amounts. Results showed that the crude LVA-containing flasks produced more PHA at higher LVA concentrations than the flasks containing standard LVA. The PHA biopolymers produced from crude LVA had 3HV contents as high as 81 mol%. Interestingly, when standard LVA was used in combination with crude glycerine, an inhibitory effect was seen on the growth of the bacterial strain even at LVA concentrations as low as 0.06%, which had a detrimental effect on the PHA yield. Unfortunately, the authors did not report the compositions of the PHA biopolymers derived from standard LVA, but the yield difference between the two sets of cultures seems to point to a possible role of other unidentified constituents of the crude LVA in the stimulation of greater PHA production at increased LVA concentrations. Another report focused on the search for valid nucleating agents to help the crystallization of P-3HB-co-3HV (21) (Table 1, Entry 2). In that paper Burkholderia cepacia ATCC 17759 (formerly Pseudomonas cepacia) was used to produce various copolymers using a two-stage, fed-batch fermentation procedure in which the glycerine content was maintained between 1 and 3%. The first stage of the fermentation involved the growth of B. cepacia to high concentrations using glycerine as the sole carbon source (120 h). During the second stage, LVA was introduced into the fermenter at a rate approaching 0.5 g/L·h thus maintaining an LVA concentration that was less than 0.1%, which was inhibitory to bacterial growth. By varying the time at which LVA introduction was initiated (72 h, 96 h, or 120 h), P-3HB-co-3HV copolymers were produced with 3HV contents ranging from 5.6 to 32.6 mol%. 345 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Table 1. Conditions for Successful Short-Chain PHA Biosynthesis from Glycerine and LVA Entry

Bacterial strain

Titer (g/L)

Conditions

Compositions 3HB

1

2

3

Halomonas hydrothermalis

Burkholderia cepacia

Pseudomonas oleovorans

Shake Flask: • 100-mL volume • crude glycerinea • LVA concentration = 0 to 1.0% Fermenter: • 5-L volume • Reagent-grade glycerinec • Two-stage fermentation 1) Stage 1 = growth 2) Stage 2 = LVA supplement Shake Flask: • 1-L volume • Reagent-grade glycerinec • 1 wt% Total C • LVA concentration = 0 to 1.0% Fermenter: • 10-L volume •c • 1 wt% Total C • LVA concentration = 0 to 0.75%

0.3–3.5

NRb

0.5–0.8

0.7–1.1

Ref.

3HV

NRb (for standard LVA) 100 94.4 88.6 85.3 82.1 80.0 69.5 67.4

0 5.6 11.4 14.7 17.9 20.0 30.5 32.6

100 63 42 3 0

0 37 58 97 100

100 73 30 0

0 27 70 100

20

21

22

a

Crude glycerine in Entry 1 was obtained as a Jatropha biodiesel byproduct and was composed of 95% glycerine, 0.3% moisture and trace amounts of methanol, FFA, and metal impurities (19). b NR = Not Reported. c Reagent grade glycerine = 99.5%.

Perhaps the most focused paper on the use of glycerine and LVA as cosubstrates for the production of compositionally distinct short-chain PHA came from the laboratory of Ashby et al. 2012 (22). In that study both shake flask (1-L volumes) and batch fermentation (10-L volumes conducted in a 12-L capacity bioreactor) strategies were used to produce PHA polymers with widely varying monomeric compositions. Pseudomonas oleovorans NRRL B-14682 was used as the producing strain, and by varying the glycerine:LVA ratios (total carbon source concentration remained at 1 wt% for all cultures), PHA polymers were 346 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

produced with compositions ranging from P-3HB to P-3HB-co-3HV with varying 3HB:3HV ratios to poly-(3HV) (for specific compositions see Table 1, Entry 3). In contrast to the Zhu et al. study (21), it was determined that P. oleovorans could grow and produce PHA polymers in cultures with LVA concentrations as high as 1 weight percent (wt%). However, bacterial growth was reduced at LVA concentrations above 0.8 wt% as carbon source utilization at these higher LVA concentrations was retarded. At lower LVA concentrations (and hence higher glycerine concentrations), the molecular weights of the resulting PHA polymers were reduced. This reduction was due to glycerine serving as a chain-terminating agent: the higher the initial glycerine content in the media, the more likely chain termination would occur earlier in the synthetic process. This resulted in PHA polymers with smaller molecular weights from cultures with higher glycerine content. This phenomenon was reported previously for PHB produced from both crude (23) and refined glycerine (24) using nuclear magnetic resonance (NMR) spectroscopy.

Whey Permeate Whey is the liquid material left over from the curdling of milk in cheese-making. Once protein and other solids are removed from the whey, the material is considered whey permeate whose carbohydrate (lactose) content is typically no less than 76%. In a 2017 report, Koller et al. described efforts to produce PHA polymers from whey permeate and LVA using the bacterium Hydrogenophaga pseudoflava DSM 1034 (25). Previous work by this same group and others had demonstrated that this bacterial strain had the capability to utilize either hydrolyzed or intact whey lactose for PHA polymer synthesis (26, 27) and that it possessed the genetic capability to produce 3HV precursors under suitable growth conditions (26, 28). When using LVA (at concentrations up to 1 g/L) in combination with glucose, P-3HB-co-3HV copolymers were produced with 3HV contents as high as 100% (PHV homopolyester). When hydrolyzed whey permeate was added to the medium such that glucose and galactose were present in equimolar concentrations, it was found that glucose was preferred over galactose for bacterial growth and that higher LVA media concentrations (up to 1 g/L) caused sugar utilization to slow but resulted in P-3HB-co-3HV copolymers with higher 3HV content. When sodium valerate (another well-known precursor for 3HV synthesis) was added in addition to LVA (final concentration = 1 g/L sodium valerate: 0.5 g/L LVA: 5 g/L hydrolyzed whey permeate), higher polymer yields were achieved as well as higher (up to ~55 mol%) 3HV contents. These results demonstrated that while sodium valerate seemed to be a better precursor for 3HV monomer synthesis, LVA can also be used in combination with hydrolyzed whey permeate under fluctuating carbon source ratios to produce P-3HB-co-3HV copolymers with varying 3HB:3HV ratios.


Table 2. Conditions for Successful Short-Chain PHA Biosynthesis from Xylose and LVA Entry

Bacterial strain

Titer (g/L)

Conditions

Compositions 3HB

1

2

Burkholderia cepacia

Burkholderia sacchari

Shake Flask: • 585-mL volume • Xylose concentration = 2.2% • LVA concentration = 0.07 to 0.67% • 68 to 70 h total time

Shake Flask: • 1-L volume • Xylose concentration = 2% • LVA concentration = 0 to 0.8% • 24 to 96 h total time Shake Flask: • 1-L volume • Xylose concentration = 1% • Glucose concentration = 1% • LVA concentration = 0 to 0.8% • 24 to 96 h total time

Ref.

HV

up to 61 mol% 3HV up to 4.2

(LVA concentration >0.5% resulted in decreases in cell and PHA yields)

1.0–2.2 up to 65 mol% @ 3HV 72 h (for specific compositions see Table 3)

37

44

0.8–2.6 @ 72 up to 76 mol% h 3HV

Xylose Lignocellulosic biomass is an abundant, low-cost material and has been substituted for the more traditional carbon feedstocks in an effort to ease PHA production costs (29), but the existence of phenolic compounds in the lignocellulosic hydrolysates negatively affects PHA production (30–32). Xylose is an abundant pentose sugar that is present in the hemicellulosic fraction of lignocellulosic materials. It can be produced from plant biomass by dilute acid and/or enzymatic hydrolysis and has been established as an effective low-cost feedstock for short-chain PHA polymer synthesis. In fact, a 2004 study documented the isolation of 54 bacterial strains from soil capable of utilizing xylose for growth and PHA biopolymer production (33) while a follow-up study in 2009 reported the isolation of 13 additional isolates able to metabolize xylose (34). Unfortunately, the authors did not specifically identify these isolates. However, a few known xylose-utilizing strains have been identified. Burkholderia 348 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

cepacia ATCC 17759 (formerly Pseudomonas cepacia) (35) and Burkholderia sacchari DSM 17165 (IPT101) (36) are both known to synthesize short-chain PHA polymers from xylose and as such, these strains have been studied for the successful utilization of xylose in combination with LVA in the synthesis of PHA polymers (Table 2). The 2004 paper by Keenan et al. (Table 2, Entry 1) was the first documented study on the possibility of utilizing xylose in combination with LVA to produce P-3HB-co-3HV copolymers (37). In that study B. cepacia ATCC 17759 was used as the producing strain in a two-stage shake-flask culture protocol. The first stage (seed cultures) focused on cell growth in order to increase bacterial biomass prior to the PHA production (second) stage. Polymer production was accomplished in the presence of 2.2% xylose and LVA concentrations varying from 0.07 to 0.67%. The results showed that LVA enhanced both bacterial growth and PHA polymer accumulation up to 0.52% LVA media concentration. Under these conditions P-3HB-co-3HV copolymers were produced with an average 3HV content of 43 mol%. At LVA concentrations higher than 0.52%, copolymers were produced with 3HV contents as high as 61 mol% but cell and PHA polymer yields were drastically reduced. In addition, the chemical shifts and relative intensities of the expanded carbonyl resonance peaks in the 13C NMR corresponding to the 3HB-3HB, 3HB-3HV, and 3HV-3HV dyads were similar to those previously reported (38, 39) for statistically random copolymers. From this the authors supposed that the copolymers derived from B. cepacia in this study also had a random sequence distribution. In fact, most copolymeric PHAs are reportedly sequenced in random distributions with the specific monomeric units spaced arbitrarily along the polymer chain. Another possibility is the formation of PHA copolymers containing block regions, which may provide an advantage in preserving the properties of the homopolymeric regions thus providing new copolymers with distinct physical qualities. Synthetic block copolymers typically consist of homopolymeric regions that are covalently linked together. These regions normally originate as two or more thermodynamically dissimilar polymeric chains that can be linked through a number of polymerization techniques including but not limited to anionic polymerization, cationic polymerization, free radical polymerization, and group transfer polymerization (40). Bacterial synthesis of copolymers containing block regions has lagged due to the lack of specificity in many PHA synthase enzymes. In a 1996 paper, Curley et al. described the sequential production of two different PHA biopolymers by the same bacterial strain (P. oleovorans ATCC 29347) when grown on mixtures of 5-phenylvaleric acid and nonanoic acid (41). In that study it was demonstrated that each carbon source was utilized separately in the production of poly-3-hydroxy-5-phenylvalerate homopolymer from 5-phenylvaleric acid and a copolymer composed of 3-hydroxyheptanoate, 3-hydroxynonanoate, and 3-hydroxyundecanoate from nonanoic acid. This proved that there is some determining factor present to allow the selective utilization of mixed substrates by the same enzyme system to produce separate biopolymers within the same bacterial strain. To date only a few block copolymers have been reportedly produced via fermentation. In a 2006 paper, Pederson et al. synthesized block 349 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

copolymers of poly-3HB-block-3HB/3HV by using fructose and pentanoic acid with Cupriavidus necator strain H16 (ATCC 17699) (42). In 2011 Li et al. reportedly used a mutant strain of Pseudomonas putida KT2442 (designated KTOY06ΔC) to produce a block copolymer of 3HB and a random copolymer of 3HV and 3-hydroxyheptanoic (3HHp) acid (poly-3HB-block-3HV/3HHp) (43). In both of those instances sequential feeding strategies were necessary to induce the formation of one homopolymeric block then, by feeding the cultures with an unrelated carbon source(s), the formation of the second block could be formed. In a 2018 report by Ashby et al. (Table 2, Entry 2) it was determined that B. sacchari DSM 17165 could produce poly-3HB-block-3HV copolymers from xylose and LVA without the need for sequential substrate feeding strategies (44). Whether in the presence of 2% xylose or 1% xylose:1% glucose, by varying the LVA concentration within the media, it was determined that copolymers could be produced that contained block regions. It was found that early in the shake-flask cultures, LVA was the primary carbon source used by the bacterium, which resulted in copolymers with high 3HV contents. As the cultures progressed, higher 3HB contents were found within the maturing copolymers (Table 3).

Table 3. Polymer Compositions for the poly-3HB-co-3HV Copolymers Derived from B. sacchari DSM 17165 Grown on 2% Xylose and 0–0.8% LVA Sample

0 LVA

0.2% LVA

0.4% LVA

0.6% LVA

0.8% LVA

Time (h)

Composition (mol%) 3HB

3HV

24

100

0

72

100

0

24

36

64

72

84

16

24

12

88

72

57

43

24

8

92

72

44

56

24

5

95

72

41

59

Thermal analysis revealed that the 24-h polymer samples displayed melting temperatures (Tm) between 77 and 105 °C, approximating the Tm values of poly-3HV homopolymers, and glass transition temperatures (Tg) between -13 and -15 °C (Tg of poly-3HV = -16 °C). These results hinted that the polymers were in fact composed of extensive regions of 3HV blocks. Thermal properties of the polymers produced in the presence of 2% xylose and 0.4%, 0.6%, and 350 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

0.8% LVA and harvested at 72 h showed two distinct Tg values, one between -4 and 2 °C, approximating that of poly-3HB (3 °C), and the other at -13 to -14 °C. These results showed that later in the production process homopolymeric blocks of both 3HB and 3HV were present. The existence of a block structure was supported by the carbonyl regions in the 13C NMR analysis of each copolymer produced (Figure 5). At 24 h the majority of the sequences favored the 3HV-3HV dyads (at 169.51 ppm) (Figure 5A). The only exception was in the polymers derived from xylose in the absence of LVA, which, logically, would not be expected to contain any 3HV monomers. In comparison, at 72 h the 3HB-3HB sequences (at 169.13 ppm) were more prevalent (Figure 5B), indicating that as the cultures progressed, more 3HB homopolymeric regions were formed, resulting in copolymers with blocks of homopolymeric regions of 3HV and 3HB monomers. Subsequent mass spectrometry analysis was conducted using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) spectrometry and showed that for the individual oligomers a block of 3HB and 3HV were especially noticeable at the smaller length oligomers (heptamer and octamer). In fact, when the Bernoullian statistical analysis was applied to the samples, there was a poor match between the calculated compositional data and the experimentally derived results, resulting in poor Hamilton agreement factors. These data further proved that these polymers did contain some level of organization in their sequence distributions. However, further analysis using tandem mass spectrometry (MS/MS) showed the loss of either 86 amu or 100 amu corresponding to either 3HB or 3HV, respectively. This result, combined with the results from the other experiments, showed that the PHA biopolymers produced by B. sacchari DSM 17165 in the presence of 2% xylose and various concentrations of LVA were composed primarily of block structures with some relatively random regions within the polymer chains.

Conclusions LVA is a well-known platform chemical that has been used for the chemical synthesis of many beneficial materials. In this chapter we have demonstrated that LVA can also be used as a viable feedstock for PHA polymer biosynthesis. Typically, LVA has been used along with other conventional feedstocks (e.g., glucose) to produce PHA polymers; however, with the relatively high price of PHA polymer synthesis when compared to other similar petrochemical polymers, conventional feedstocks do not benefit the economics of PHA polymer production. Because LVA is easily produced by acid catalysis from an abundant renewable material, its cost is low compared to other commonly used feedstocks such as propionic acid or pentanoic (valeric) acid. By using LVA in combination with other inexpensive carbon sources, such as glycerine, whey permeate, and xylose, unique PHA polymers have been produced with widely varying compositions and organization. By controlling these parameters the properties of these polymers can be better tuned to specific applications and the cost to produce these renewable polymers reduced, making them more favorable for large-scale applications. 351 Cheng et al.; Green Polymer Chemistry: New Products, Processes, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Figure 5. Expanded carbonyl region of the 13C NMR analyses of the polymers produced by B. sacchari DSM 17165 on 2% xylose and varying concentrations (0–0.8%) LVA at 24 h (A) and 72 h (B). (Resonances at 169.51 ppm correspond to the 3HV-3HV dyads, at 169.31 ppm and 169.33 ppm correspond to the 3HB-3HV dyads, and those at 169.13 ppm correspond to the 3HB-3HB dyads.)

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