A Critical Review on the Economically Feasible and Sustainable Poly(3-Hydroxybutyrate-co-3-hydroxyvalerate) Production from Alkyl Alcohols

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) is the most studied short-chain-length polyhydroxyalkanoates (PHA) with high application importance in various fields. The domination of high-cost propionate and valerate over other 3-hydroxyvalerate (3HV) precursors owing to their wide preference among PHA-producing bacteria has hindered the development of diverse production processes. As alkyl alcohols are mainly produced from inexpensive starting materials through oxo synthesis, they contribute a cost-effective advantage over propionate and valerate. Moreover, alkyl alcohols can be biosynthesized from natural substrates and organic wastes. Despite their great potential, their toxicity to most PHA-producing bacteria has been the major drawback for their wide implementation as 3HV precursors for decades. Although the standard PHA-producing bacteria Cupriavidus necator showed promising alcohol tolerance, the 3HV yield was discouraging. Continuous discovery of alkyl alcohols-utilizing PHA-producing bacteria has enabled broader choices in 3HV precursor selection for diverse P(3HB-co-3HV) production processes with higher economic feasibility. Besides continuous effort in searching for promising wild-type strains, genetic engineering to construct promising recombinant strains based on the understanding of the mechanisms involved in alkyl alcohols toxicity and tolerance is an alternative approach. However, more studies are required for techno-economic assessment to analyze the economic performance of alkyl alcohol-based production compared to that of organic acids.


General Overview
Polyhydroxyalkanoates (PHA) are emerging as the next generation plastics owing to their plastic-like properties, renewability, biodegradability, and biocompatibility [1]. PHA are accumulated by bacteria under carbon excess but nitrogen-limiting conditions and stored as a reserved energy source in the form of single or multiple granules in the cytoplasm [2]. PHA have gained much industrial interest in the last few decades due to their potential as substitutes for conventional plastics, and various fermentation strategies have been developed to establish microbial PHA production for commercialization. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) is the most studied PHA copolymer with mechanical properties comparable to that of polypropylene. The 3hydroxyvalerate (3HV) monomer provides elastomeric property to the copolymer, enabling broader application compared to the homopolymer poly(3-hydroxybutyrate) (P(3HB)) [3].  Table 1. Properties improvement after the incorporation of a secondary (and tertiary) component into P(3HB-co-3HV) and their potential applications.

Incorporated Components A Changes in the Properties Potential Applications
Ref.

Incorporated Components A Changes in the Properties Potential Applications
Ref.

α-P(3HB) Incorporation method:
Solvent casting 3HV fraction: 10 mol% P(3HB-co-3HV):α-P(3HB) (  P(3HB-co-3HV) is a potential substitute for petroleum-based plastic packaging material as it possesses high water and aroma (limonene and linalool) barrier properties while having comparable thermal and mechanical properties to that of polypropylene (PP) and lowdensity polyethylene (LDPE) [15]. As PP and LDPE are applied extensively for packaging and consumables, which are highly disposable, the substitution with P(3HB-co-3HV) can contribute to reduced stable solid waste creation of petroleum-based plastics [47,48]. Unlike the augmented cytotoxicity by higher 3HV molar fraction, lower 3HV molar fraction causes high stereoregularity, slow crystallization rate, formation of large size spherulites, and secondary crystallization that are discouraging for packaging purposes [24,47,48]. Poly(butylene succinate), poly(butylene adipate-co-terephthalate), natural rubber, or other polymers with plasticizer or toughness properties can be incorporated to overcome the limitations and extend its application as packaging materials (Table 1).
Moreover, PHA-based mulch films are potential substitutes for conventional plastic mulch films. Mulching increases crops productivity, increases horticulture products, prevents water evaporation from the soil, prevents soil erosion, reduces water consumption, and controls weeds [49]. PHA-based mulch films overcome the environmental problems caused by the post-consumption of plastic mulch films made from LDPE, linear low-density polyethylene (LLDPE), and high-density polyethylene (HDPE) due to their poor degradability [50]. Moreover, the physicochemical properties of P(3HB-co-3HV) enable the controlled release of herbicides and insecticides. Herbicides and insecticides can be integrated into P(3HB-co-3HV)-containing pellets and sown along the plantation to be released upon degradation from the pellets depending on the level of pest activity [51,52].
On the other hand, endogenous P(3HB-co-3HV) acts as the electron donor for the denitrification of wastewater in the aquaculture industry. Biomass with PHA-accumulating ability, generally P(3HB) and poly(3-hydroxyvalerate) (P(3HV)), from activated sludge, is employed to remove resulting ammonia from fish excretion and dead animal bodies in circulating water. Unlike the conventional techniques that involve the addition of acetate and ethanol to promote microbial activity, the biomass is precultured for PHA accumulation. The endogenous PHA is used for denitrification that accurately couples with slow metabolic activity in the absence of exogenous carbon source and in the presence of nitrogen [53,54]. The exclusion of volatile fatty acids feeding during the denitrification process prevents the contamination with the dissolved organic carbon that lowers the effluent water quality, and the employment of endogenous PHA is more cost-effective compared to feeding extracted PHA to denitrifying bacteria [55].

Bioconversion of Alkyl Alcohols and Organic Acids into P(3HB-co-3HV)
The conversion of organic acid into 3HV starts with β-oxidation, where propionic acid (C3) is converted into propionyl-CoA, whereas valeric acid (C5) is converted into propionyl-CoA and acetyl-CoA, respectively [56]. The 3HV monomer is formed from the resulting propionyl-CoA couples with acetyl-CoA and is polymerized to P(3HB-co-3HV) copolymer with the 3HB monomer. The 3HB monomer is formed from the resulting acetyl-CoA provided majorly by the main carbon source such as oils or sugars ( Figure 2) [14,[57][58][59][60]. The employment of alkyl alcohols as 3HV precursors is limited to odd carbon number primary alcohols. Primary alcohols are oxidized to aldehydes that can be further oxidized more easily to their respective carboxylic acids. The oxidation processes can occur chemically with the presence of oxidizing agents or biologically with the presence of alcohol dehydrogenase and aldehyde dehydrogenase [61]. Oxidation of secondary alcohols liberates ketones with no further oxidation due to the oxidatively stable nature of ketones [62,63]. Odd carbon number primary alcohols such as 1-propanol or 1-pentanol are oxidized to 1-propanal and 1-pentanal that further oxidized to propanoic acid and valeric acid, respectively. The resulting propionic acid or valeric acid enters β-oxidation to liberate propionyl-CoA for 3HV formation ( Figure 2) [14,[57][58][59][60].
Although levulinic acid is a cost-effective 3HV precursor, the catabolic pathway involved is undetermined. Generally, levulinic acid catabolism releases intermediates that are converted via β-oxidation to release acetyl-CoA and propionyl-CoA for P(3HB-co-3HV) biosynthesis [64]. Bacteria capable of using levulinic acid as the 3HV precursor are rare and are mainly C. necator, with the exception of Burkholderis sp. IS-01 and Hydrogenophaga pseudoflava DSM 1034 [10,11,[65][66][67][68]. C. necator KHB-8862 and H. The employment of alkyl alcohols as 3HV precursors is limited to odd carbon number primary alcohols. Primary alcohols are oxidized to aldehydes that can be further oxidized more easily to their respective carboxylic acids. The oxidation processes can occur chemically with the presence of oxidizing agents or biologically with the presence of alcohol dehydrogenase and aldehyde dehydrogenase [61]. Oxidation of secondary alcohols liberates ketones with no further oxidation due to the oxidatively stable nature of ketones [62,63]. Odd carbon number primary alcohols such as 1-propanol or 1-pentanol are oxidized to 1-propanal and 1-pentanal that further oxidized to propanoic acid and valeric acid, respectively. The resulting propionic acid or valeric acid enters β-oxidation to liberate propionyl-CoA for 3HV formation ( Figure 2) [14,[57][58][59][60].

Microorganisms and Carbon Sources
Biomass

Techno-Economic and Sustainability Assessment
The annual operating costs in PHA production generally include the direct fixed capital-dependent items, labor-dependent items, administration, and overhead expenses, raw materials, utilities, and downstream processing such as waste management. According to the techno-economic analysis conducted by Choi and Lee (1999) for various pure carbon sources, the substrate cost accounted for 48%-60% of the total costs ( Figure 3) [93]. After excluding the trace elements, which are essentials, pure carbon sources that possess high nutritional value such as glucose, glycerol, starch, methane, oils, and volatile fatty acids are commercial products, and their employment leads to higher substrate cost compared to that of industrial or domestic wastes. Due to higher economic advantage and increasing emphasis on sustainability, the employment of wastes as carbon sources is widely attempted. Theoretically, substituting pure substrates with wastes contributes to a huge reduction in raw material expenses. However, pretreatments are needed for certain wastes to remove impurities and toxins or to adjust pH [94]. Pretreatments impose additional costs whereby extra chemicals or equipment are necessary with possible individual optimization. Bhattacharyya and co-workers (2015) reported decreased raw material cost to 39% with the employment of wheat stillage, but the utilities cost increased to 21% as compared to that reported by Choi and Lee (1999) (Figure 3) [93,95]. wastes to remove impurities and toxins or to adjust pH [94]. Pretreatments impose additional costs whereby extra chemicals or equipment are necessary with possible individual optimization. Bhattacharyya and co-workers (2015) reported decreased raw material cost to 39% with the employment of wheat stillage, but the utilities cost increased to 21% as compared to that reported by Choi and Lee (1999) (Figure 3) [93,95]. Techno-economic analysis on PHA production annual costs using pure carbon sources and wastes [93,95].
As opposed to main carbon sources, where numerous studies have been conducted on various wastes, employing wastes as 3HV precursors is not practical due to the composition inconsistency [95]. Due to the necessity of propionyl-CoA for 3HV formation, sole reliance on wastes results in the narrow choice to those with propionate or valerate related components; thus, in most cases, a 3HV precursor is still required to achieve sufficient 3HV fraction for the copolymer to be practically useful [64,95]. This leads to increased raw material cost as propionic acid and valeric acid, which are widely preferred by PHA-producing bacteria, are high-cost precursors ( Table 2). The potential of 1propanol and 1-pentanol as alternatives for propionic acid and valeric acid is well-known but lack practicality due to its high toxicity to the majority of bacteria. Since 1996, several PHA-producing bacteria from different genera have been reported to use 1-propanol or/and 1-pentanol as 3HV precursors ( Table 2). The emergence of these bacteria bypasses the bottleneck of precursor dominance by organic acids and enables further innovation in fermentation strategies to develop economically feasible and sustainable production processes. Furthermore, 1-propanol and 1-pentanol are manufactured through wellestablished oxo synthesis and can be biosynthesized by bacteria from sustainable carbon sources such as glucose, glycerol, and organic wastes, which are abundant in nature.

Oxo Synthesis of Alkyl Alcohols
Oxo synthesis is an established process for the manufacture of alkyl alcohols at an industrial scale with simple operational requirements and low specificity in raw materials, including branched-chain, long-chain, and cyclic olefins [96,97]. It is thoroughly investigated for the production of a wide variety of industrial chemicals. The synthesis involves hydroformylation to convert olefins (also known as alkenes) into aldehydes to be further converted into alcohols through hydrogenation. Homogeneous catalysts are employed in hydroformylation, while heterogenous catalysts are employed in hydrogenation for reaction induction. Generally, these reactions are carried out in Figure 3. Techno-economic analysis on PHA production annual costs using pure carbon sources and wastes [93,95].
As opposed to main carbon sources, where numerous studies have been conducted on various wastes, employing wastes as 3HV precursors is not practical due to the composition inconsistency [95]. Due to the necessity of propionyl-CoA for 3HV formation, sole reliance on wastes results in the narrow choice to those with propionate or valerate related components; thus, in most cases, a 3HV precursor is still required to achieve sufficient 3HV fraction for the copolymer to be practically useful [64,95]. This leads to increased raw material cost as propionic acid and valeric acid, which are widely preferred by PHA-producing bacteria, are high-cost precursors ( Table 2). The potential of 1-propanol and 1-pentanol as alternatives for propionic acid and valeric acid is well-known but lack practicality due to its high toxicity to the majority of bacteria. Since 1996, several PHA-producing bacteria from different genera have been reported to use 1-propanol or/and 1-pentanol as 3HV precursors ( Table 2). The emergence of these bacteria bypasses the bottleneck of precursor dominance by organic acids and enables further innovation in fermentation strategies to develop economically feasible and sustainable production processes. Furthermore, 1propanol and 1-pentanol are manufactured through well-established oxo synthesis and can be biosynthesized by bacteria from sustainable carbon sources such as glucose, glycerol, and organic wastes, which are abundant in nature.

Oxo Synthesis of Alkyl Alcohols
Oxo synthesis is an established process for the manufacture of alkyl alcohols at an industrial scale with simple operational requirements and low specificity in raw materials, including branched-chain, long-chain, and cyclic olefins [96,97]. It is thoroughly investigated for the production of a wide variety of industrial chemicals. The synthesis involves hydroformylation to convert olefins (also known as alkenes) into aldehydes to be further converted into alcohols through hydrogenation. Homogeneous catalysts are employed in hydroformylation, while heterogenous catalysts are employed in hydrogenation for reaction induction. Generally, these reactions are carried out in separate reactors where the resulting aldehydes from the primary reactor are transferred into the second reactor to be hydrogenated. Catalysts and carbon monoxide in the primary reactor are removed either by decobalting or been recycled back to the primary reactor to prevent entry into the second reactor as a precautious measure to extend the shelf life of hydrogenation catalysts. Recycling the catalysts contributes to high economic feasibility as high-cost catalysts such as rhodium-based catalysts can be reused for subsequent batches. However, an 8-55% decrease in catalyst yield after repeated recycling is expected [98]. The resulting alcohols are purified from the mixture via distillation (Figure 4) [97]. separate reactors where the resulting aldehydes from the primary reactor are transferred into the second reactor to be hydrogenated. Catalysts and carbon monoxide in the primary reactor are removed either by decobalting or been recycled back to the primary reactor to prevent entry into the second reactor as a precautious measure to extend the shelf life of hydrogenation catalysts. Recycling the catalysts contributes to high economic feasibility as high-cost catalysts such as rhodium-based catalysts can be reused for subsequent batches. However, an 8%-55% decrease in catalyst yield after repeated recycling is expected [98]. The resulting alcohols are purified from the mixture via distillation ( Figure  4) [97].

1-Propanol
Oxo synthesis of 1-propanol begins with the rhodium-catalyzed hydroformylation of ethylene (also known as ethene) to propanal with the aid of rhodium-triphenylphosphine catalysts. The resulting 1-propanal is distilled from the catalyst-containing solution, and carbon monoxide is removed. Hydrogenation can be carried out in either the heterogenous vapor phase or the heterogenous liquid phase. Heterogeneous vapor phase hydrogenation takes place at 110-150 °C and 0.14-1.00 MPa with the aid of copper, zinc, nickel, and chromium catalysts supported on alumina (CAS:1344-28-1) or kieselguhr (CAS:91053-39-3) [62]. Heat is removed either by an external heat exchange device or an internal cooler [100]. This process produces impurities such as dipropyl ether, ethane, and propyl propionate. Selectivity enhancers such as alkali or transition metals are added to reduce the formation of esters, while an additional 1%-10% water could suppress the formation of ether [62,101]. Propyl propionate is separated from the product mixture and hydrogenolyzed with the aid of reduced CuO-ZnO catalysts at 75-300 °C and 9.8 kPa-9.8 MPa to produce 1-propanol as the major product [62]. Heterologous liquid phase hydrogenation involved nickel or copper catalysts at a lower temperature of 95-120 °C and a higher pressure of 3.5 MPa. Crude 1-propanol is purified via distillation with the aid of an azeotroping agent such as dipropyl ether or cyclo-hexane to remove water for highly pure 1-propanol yield (>99%) (Figure 4) [62,102].

1-Propanol
Oxo synthesis of 1-propanol begins with the rhodium-catalyzed hydroformylation of ethylene (also known as ethene) to propanal with the aid of rhodium-triphenylphosphine catalysts. The resulting 1-propanal is distilled from the catalyst-containing solution, and carbon monoxide is removed. Hydrogenation can be carried out in either the heterogenous vapor phase or the heterogenous liquid phase. Heterogeneous vapor phase hydrogenation takes place at 110-150 • C and 0.14-1.00 MPa with the aid of copper, zinc, nickel, and chromium catalysts supported on alumina (CAS:1344-28-1) or kieselguhr (CAS:91053-39-3) [62]. Heat is removed either by an external heat exchange device or an internal cooler [100]. This process produces impurities such as dipropyl ether, ethane, and propyl propionate. Selectivity enhancers such as alkali or transition metals are added to reduce the formation of esters, while an additional 1%-10% water could suppress the formation of ether [62,101]. Propyl propionate is separated from the product mixture and hydrogenolyzed with the aid of reduced CuO-ZnO catalysts at 75-300 • C and 9.8 kPa-9.8 MPa to produce 1-propanol as the major product [62]. Heterologous liquid phase hydrogenation involved nickel or copper catalysts at a lower temperature of 95-120 • C and a higher pressure of 3.5 MPa. Crude 1-propanol is purified via distillation with the aid of an azeotroping agent such as dipropyl ether or cyclo-hexane to remove water for highly pure 1-propanol yield (>99%) (Figure 4) [62,102].

The Wood-Werkman Pathway in Propionibacteria
Biosynthesis of 1-propanol by wild-type bacteria is inefficient as 1-propanol is synthesized as a byproduct through propionic acid synthesis processes. Propionibacteria such as Propionibacterium acidipropionici and Propionibacterium freudenreichii are able to produce 1-propanol through the Wood-Werkman pathway (also known as the dicarboxylic pathway, or the methylmalonyl-CoA pathway). The synthesis process requires an anaerobic condition where the carbon source is converted into pyruvate and enters the Wood-Werkman pathway to produce propionic acid as the main product [103,104]. The 1-propanol yield reported was in the range of 0.04-0.14 mol/mol, equivalent to 0.6-1.8 g/L. The 1-propanol production was found to be higher when glycerol was employed, compared to glucose [105,106]. The precise processes involved in 1-propanol formation are undetermined but could probably be by two-step reduction from propionyl-CoA to 1-propanol aided by acylating propionaldehyde dehydrogenase and propanol dehydrogenase ( Figure 5) [107].

The Wood-Werkman Pathway in Propionibacteria
Biosynthesis of 1-propanol by wild-type bacteria is inefficient as 1-propanol is synthesized as a byproduct through propionic acid synthesis processes. Propionibacteria such as Propionibacterium acidipropionici and Propionibacterium freudenreichii are able to produce 1-propanol through the Wood-Werkman pathway (also known as the dicarboxylic pathway, or the methylmalonyl-CoA pathway). The synthesis process requires an anaerobic condition where the carbon source is converted into pyruvate and enters the Wood-Werkman pathway to produce propionic acid as the main product [103,104]. The 1-propanol yield reported was in the range of 0.04-0.14 mol/mol, equivalent to 0.6-1.8 g/L. The 1-propanol production was found to be higher when glycerol was employed, compared to glucose [105,106]. The precise processes involved in 1-propanol formation are undetermined but could probably be by two-step reduction from propionyl-CoA to 1-propanol aided by acylating propionaldehyde dehydrogenase and propanol dehydrogenase ( Figure 5) [107].

The Acrylate Pathway in Clostridium
Clostridium propionicumable and Clostridium neopropionicumable are able to use amino acids (alanine and serine), lactate, and ethanol as growth-promoting substances under anaerobic conditions [108,109]. C. neopropionicumable synthesizes a small amount of 1propanol (0.06 g/L, 0.03 mol/mol) from ethanol with propionate and acetate as the main products [109]. By employing the bacterial mixture dominated by Alkalibaculum bacchi (34%) and C. propionicumable (54%), C. propionicumable produced 6.0 g/L 1-propanol and 1.0 g/L 1-butanol, whereas A. bacchi produced 8.0 g/L ethanol from syngas (the carbon source) and corn-steep liquor (the source of amino acids and minerals) [110]. The resulting 1-propanol was proposed to be the product from a two-step reduction in propionyl-CoA produced through the acrylate pathway by using the lactoyl-CoA that is not used for

The Acrylate Pathway in Clostridium
Clostridium propionicumable and Clostridium neopropionicumable are able to use amino acids (alanine and serine), lactate, and ethanol as growth-promoting substances under anaerobic conditions [108,109]. C. neopropionicumable synthesizes a small amount of 1propanol (0.06 g/L, 0.03 mol/mol) from ethanol with propionate and acetate as the main products [109]. By employing the bacterial mixture dominated by Alkalibaculum bacchi (34%) and C. propionicumable (54%), C. propionicumable produced 6.0 g/L 1-propanol and 1.0 g/L 1-butanol, whereas A. bacchi produced 8.0 g/L ethanol from syngas (the carbon source) and corn-steep liquor (the source of amino acids and minerals) [110]. The resulting 1-propanol was proposed to be the product from a two-step reduction in propionyl-CoA produced through the acrylate pathway by using the lactoyl-CoA that is not used for propionic acid synthesis ( Figure 6) [109]. However, further experimentations are needed to provide essential information for a complete view of the biosynthesis pathway. propionic acid synthesis ( Figure 6) [109]. However, further experimentations are needed to provide essential information for a complete view of the biosynthesis pathway.

The Carboxylate Reduction Pathway in Clostridium
Anaerobic digestion by microbial consortia is a promising hydrogen production process where the members in the microbial community play different roles to convert raw materials into hydrogen under anaerobic conditions. As sterilization is commonly excluded from anaerobic digestion, organic acids produced by acetogens in the consortia cause decreased pH that disrupts the metabolic activity of hydrogen-producing bacteria [111]. Clostridium ragsdalei (ATCC BAA-622, DSM 15248) is an acetogen capable of synthesizing alcohols by ferredoxin-mediated carboxylate reduction. With the involvement of exogenous CO and ferredoxin, n-fatty acids up to six carbons in length can be reduced to corresponding alcohols (Figure 7). The concentration of produced 1propanol reported was 1.7 g/L 1-propanol from propionic acid, with a conversion efficiency of 97%. However, the concentration of 1-pentanol obtained was merely 0.2 g/L, with a conversion efficiency of 82% [112].

The Carboxylate Reduction Pathway in Clostridium
Anaerobic digestion by microbial consortia is a promising hydrogen production process where the members in the microbial community play different roles to convert raw materials into hydrogen under anaerobic conditions. As sterilization is commonly excluded from anaerobic digestion, organic acids produced by acetogens in the consortia cause decreased pH that disrupts the metabolic activity of hydrogen-producing bacteria [111]. Clostridium ragsdalei (ATCC BAA-622, DSM 15248) is an acetogen capable of synthesizing alcohols by ferredoxin-mediated carboxylate reduction. With the involvement of exogenous CO and ferredoxin, n-fatty acids up to six carbons in length can be reduced to corresponding alcohols (Figure 7). The concentration of produced 1-propanol reported was 1.7 g/L 1-propanol from propionic acid, with a conversion efficiency of 97%. However, the concentration of 1-pentanol obtained was merely 0.2 g/L, with a conversion efficiency of 82% [112]. propionic acid synthesis ( Figure 6) [109]. However, further experimentations are needed to provide essential information for a complete view of the biosynthesis pathway.

The Carboxylate Reduction Pathway in Clostridium
Anaerobic digestion by microbial consortia is a promising hydrogen production process where the members in the microbial community play different roles to convert raw materials into hydrogen under anaerobic conditions. As sterilization is commonly excluded from anaerobic digestion, organic acids produced by acetogens in the consortia cause decreased pH that disrupts the metabolic activity of hydrogen-producing bacteria [111]. Clostridium ragsdalei (ATCC BAA-622, DSM 15248) is an acetogen capable of synthesizing alcohols by ferredoxin-mediated carboxylate reduction. With the involvement of exogenous CO and ferredoxin, n-fatty acids up to six carbons in length can be reduced to corresponding alcohols (Figure 7). The concentration of produced 1propanol reported was 1.7 g/L 1-propanol from propionic acid, with a conversion efficiency of 97%. However, the concentration of 1-pentanol obtained was merely 0.2 g/L, with a conversion efficiency of 82% [112].

Biosynthesis of 1-Propanol and 1-Pentanol by Genetic-Engineered E. coli 7.1. Co-Expression of the Citramalate and Threonine Pathway
Numerous genetic engineering attempts were carried out for alkyl alcohols biosynthesis through the individual threonine or citramalate pathway and showed successful biosynthesis of 1-propanol from the intermediate 2-ketobutyrate in the pathways [113,114]. For greater industrial applicability, co-expression of both pathways was attempted in E. coli BW25113. The simultaneous operation of the pathways in a single host showed a synergic effect on 1-propanol production. The co-expression provided a larger 2-ketobutyrate pool for decarboxylation and reduction to 1-propanol (Figure 8). A high 1-propanol concentration of 8.0 g/L was reported with a 1-propanol yield of 0.15 g/g from glucose, which was higher than 0.09 and 0.11 g/g for individual threonine and citramalate pathway, respectively [115].  Figure 7. Biosynthesis of 1-propanol and 1-pentanol by wild-type C. ragsdalei through the carboxylate reduction pathway [112].

Co-Expression of the Citramalate and Threonine Pathway
Numerous genetic engineering attempts were carried out for alkyl alcohols biosynthesis through the individual threonine or citramalate pathway and showed successful biosynthesis of 1-propanol from the intermediate 2-ketobutyrate in the pathways [113,114]. For greater industrial applicability, co-expression of both pathways was attempted in E. coli BW25113. The simultaneous operation of the pathways in a single host showed a synergic effect on 1-propanol production. The co-expression provided a larger 2-ketobutyrate pool for decarboxylation and reduction to 1-propanol (Figure 8). A high 1-propanol concentration of 8.0 g/L was reported with a 1-propanol yield of 0.15 g/g from glucose, which was higher than 0.09 and 0.11 g/g for individual threonine and citramalate pathway, respectively [115].

Interactive Elongation Cycle of 2-Ketoacids
Biosynthesis of 1-pentanol was made possible by introducing Lactococcus lactis ketoisovalerate decarboxylase (Kivd) modified via saturated mutagenesis of the V461 key residue of the enzyme with glycine and serine into E. coli BW25113 to promote its selectivity toward 2-ketocaproate, which is the precursor for 1-pentanol. Besides lowered catalytic efficiency of the modified Kivd toward 2-ketoacids upstream of 2-ketocaproante, the increased supply of acetyl-CoA by acetate feeding encouraged 2-ketoacid elongation cycle for enhanced 1-pentanol production ( Figure 9). The high specificity of this approach was implied by 90% 1-pentanol in the alcohol product mixture, equivalent to 2.2-2.4 g/L upon production harvest. The synthesis of alcohols with a longer alkyl chain was found to be minimized as further elongation of the 2-ketoacid was discouraged due to the active use of 2-kerocaproate for 1-pentanol synthesis [116].

Interactive Elongation Cycle of 2-Ketoacids
Biosynthesis of 1-pentanol was made possible by introducing Lactococcus lactis ketoisovalerate decarboxylase (Kivd) modified via saturated mutagenesis of the V461 key residue of the enzyme with glycine and serine into E. coli BW25113 to promote its selectivity toward 2-ketocaproate, which is the precursor for 1-pentanol. Besides lowered catalytic efficiency of the modified Kivd toward 2-ketoacids upstream of 2-ketocaproante, the increased supply of acetyl-CoA by acetate feeding encouraged 2-ketoacid elongation cycle for enhanced 1-pentanol production ( Figure 9). The high specificity of this approach was implied by 90% 1-pentanol in the alcohol product mixture, equivalent to 2.2-2.4 g/L upon production harvest. The synthesis of alcohols with a longer alkyl chain was found to be minimized as further elongation of the 2-ketoacid was discouraged due to the active use of 2-kerocaproate for 1-pentanol synthesis [116]. Polymers 2022, 14, x FOR PEER REVIEW 20 of 33 Figure 9. Biosynthesis of 1-pentanol by genetically engineered E. coli BW25113 through interactive elongation cycles of 2-letoacids [116]. Dash arrow indicates lower selectivity toward the reaction.

Extended Dissimilation of Succinate
The sleeping beauty mutase (SBM) operon in E. coli is a four-gene operon (sbm-ygfD-ygfG-ygfH) that encodes various enzymes required in a cobalamin-dependent metabolic pathway for decarboxylation of succinate into propionate [117]. An activated chromosomal SBM operon encodes methylmalonyl-mutase (by sbm), methylmalonyl-CoA decarboxylase (by ygfG), and propionyl-CoA:succinate CoA transferase (by ygfH) in plasmid-free propanogenic E. coli BW25113 enabled extended dissimilation of succinate to synthesis 1-propanol ( Figure 10). Glycerol favored solventogenesis over glucose due to the necessity of a solventogenic pathway as an auxiliary channel for redox balance upon glycerol dissimilation under anaerobic conditions. An anaerobic fed-batch strategy established by using the engineered E. coli strain produced high titers of 7.0 g/L 1propanol, thus implying its high industrial applicability [118].

Extended Dissimilation of Succinate
The sleeping beauty mutase (SBM) operon in E. coli is a four-gene operon (sbm-ygfD-ygfG-ygfH) that encodes various enzymes required in a cobalamin-dependent metabolic pathway for decarboxylation of succinate into propionate [117]. An activated chromosomal SBM operon encodes methylmalonyl-mutase (by sbm), methylmalonyl-CoA decarboxylase (by ygfG), and propionyl-CoA:succinate CoA transferase (by ygfH) in plasmid-free propanogenic E. coli BW25113 enabled extended dissimilation of succinate to synthesis 1-propanol ( Figure 10). Glycerol favored solventogenesis over glucose due to the necessity of a solventogenic pathway as an auxiliary channel for redox balance upon glycerol dissimilation under anaerobic conditions. An anaerobic fed-batch strategy established by using the engineered E. coli strain produced high titers of 7.0 g/L 1-propanol, thus implying its high industrial applicability [118].

Extended Dissimilation of Succinate
The sleeping beauty mutase (SBM) operon in E. coli is a four-gene operon (sbm-ygfD-ygfG-ygfH) that encodes various enzymes required in a cobalamin-dependent metabolic pathway for decarboxylation of succinate into propionate [117]. An activated chromosomal SBM operon encodes methylmalonyl-mutase (by sbm), methylmalonyl-CoA decarboxylase (by ygfG), and propionyl-CoA:succinate CoA transferase (by ygfH) in plasmid-free propanogenic E. coli BW25113 enabled extended dissimilation of succinate to synthesis 1-propanol ( Figure 10). Glycerol favored solventogenesis over glucose due to the necessity of a solventogenic pathway as an auxiliary channel for redox balance upon glycerol dissimilation under anaerobic conditions. An anaerobic fed-batch strategy established by using the engineered E. coli strain produced high titers of 7.0 g/L 1propanol, thus implying its high industrial applicability [118].

Acquired Carboxylate Reduction Pathway
Conversion of organic acids produced by acetogens during anaerobic digestion into other useful products is suggested to be beneficial as a solution to maintain the stability of the biogas production process. An E. coli BL21(DE3) strain harboring Clostridium acetobutylicum alcohol dehydrogenase (AdhE2) and Megasphaera hexanoica acyl-CoA transferase (ACT01_02765) was developed for conversion of the C 2 -C 8 organic acids commonly found in anaerobic digestion into corresponding primary alcohols. The metabolic pathway is relatively simpler as it only involves two steps aided by two enzymes (Figure 11). Following the conversion rate of 1.1 for C 4 acid into 1-butanol, the functional alcohol dehydrogenase and acyl-CoA transferase resulted in a promising conversion rate of 0.8 for both 1-propanol and 1-pentanol [119].

Acquired Carboxylate Reduction Pathway
Conversion of organic acids produced by acetogens during anaerobic digestion into other useful products is suggested to be beneficial as a solution to maintain the stability of the biogas production process. An E. coli BL21(DE3) strain harboring Clostridium acetobutylicum alcohol dehydrogenase (AdhE2) and Megasphaera hexanoica acyl-CoA transferase (ACT01_02765) was developed for conversion of the C2₋C8 organic acids commonly found in anaerobic digestion into corresponding primary alcohols. The metabolic pathway is relatively simpler as it only involves two steps aided by two enzymes (Figure 11). Following the conversion rate of 1.1 for C4 acid into 1-butanol, the functional alcohol dehydrogenase and acyl-CoA transferase resulted in a promising conversion rate of 0.8 for both 1-propanol and 1-pentanol [119].

Alkyl Alcohol-Tolerant P(3HB-co-3HV)-Producing Bacteria
Alcohols are unsuitable to be employed as 3HV precursors for C. necator (also known as Ralstonia eutropha, Alcaligenes eutrophus, or Wautersia eutropha), which is the standard PHA-producing bacteria. Although C. necator H16 is capable of surviving methanol, ethanol, and propanol, extensive exposure to these alcohols is detrimental to PHA accumulation, thus resulting in lower biomass. The employment of 8.0 g/L 1-propanol, which is convertible into propionyl-CoA, contributed to merely 3 mol% 3HV with a 3HV yield of 0.14 g/g [13,120] (Table 2). The individual employment of 1-propanol and 1pentanol also caused a remarkably high reduction in biomass and PHA content of C. necator DSM 545. The employment of 1-pentanol caused C. necator DSM 545 biomass and PHA content to decrease by 40% and 20%, respectively. Comparatively,1-propanol exerted a lower adverse effect compared to 1-pentanol, whereby its employment decreased C. necator DSM 545 PHA content by 10% with no negative influence on bacterial biomass [10] (Table 2). To overcome the limitation in 3HV precursor selection, isolation of alkyl alcohol-tolerant P(3HB-co-3HV)-producing bacteria is continuously attempted and has led to the discovery of various promising bacteria with the capability to use alkyl alcohols as 3HV precursors (Figure 12).

Alkyl Alcohol-Tolerant P(3HB-co-3HV)-Producing Bacteria
Alcohols are unsuitable to be employed as 3HV precursors for C. necator (also known as Ralstonia eutropha, Alcaligenes eutrophus, or Wautersia eutropha), which is the standard PHAproducing bacteria. Although C. necator H16 is capable of surviving methanol, ethanol, and propanol, extensive exposure to these alcohols is detrimental to PHA accumulation, thus resulting in lower biomass. The employment of 8.0 g/L 1-propanol, which is convertible into propionyl-CoA, contributed to merely 3 mol% 3HV with a 3HV yield of 0.14 g/g [13,120] ( Table 2). The individual employment of 1-propanol and 1-pentanol also caused a remarkably high reduction in biomass and PHA content of C. necator DSM 545. The employment of 1-pentanol caused C. necator DSM 545 biomass and PHA content to decrease by 40% and 20%, respectively. Comparatively,1-propanol exerted a lower adverse effect compared to 1-pentanol, whereby its employment decreased C. necator DSM 545 PHA content by 10% with no negative influence on bacterial biomass [10] (Table 2). To overcome the limitation in 3HV precursor selection, isolation of alkyl alcohol-tolerant P(3HB-co-3HV)-producing bacteria is continuously attempted and has led to the discovery of various promising bacteria with the capability to use alkyl alcohols as 3HV precursors ( Figure 12 P. denitrificans ATCC 17741 was the first bacteria reported in 1996 for the use of alkyl alcohol as the 3HV precursor. P. denitrificans ATCC 17741 is a mixotrophic colorless sulfur bacterium capable of using 1-pentanol as the sole carbon source for growth and P(3HV) accumulation [14,121]. The study was conducted by maintaining the concentration of 1pentanol at 1.6 g/L for 24 h. Approximately 6.8 g/L biomass with 1.2 g/L P(3HV) homopolymer was achieved [14] (Table 2).
Erwinia sp. USMI-20 was reported with its preference for alkyl alcohols instead of organic acids as 3HV precursors. Erwinia sp. USMI-20 achieved higher biomass with the co-employment of 1-propanol and 1-pentanol compared to that when palm oil was employed solely. A higher PHA content of 50 and 62 wt% was also achieved for 1propanol and 1-pentanol, respectively, compared to 40 wt% and 34 wt% for propionic acid and valeric acid. 1-pentanol was more promising compared to 1-propanol as Erwinia sp. USMI-20 accumulated a higher 3HV fraction of 20 mol% from 1-pentanol compared to 6 mol% from 1-propanol. 1-pentanol can be employed as a substitute for valeric for Erwinia sp. USMI-20 owing to the higher 3HV yield of 0.43 g/g for 1-pentanol, which was 2-fold higher than that for valeric acid [60]. The production was scaled up to 10 L by employing 4.6 g/L palm oil and 1.4 g/L 1-pentanol, where 1-pentanol was added at 20 h post P. denitrificans ATCC 17741 was the first bacteria reported in 1996 for the use of alkyl alcohol as the 3HV precursor. P. denitrificans ATCC 17741 is a mixotrophic colorless sulfur bacterium capable of using 1-pentanol as the sole carbon source for growth and P(3HV) accumulation [14,121]. The study was conducted by maintaining the concentration of 1-pentanol at 1.6 g/L for 24 h. Approximately 6.8 g/L biomass with 1.2 g/L P(3HV) homopolymer was achieved [14] (Table 2).
Erwinia sp. USMI-20 was reported with its preference for alkyl alcohols instead of organic acids as 3HV precursors. Erwinia sp. USMI-20 achieved higher biomass with the co-employment of 1-propanol and 1-pentanol compared to that when palm oil was employed solely. A higher PHA content of 50 and 62 wt% was also achieved for 1-propanol and 1-pentanol, respectively, compared to 40 wt% and 34 wt% for propionic acid and valeric acid. 1-pentanol was more promising compared to 1-propanol as Erwinia sp. USMI-20 accumulated a higher 3HV fraction of 20 mol% from 1-pentanol compared to 6 mol% from 1-propanol. 1-pentanol can be employed as a substitute for valeric for Erwinia sp. USMI-20 owing to the higher 3HV yield of 0.43 g/g for 1-pentanol, which was 2-fold higher than that for valeric acid [60]. The production was scaled up to 10 L by employing 4.6 g/L palm oil and 1.4 g/L 1-pentanol, where 1-pentanol was added at 20 h post incubation. The 3HV fraction achieved was 20 mol% in 56 wt% PHA content of 5.4 g/L biomass, with 0.43 g/g 3HV yield [122] (Table 2).
M. extorquens G10 demonstrated the production of P(3HB-co-3HV) from an alkyl alcohol mixture of C1 and C5 alcohol. A 4 L production of P(3HB-co-3HV) from a methanolpentanol mixture by M. extorquens G10 showed a promisingly high PHA concentration of 7.5-18.0 g/L. The carbon mixture was supplemented fractionally based on the dissolved oxygen peaks observed. With an increased portion of 1-pentanol from 2 to 20 mol%, the biomass decreased with association to reduction in PHA content from 40.0 to 25.0 g/L and 45 to 30 wt%, respectively. Despite the negative influence on biomass and PHA content, 3HV composition of 14-50 mol% was achieved [90] (Table 2).
M. halotolerans C2 demonstrated P(3HB-co-3HV) production from C1, C2, and C5 alkyl alcohol. P(3HB-co-3HV) production by M. halotolerans C2 through fractional feeding of methanol-ethanol mixture resulted in increased 3HV composition from 2 to 51 mol% parallel to increased 1-pentanol supply from 5 to 15 % v/v methanol. A considerably high PHA content of 73-98 wt% was accumulated by the bacterium [91] (Table 2). P(3HB-co-3HV) production by Methylocystis sp. WRRC1 from methane and 1-pentanol demonstrated a 0.17 g/g 3HV yield from 1.0 g/L 1-pentanol. The 6-fold lower consumption of methane by the bacteria with the co-employment of 1-pentanol compared to that of sole employment of methane denoted the preference of the bacteria for 1-pentanol over methane. However, 1-pentanol is non-competitive against valerate where Methylocystis sp. WRRC1 achieved a 1-fold higher 3HV concentration with the co-employment of sodium valerate compared to that of 1-pentanol. On the other hand, the co-employment of sodium valerate did not cause reduced methane consumption and contributed to a higher 3HB concentration [86] (Table 2).
M. haematophila (also known as Naxibacter haematophila) UMTKB-2, a slow-growing bacterium, was also reported with the capability to use 1-pentanol for 3HV accumulation with a preference for 1-pentanol over valeric acid and sodium valerate. The co-employment of 1-pentanol resulted in 2-fold and 11-fold higher biomass and PHA content compared to that of valeric acid and sodium valerate, respectively. Upon optimization by using response surface methodology, M. haematophila UMTKB-2 achieved 7 mol% 3HV with 0.40 g/g 3HV yield. Unlike the PHA accumulation process of Cupriavidus sp. that ends within 48-72 h, 122 h was needed for optimum P(3HB-co-3HV) accumulation by M. haematophila UMTKB-2 [89] (Table 2).

Mode of Action of 1-Propanol and 1-Pentanol on Proteins
Short-chain alcohols exert a hydrophobic effect by interacting with proteins and lead to the structural unfolding of the protein [126]. Changes in membrane fluidity ensue due to the direct insertion of lipophilic agents into the cellular membrane after direct physicochemical interaction with alcohols. This induces adaptive membrane alteration by changing the fatty acid composition of the membrane [127]. Impaired inner membrane integrity associated with depletion in proton motive force due to the increased proton motive force demand for chemical, osmotic and mechanical adjustment induces the psp operon to prevent proton loss. As a result, the cells experience a metabolic shift to anaerobic respiration together with downregulation of motility for adjustment and maintenance of energy as well as for proton motive force usage [128]. The extent of water exclusion is greater with increasing alkyl groups of the alcohol, which is non-polar. By considering the hydrophobic effect of methanol < ethanol < propanol = butanol, pentanol may exert a similar hydrophobic effect on protein and result in pentanol-induced protein unfolding [126]. Furthermore, pentanol is capable of inactivating membrane proteins such as transporters but rarely causes structural changes to the cell membrane [129].

Mechanisms Involved in Alcohols Tolerance
Aliphatic alcohols, aromatic compounds, or other organic solvents are toxic to bacteria when present in high concentrations. Nevertheless, certain bacteria are able to thrive in the high concentration of such toxic organic chemicals. Bacterial solvent tolerance is a multifactorial process that involves gene expression and subsequent physiological changes to respond to stress conditions. Extrusion of the toxic compounds from the cell to the external environment and reduced cell membrane permeability to prevent further influx of toxic compounds are the relevant mechanisms to survive alcohol stress.

Changes in the Cell Membrane
Alcohol-induced cell leakage of magnesium and nucleotides is the primary damaging action that affects bacterial viability in alcohols [130]. As alcohols interact with the cell membrane and decrease the degree of membrane organization, proteins that participate in membrane structure organization and surface stabilization are critical in alcohol tolerance [131]. Isomerase incorporates fatty acids into the phospholipid headgroups of the phospholipid bilayer and causes isomerization of cis unsaturated fatty acids to trans unsaturated fatty acids to form a denser membrane, as demonstrated by Pseudomonas and Vibiro [132]. Changes in cell membrane composition that attributed to increased cis-11 vaccenic acid (18:1) or cis-9 oleic acid (18:1) with a corresponding decrease in palmitic acid (16:0) were demonstrated for E. coli, Lactobacillus homohiochii, and Saccharomyces cerevisiae [131,133,134]. The synthesis of phosphatidylethanolamine by Zymomonas mobilis was partially inhibited in the presence of alcohols. As a result, a membrane with an elevated proportion of acidic phospholipids (phosphatidylglycerol and cardiolipin) and an overall reduction in the phospholipid:protein ratio is synthesized, thus increasing the efficiency of efflux pumps in alcohol extrusion [135,136].

Stress Response System
Exposure to alkyl alcohols leads to changes in the level of expression of certain genes as responses to stress for adaptation. As demonstrated in E. coli, exposure to 1-butanol causes downregulation of several genes related to histidine, leucine, arginine, tryptophan, and methionine biosynthesis and transport, thus leading to a significantly lower level of related proteins. Downregulation of genes related to amino acids metabolism is an indicator for bacterial growth inhibition in alcohols. As opposed to that, opp operon (oppABCDF) that encodes the components in a polyamine-induced oligopeptide ABC transport system is upregulated for the transport of hydrophilic substances to compensate for the hydrophobic pressure exerted by alcohols [137,138]. Genes responsible for response to heat shock and extracytoplasmic stress (cpx regulon) are upregulated, and periplasmic chaperone Spy is encoded to respond to protein misfolding activity [139][140][141][142]. Increased isobutanol tolerance of C. acetobutylicum is also conferred to overexpression of genes related to heat shock [139,143]. Genes related to the membrane and periplasmic space carbohydrate transport and metabolisms are upregulated to transport and phosphorylate hexoses and release the phosphate esters into the cytoplasm, probably as a repair mechanism for damaged bilayer [139,144]. Furthermore, the upregulation of genes from the 13-member nuo operon and 5-member cyo operon is also an indicator for the increased requirement of energy or disruption of respiratory efficiency upon exposure to 1-butanol [139]. However, the operons are downregulated when exposed to isobutanol [137]. Exposure to ethanol causes induced expression of psp operon to restore proton motive force lost due to disruption of the cell membrane by ethanol, but the expression level remains unchanged for isobutanol [128,137].

Challenges in Wide Implementation of Alkyl Alcohols as 3HV Precursors
Low alcohol tolerance due to alcohol toxicity is the major drawback for the employment of alkyl alcohols as 3HV precursors. Isolation of novel PHA-producing bacteria with substantial alcohol tolerance is a continuous effort in developing production processes with higher economic feasibility. With established primary alkyl alcohol bioproduction processes, the employment of alkyl alcohols also contributes to sustainability. Alcohol tolerance involves complex regulatory systems, and knowledge from cell-wide stress response is still in demand. Theoretically, genetic engineering can be adopted to create an alkyl alcohol-tolerant PHA-producing bacteria by either introducing pha genes into an alkyl alcohol-tolerant host or modulating alcohol tolerance of a non-alkyl alcohol-tolerant PHA-producing bacteria. Comparatively, the former approach is more rational as alcohol tolerance involves complex systems and is not economically feasible for commercial importance.
Although genetic-engineered E. coli with mutated rpoA gene was constructed successfully to produce products with commercial importance such as 1-butanol, the attempt was based on extensive studies on the rpoA gene and its roles in phenotypic changes of E. coli [145][146][147][148][149]. Owing to numerous studies on the incorporation of pha genes into E. coli, which demonstrated successful production of various PHA, such approaches can be adopted for the construction of alkyl alcohol-tolerant strains with acquired PHA-producing ability [150][151][152]. However, a candidate strain with broad substrate preference is preferred for production process establishment with different substrates and fermentation strategies. The capability to use wastes with high carbon content will be an added value for higher industrial applicability owing to its sustainability and higher economic feasibility compared to pure carbon sources [87].
Despite the promising potential shown by the known alkyl alcohol-tolerant P(3HBco-3HV)-producing bacteria, scaling up the production remains challenging. As low 3HV compositions are commonly reported for shake flask scale production, various production strategies have to be adopted to increase the molar fraction of 3HV. Fed-batch production strategies that enable the addition of alkyl alcohols eventually are practically preferred to achieve high 3HV composition of P(3HB-co-3HV) and at the same time minimize the negative influences caused by the relative toxicity of alkyl alcohol. However, some of the bacteria that depicted decreased biomass and PHA content with the employment of alkyl alcohol at low concentration or with a preference for organic acid sodium salt over alkyl alcohol have low applicability as candidate P(3HB-co-3HV) producers when alkyl alcohols are to be employed. In addition, more studies on large-scale P(3HB-co-3HV) production involving alkyl alcohols are still in demand to compare their industrial practicality as alternative 3HV precursors for organic acids in terms of sustainability and economic feasibility.

Concluding Remark
The high sale price of P(3HB-co-3HV) has been the major obstacle to commercialization. Although various carbon sources have been explored, limited precursor choice due to the domination by propionate and valerate has caused the development of diverse P(3HB-co-3HV) production to reach a bottleneck. With increasing studies reporting the discovery of alkyl alcohol-utilizing PHA-producing bacteria with promising bioconversion efficiency of 1-propanol and 1-pentanol into 3HV, the toxicity of alkyl alcohols and low 3HV yield are no longer the major concern. Future attempts should focus on continuous searching of alkyl alcohols tolerant PHA-producing bacteria to discover more promising wild-type strains.
Moreover, genetic engineering of bacterial metabolic pathways to achieve successful or higher bioconversion rate of alkyl alcohols into 3HV is also important to overcome low bacterial viability and alcohol-3HV bioconversion efficiency. However, more studies are required for techno-economic assessment to compare to what extent 1-propanol and 1pentanol could contribute to higher economic feasibility than propionate and valerate.

Conflicts of Interest:
The authors declare no conflict of interest to disclose.