Integrated production of microbial biopolymer (PHA) with other value-added bioproducts: an innovative approach for sustainable production

ABSTRACT

Polyhydroxyalkanoates (PHA), due to their remarkable biodegradability, biocompatibility, and favorable physical and chemical properties, are tested for applications in the agricultural, industrial, and medical fields in place of conventional plastic. Sustainable microbial PHA production is a challenge because of the economic aspects accrued due to the cost-effective substrates and downstream processing. Different approaches are adopted to circumvent the high economic price of PHA production by co-production, employing recombinant strain, mixed cultures, and low-cost feedstock. The review focuses on the co-production of intracellular microbial PHA with an extracellular value-added product. The present review highlights the feasibility of other bio-based products from the feedstock to offset the cost of PHA production with emphasis on another viable option. The paper discusses the recent progress in the simultaneous production of PHA with exopolysaccharides, pigments, enzymes, bio-surfactants, biofuels which primarily fulfill the criteria of higher yield of PHA, and another high-value product that can be achieved. This approach not only maximizes resource utilization but also provides opportunities for the development of a wide range of sustainable bio-based products to improve the overall feasibility of the process.

GRAPHICAL ABSTRACT

KEYWORDS:

• Polyhydroxyalkanoates
• co-production
• extracellular product
• feedstock
• carbon source

1. Introduction

Polyhydroxyalkanoates (PHAs) are biological polyesters that are tested for their potential use as bioplastics. PHAs are deposited as a unique water-insoluble intracellular inclusion in the prokaryotic cells endowed with the features of being bio-based, biodegradable, biocompatible, and piezoelectric. These are naturally synthesized by more than 150 genera of bacteria and archaea (1–31;2;). PHA is a non-nitrogenous carbon reserve or energy reserve accumulated in stress conditions. The polymer exhibits a wide range of physical and mechanical properties which are similar to conventional oil-based plastics, therefore, providing a high technical replacement possibility (4–7). The inherent ability of microbes to synthesize eco-friendly plastic material has received ample attention from academia and industry over the past four decades (2,8–10). The physical, chemical, and biological properties displayed by PHA have resulted in applications of it in the agricultural, industrial, and medical fields (11–14).

Commercialization of PHA on a large scale remains the key aspect (15). The major hindrance to PHA being universally accepted in the marketplace is its exorbitant production cost. The high production cost is largely due to the cost of the substrate and effective PHA recovery (16,17). Due to the high production cost, lack of a robust strain, and effective mode of polymer recovery, presently PHA is not the prevalent choice in the marketplace as a widespread commodity. Several approaches are adopted to cut its price down by using cheaper substrates, identification of suitable feedstock, selection of a robust strain for the bioconversion, improving the bioprocess conditions, and employing recombinant strain (18,19). This is reflected in the surge of research papers published on PHA in a span of two decades. 2000–2020, which is a total of 17,478, and from 2021–2023 around 1,522 publications have been published as collected from the Scopus database (Figure 1).

Figure 1. Total number of papers published on polyhydroxyalkanoates obtained from the Scopus database from 2001 to 2020 using the keyword ‘polyhydroxyalkanoates’.

With the above challenges faced in the commercialization of PHA, recent experiments have focused on obtaining two products in the fermentation medium. The novel strategy adopted is the localization of PHA being intracellular, to couple another viable extracellular product that can be synthesized from the medium is the simultaneous production of two high-value products. The approach of intracellular PHA and extracellular products has gained significance in recent years, and several ongoing attempts are made in this direction (20). As PHA synthesis occurs intracellularly, the medium can be potentially harnessed for another value-added product. Hence, a well-formulated medium is exploited for the microbial synthesis of another product that is necessarily extracellular.

In line with the recent trends, as indicated in the literature, the present work is primarily focused on the simultaneous production of microbial PHAs and a value-added product.

2. Biology of PHA

In a stressed condition like depletion of an essential nutrient, namely oxygen, nitrogen, phosphorus, sulfur, or magnesium, and excess of carbon-rich substrate, the bacteria accumulate PHA intracellularly as discrete granules and store upto 90% of the cell dry weight. PHA occurs in the cytoplasmic fluid as granules in the size of 0.3–1.0 µm (9,21,22). Apart from storage function, recent experimental studies have proved protection against environmental stress factors and enhanced robustness to the PHA accumulating microorganism. The protective function of PHA concerning heat shock, osmotic shock, and ultraviolet protection is studied in Aeromonas hydrophila, Cupriavidus necator, Pseudomonas extremaustralis, and Rhizobium sp. Obruca et al. have summarized the novel functions of PHA granules in microorganisms and stress factors in which the protective function of PHA was described (23,24). There are more than 150 different hydroxyalkanoic acids that form the constituent of biosynthetic PHA (25). In the synthesis of PHA, due to the broad specificity of PHA synthase, any organic molecule with a carboxyl and hydroxyl moiety is catalyzed to the respective CoA thioester, which can be accumulated into a high-molecular-mass PHA (26,27).

PHA is categorized as short chain length PHA (scl-PHA) with 3–5 carbon monomers, medium chain length PHA (mcl-PHA) with 6–14 carbon monomers (Figure 2), and long chain length PHA (lcl-PHA) with greater than 14 carbon monomers in the hydroxyalkanoate units (28–30). The key factors affecting the physical properties of the polymer are monomeric compositions, microstructure, chemical structure, and molecular weight. PHA’s exact biomaterial features are determined during biosynthesis. The production of homo, hetero, and copolymer of PHA is from the various monomers of scl, mcl, or lcl-PHA. The microstructure is another critical factor for the material properties of PHA or aptly referred to as block-structured PHA (b-PHA). The block polyester PHA is constituted of diblock, triblock, or multiblock repeating units, which are considered less prone to polymer aging, and have better physical properties compared to the randomly distributed monomer units (31). Control over the composition and microstructure of PHA facilitates the production of biopolymers capable of meeting industrial criteria for the specific application (32).

Figure 2. Structure and classification of the PHA with its diversity.

The molecular weight of bacterial PHA ranges from 2 × 10⁵ to 3 × 10⁶ Da (1,33,34). The characteristic property of the polymer is entirely determined by the producing bacteria, culture conditions, metabolic pathways aimed at PHA biosynthesis, substrate specificity, and bioprocess strategy which ultimately makes the quality and quantity of the biopolymer (35,36).

Experimental studies have revealed that the synthesis and constituents of bacterial PHAs are regulated by various genomic or functional factors (28). To date, the prokaryotic microbe employed, the process conditions, the carbon source, and its concentration are decisive parameters that greatly influence the content and composition of PHA, which also holds good for other high-value product bioconversion and properties (37). Essentially a media is to be devised harnessing the bio-transformation property of the microbe to utilize the carbon source and produce PHA with a parallel extracellular product.

Microorganisms with the potential to produce PHA can be found in different habitats ranging from soil, water, wastes of vegetable oils, and dairy product industries to soil contaminated with oilseeds, deteriorated food, etc. (38–41). In the metabolic pathway of biosynthesis of PHA, a lack of PHA synthase enzyme will not accumulate PHA. PHA accumulation in the bacterial cell can be determined easily by Sudan black staining and Nile blue staining. Most PHA has been produced by bacteria and archaea, although transgenic plants were reported to produce PHA. PHAs have been detected in photosynthetic and aerobic groups, lithotrophes, organotrophes, and cyanobacteria.

PHA producers can be distinguished based on natural PHA producers and metabolically engineered microbes. Genetic engineering has resulted in the modification of non-PHA-producing bacteria to produce PHA at high levels. For example, Escherichia coli, a non-PHA accumulating bacterium (not an innate producer of PHA) is intensively studied for PHA production.

Koller et al., in the Book in Plastics from Bacteria, provided an overview of the PHA synthesizing microorganisms reported in the literature (42).

3. Co-production of PHA with value-added product

3.1. Enzyme

Enzymes are special proteins that catalyze reactions with specificity and rate enhancements. The enzyme has applications in all fields, from food and leather to textile industries, etc. The impending factor in the application of enzymes in the industrial process is linked to the cost. In an annual operating cost of an enzyme production facility, 28% of the operating cost is spent on raw materials (43). Agricultural practices result in the generation of huge quantities of agricultural residues. The wastes sometimes are burnt or dumped to rot, with the overall effect being hazardous to the environment.

Every carbon-rich residual feedstock comes with a profile of characteristics, including nutrient composition and concentration. PHA production from residual feedstock requires the reliable production of a PHA accumulating biomass and the reliable accumulation of PHA in this biomass. It is most common to consider a given feedstock of carbon for achieving both biomass and PHA production (44). Renewable and inexpensive agro wastes with abundant C and N components form an ideal starting material for the cost-effective generation of different industrial products. Wheat bran, wheat straw, rice straw, sugarcane bagasse, fruit pomace, and oil cake represent the major agro residues. Table 1 lists the lignocellulosic feedstock used in the production of important enzymes (45).

Table 1. A partial list of enzymes produced from various lignocellulosic waste feedstock.

Lignocellulosic waste feedstock	Enzymes produced
Wheat bran, Lentil husk, Starchy substrates	Alpha amylase
Sugar beet, Apple and grape pomace, Wheat bran	Lipase
Wheat Bran, Sugarcane bagasse, Orange peel	Pectinase
Wheat Bran, Wheat straw	Xylanase
Coffee husk	Tannase
Wheat bran, Lentil husk, Green gram husk, potato peel, wheat straw	Protease
Banana fruit stalk, Cow dung	Cellulase
Whey and cauliflower waste	Beta galactosidase

3.1.1. Amylase

Amylases comprise a group of industrial enzymes, making up approximately 25% of the enzyme market (46). Amylases are enzymes that hydrolyze starch molecules, producing dextrin and progressively smaller polymers composed of glucose units. Alpha amylase is an industrially important enzyme, and it finds applications in the food and textile industries. This enzyme can be coproduced along with PHA using starch based substrates (Figure 3).

Figure 3. Integrated Production of Polyhydroxyalkanoate and Amylase in bacteria using starch as substrate.

In Shamala et al., corn starch was used as the substrate for the production of PHA copolymer and amylase. The media was also supplemented with wheat bran and rice bran hydrolysates. The hydrolysates of wheat and rice bran were used for growth and metabolite production as it is a source of carbohydrates, amino acids, and fats. Wheat bran media and rice bran media individually and in combination were tested for PHA production and alpha-amylase. Maximum biomass and PHA production were observed in wheat and rice bran hydrolysate (1:1), with ammonium acetate and corn starch. The biomass and PHA production yield reported was 10 and 5.9 g/L respectively. The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate. Maximum activity of amylase was observed in wheat bran hydrolysate medium with corn starch 2–40 U/mL . Bacillus sp. CFR 67 was employed for the study (47).

Sreekanth et al., reported on the PHA and amylase yield with varying different carbon sources, nitrogen sources, and supplementation of wheat bran and rice bran hydrolysates using Bacillus sp. CFR 67. With glucose as the carbon source the yield of PHA was 444 mg/L and amylase 66 U/mL whereas, with sucrose, the yield of PHA was 480 mg/L and amylase 42 U/mL. Among the inorganic N source, ammonium acetate gave the best result of 192 mg/L PHA and 36.6 U/mL amylase. Among the organic sources, beef extract supported the higher synthesis of PHA 592 mg/L and amylase 17.7 U/mL. Supplementation with wheat bran produced 524 mg/L PHA and 73 U/mL of amylase. Rice bran produced 20% less amylase in comparison to wheat bran (48) as given in Table 1.

3.2. Biosurfactants

Biosurfactants structurally are amphiphilic compounds playing a key role in decreasing surface tension. Rhamnolipid is the most widely studied microbially produced low molecular weight glycolipid surfactant. The hydrophilic region of one or two rhamnose moieties linked through a glycosidic bond, and a hydrophobic region consisting of one or two beta-hydroxy fatty acids with varying carbon atoms from 6 to 24 in the chain (49,50). Around 60 different rhamnolipid homologs have been reported and produced by the species of Pseudomonas, Burkholderia, Acinetobacter, and Enterobacter (49,51,52). Biosurfactants increase the surface area, solubility and the bioavailability of hydrophobic water insoluble substrates. Biosurfactants also enhances the formation of stable water–oil emulsions and the breakdown of the oil film They also have a role in removal of heavy metal from contaminated sediments by solidification, stabilization and washing (53). Concerning the physicochemical and biological properties, it finds its application in emulsification, foaming, solubilization purposes, detergents, formulation of cosmetics and pharmaceuticals, bioremediation, water treatment, and environmental control and management (54–57). As rhamnolipids are secreted into the medium, they can be utilized for PHA production. Hydrophobic substrates induce the production of rhamnolipids, and hence the carbon source is accordingly selected. Since rhamnolipids are an extracellular component, it is comparatively easy to the employment of downstream techniques in extracting the two compounds of rhamnolipid from the supernatant after centrifugation and the intracellular PHA from the pellet (Figure 4). There are a few strains of bacteria reported producing both rhamnolipid and PHA for example Pseudomonas aeruginosa. Marsudi et al. reported the intracellular production of PHA and extracellular production of rhamnolipid using low cost substrate palm oil by Pseudomonas aeruginosa. In this study, palm oil was catalyzed into fatty acids and glycerol. Fatty acids and glycerols were used in the synthesis of PHA and rhamnolipid respectively. PHA was produced till the exhaustion of fatty acids, and rhamnolipids until glycerol was present (58). The other group of bacteria that are suited for the dual synthesis is Burkholderia thailandensis, with cooking oil as the substrate, and Thermus thermophilus HB8 with glucose or sodium gluconic as feed (59). Kourmenza et al. reported the used cooking oil (sunflower oil) as the substrate, employing the non-pathogenic strain Burkholderia thailandensis E264 in a 10L bioreactor, with the biomass being 12.2 g/L, PHA 7.5 g/L, and rhamnolipid 2.2 g/L. What factors in the carbon metabolic flux direct the biosynthesis of rhamnolipid over PHA needs to be elucidated (60).

Figure 4. Simultaneous production of PHA and biosurfactant in a single fed-batch fermentation.

3.3. Polymers

3.3.1. Simultaneous production of PHA and Xanthan gum

Xanthan gum is a natural microbial polysaccharide and an important industrial polymer produced by the Xanthomonas genus. The branched hetero and exopolysaccharide have a primary structure comprising of recurring pentasaccharides, made by glucose, mannose units of two each, and one glucuronic acid unit. The molecular weight falls in the range from 2 × 10⁶ to 20 × 10⁶ Da (61,62). Analysis of the physical and chemical characteristics displays the ability to modify the rheological behavior of aqueous solutions, increasing their viscosity by intermolecular associations. The toxicological and safety properties of Xanthan gum for food and pharmaceutical properties have been comprehensively investigated. The findings demonstrated that it is safe and non-toxic and can be used as a food additive. The physicochemical properties displayed makes it a favorable choice for thickening, suspending, and emulsifying agent (63). Both polymers are value-added products. Using low-cost substrate, a study conducted by Rodrigues et al. reported simultaneous and individual production of PHA and Xanthan gum with palm oil as the substrate. The study utilized axenic cultures and pure cultures, namely Cupriavidus necator and Xanthomonas campestris. After the optimization of individual production of PHA and Xanthan gum, the simultaneous production of both polymers was executed (Figure 5). The yield of the PHA increased from 3.39 g/L to 6.43 g/L. The study introduced a process for the biotechnological integration of both polymers. Additionally, co-culture also promoted more PHA production (37). Table 2 summarizes the coproduction, the organism, the substrate, and the yield.

Figure 5. Flowchart of coproduction of PHA along with Xanthan gum.

Table 2. A partial list of co-producers of intracellular PHA and extracellular product.

Organism	Substrate	Intracellular product (PHA) in g/L or % CDW	Extracellular product	The yield of the extracellular product	References
Recombinant E.coli	Glucose and Indole	57.4%	Indigo	0.60 mM	Jung et al. (64)
Pseudomonas chlororaphis	Glycerol	mcl-PHA 2.23 g/L	EPS and Phenazines	6.10 g/L	De Meneses et al. (65)
Clostridium beijernicki ASU10	i. Glycerol ii. Sugarcane molasses	84% (CDW) 3HB 37.97%(CDW) 3HB, 3 HO	H2 gas and Acetone, Butanol and Ethanol	i. H2 5.1 ± 0.8 ml/h, ABE 9.33 ± 2.98 g/L ii. H2 11 ± 0.44 ml/h ABE 10.83 ± 4.1 g/L	Hassan et al. (66)
Arenibacter palladensis YHY2 And Ralstonia eutropha H16	Chitin	1.02 g/L PHB And 0.198 g/L PHV	Bioelectricity N acetylglucosamine	15.15 µA/cm^2 196.34 mg/g of chitin	Ranjit Gurav et al. (67)
Bacillus subtilis OK2	Orange peel	1 g/L	PGA	0.40 g/L	Sukan et al. (68)
Haloferax mediterranei DSM 1411	Glucose (10 L Bioreactor)	13.02 g/L (PHBV)	EPS	1.31 g/L	Koller et al. (69)
Sinorhizobium meliloti MTCC 100	Sucrose 10 g/L with rice bran hydrolysate	PHA 3.6 g/L	EPS	11.8 g/L	Saranya Devi et al. (47)
Bacillus sp. CFR-67	Unhydrolysed corn starch (10–50 g/L) supplemented with wheat bran hydrolysate	PHA 5.9 g/L	Alpha amylase	2–40 U ml^−1min^−1	Shamala, Vijayendra, Joshi (70)
Pseudomonas mendocina NK-01	Glucose in 200 L fermentor	0.316 g/L (mcl-PHA)	Alginate oligosaccharides	0.57 g/L	Guo et al. (71)
Pseudomonas aeruginosa IFO 3924	Palm oil Glycerol	0.59 g/L 0.324 g/L	Rhamnolipid	0.43 g/L 1.32 g/L	Marsudi et al. (58)
Azotobacter beijerinckii strain WDN-01	Glucose (3%)	PHB 2.7 g/L	Exopolysaccharide	1.2 g/L	Pal et al. (72)
Bacillus licheniformis PL 26	Jatropha biodiesel waste	1.1 g/L (PHBV)	ε- polylysine	0.2 g/L	Bhattacharya et al. (73)
Burkholderia thailandensis E264	Used cooking oil (10L Bioreactor)	7.5 g/L	Rhamnolipids	2.2 g/L	Kourmenza et al. (60)
Cupriavidus necator NCIMB 11599, Paracoccus sp. LL1, and Bacillus megaterium ALA2	Algal biomass (10% W/V) Laminaria japonica brown algae	32%	–	–	Muhammad et al. (74)
Paracocus sp LL1	defatted Chlorella biomass	1.48 g/L	Carotenoides	6.08 mg/L	Khomlaem et al. (75)
Bacillus megaterium ALA2, Cupriavidus necator KCTC 2649, and Haloferax mediterranei DSM 1411	defatted Chlorella biomass	3.79 g/L	Carotenoides	1.80 mg/L	Khomlaem et al. (76)
Bacillus megaterium ALA2 Paracoccus	Corn cob	31.3 g/L	Astaxanthin	11.95 mg/L	Khomlaem et al. (77)
Halomonas sp	Corallina mediterranea	63%	–	–	Abd El-Malek et al. (78)

3.3.2 Simultaneous production of PHB and polyglutamic acid

Poly (γ-glutamic acid) is a polymer of glutamic acid units produced by a few microorganisms. The polymer has wide applications ranging from the food industry, pharmaceuticals, and wastewater treatment. It is produced by a few of the Bacillus species. Sukan et al. reported the co-production of PHA and polyglutamic acid. The aim was to demonstrate whether simultaneous production is feasible, with not much significance given to the aspect of productivity. Bacillus subtilis OK2 yielded 1 and 0.4 g/L PHB and poly (γ-glutamic acid) respectively. Instead of glucose as the substrate, a low-cost substrate orange peel was also tested. With the orange peel carbon substrate, two important products were observed (68). A dual production medium was optimized for the study by incorporating the components of individual polymer production media, involving glutamic acid, glucose, and citric acid. The study identified the optimum culture conditions for the dual products, poly (γ-glutamic acid) from the culture broth (Extracellular) and cell autolysis with the release of PHB granules was observed. Hence, the overall study with the opportunities and challenges paves the way for using bio-waste for dual production.

3.4. Exopolysaccharides

Microorganisms synthesize a wide variety of different exopolysaccharides (EPS), composed of carbohydrate units, uronic acid, and other non-carbohydrate units such as acetate, pyruvate, succinate, and pyruvate. These polysaccharides accordingly are classified as anionic, neutral, or cationic. Different properties of the EPS ranging from gelling, viscosity, and biofilm formation are adopted for various commercial applications in food-related industries (79,80).

Some of the genera of bacteria that are identified for the production of EPS are Acetobacter, Aureobasidium, Bacillus, Halomonas, Lactobacillus, and Pseudomonas. Microbial polymers such as pullulan, kefiran, bacterial cellulose, gellan, and levan are placed under the category of EPS (81–83).

EPS plays an important role in supplying cells with energy and protection when subjected to adverse growth conditions and nutrient limitations. Both PHA and EPS are produced intracellularly and extracellularly respectively when microorganisms are under stress conditions. Simultaneous production of EPS and PHB was reported in various microorganisms (47–85). They have the potential to replace plant and algal EPS that are traditionally being used in the food, pharmaceutical, textile, and oil industries.

Members of the genus Azotobacter are known to accumulate PHB as intracellular carbon and energy reserve material when grown under nutrient limiting conditions other than the carbon source. Only a few of them who can accumulate PHB to a significant level has been employed for the production of PHB. Among these are Azotobacter vinelandii, A. salinestris, and A. chroococcum (86,87). Azotobacters have also been recognized as producers of water-soluble extracellular polysaccharides. It has been suggested that exopolysaccharides may protect the cells from desiccation, phagocytosis, and phage attack (72). Maximum PHB production with 3% (w/v) glucose was 2.7 g/L and exopolysaccharide was 1.2 g/L, as provided in Table 2.

Koller et al. reported the use of a haloarchaeon for the simultaneous production of two types of biopolymers. Poly 3(hydroxybutyrate-co-hydroxyvalerate) (PHBV) was accumulated as intracellular granules, whereas an extracellular polysaccharide was excreted in parallel to bio polyester synthesis.(Figure 6) The EPS constitutes a sulfated (anionic) polysaccharide of high molar mass, resulting in a typical mucous character on the solid medium. Such EPS of algal origin is used in cosmetics as well as therapeutic reagents. The haloarchaeon understudy was attractive due to its capacity to synthesize PBHV, from structurally unrelated resources such as carbohydrates and glycerol. In the study, the inoculum cultures were added to 7 liters of a non-sterilized medium in a 10L bio-reactor in the fed-batch mode. PHA synthesis began simultaneously with biomass production that was exhibiting growth-associated PHA production. The final titer of PHA for the entire process was found to be 13 g/L, and the extra polysaccharide was 1.31 g/L. There were many other interesting findings in the study, such that sterilization of the medium and the reactor was not required to maintain the monoseptic culture (69).

Figure 6. Production of exopolysaccharide and PHA from fermentation.

3.5. Biofuels

The term ‘biofuel’ encompasses a wide variety of products such as bioethanol, biodiesel, biohydrogen, biobutanol, bioether, biogas, and syngas (88,89). Biomass can be effectively utilized in bioconversion to biofuel providing an alternative to the negative aspects of fossil fuels. An integrative approach to produce biohydrogen and PHA has been studied, employing pure, complex substrates and also biowastes. Bacillus, Clostridium, Enterobacter, Rhodospirillum, and Rhodobacter are the most promising organisms in the production of biological hydrogen and PHA (90,91). Singh et al., reported the production of biological hydrogen and PHA using Bacillus thuringenesis with maximum hydrogen 460 mmol/mol glucose and PHA accumulation of 5.3 wt% (92).

Hassan et al. reported Clostridium beijerinckii ASU10 being a promising candidate for the recycling of organic, renewable waste materials and the synthesis of low-cost biodegradable plastics simultaneously with the production of clean liquid and gaseous fuels. The agro-industrial feedstock, sugarcane molasses, and crude glycerol waste from the biodiesel generation served as low cost, cheap substrate, for the concomitant production of extracellular metabolites of Hydrogen, Acetone-Butanol-Ethanol (ABE), and intracellular inclusion bodies PHA (66). In this study, sugarcane and molasses were used separately and the generation of ABE from glycerol and molasses was 9.33 ± 2.98 g/L and 10.831 ± 4.1 g/L, and the yield of PHA was 84% and 37% from glycerol and molasses respectively. Ebrahimian et al., in a study, investigated the simultaneous production of biohydrogen, 2,3-butanediol, ethanol, and PHA from the enzymatic hydrolysate of organic fraction of municipal solid waste. Pretreatment was included by acetic acid catalyzed organosolv at different temperatures and reaction times to increase the biological conversion. The enzymatic hydrolysates were subjected to fermentation by Enterobacter aerogenes. In this environment friendly process, each kg of organic fraction-municipal solid waste, a biofuel equivalent to 257 ml gasoline, and a PHA yield of 40 g/kg was produced (93). Therefore, the studies open avenues for the parallel production of two products with popular bacteria for bioconversion.

3.6. Concurrent production of electricity, N -acetylglucosamine, and PHA

The crisis of energy requirements from depleting fossil resources has led to the investigation of efficient, economical alternate methods. Microbial fuel cell (MFC) technology is a green choice with dual functionalities; utilization of waste biomass and reducing environmental pollution on one hand, and generation of electricity on the other hand (94). In Gurav et al., MFC with the production of PHA was studied. Crustacean shells from the shellfish processing industries are an abundant source of chitin biomass for commercial chitin. Chemical methods for chitin hydrolysis are disadvantageous both economically and environmentally. Therefore, microbial bioconversion is preferred (95). To offset the bioconversion cost associated, there is a need for the development of a strategy to utilize the chitin biomass and its degradation organic products for simultaneous value-added products. Ranjit Gurav et al., in their study, isolated the bacterium Arenibacter palladensis YHY2 that hydrolyzed crab chitin in two systems, that is, shake flask and MFC system. The quantified chitinase enzyme at 71.44 ± 1.90 U/mL, an important by-product of chitin degradation was found to be N-acetylglucosamine in a shake flask.

The potential of Cupravidus necator H16 potential to utilize N-acetylglucosamine as a substrate for the production of PHA was coupled with the Arenibacter palladensis YHY2 strain. Co-cultivation of both strains was evaluated for electricity generation. Chitin was utilized by YHY2 strain in the MFC generating electricity in the range of 4.28 µA/cm² at the start stage and 10.72 µA/cm² at 204 h with a peak of 15.15 µA/cm² at the start stage. Other important metabolites evaluated in the MFC system were N-Acetyl glucosamine, followed by butyrate, acetate, lactate, and propionate. The studies indicated that the soluble GlcNAc was metabolized for PHA accumulation by C necatoH16. The GC analysis revealed the components, 1.020 g/l PHB and 0.198 g/l of PHV in PHA (67). Thus, a one-pot hydrolysis system is a novel method to use chitin biomass with the production of a substrate, suitable for the accumulation of PHA by the microbe. Production of PHA using C necator H16 has been widely studied by several researchers. Bhatia et al. reported that the major drawback of the bacterium was carbon utilization capability limiting to GlcNAc and fructose (96).

3.7. Pigments

Natural pigments obtained from microbial sources is of high value vs synthetic pigments. Pigments have varied applications as food colorants, antimicrobial and cytotoxic activities. Microbial pigmented molecules such as, carotenoids, flavins, indigoids, melanins, pheomelanin,, phenazines, phenazostatin D, prodigiosin, violacein, glaukothalin, pycocyanin, xanthomonadin, phenazine, canthaxanthin, astaxanthin, β-carotene, etc. are produced as biproducts by several microorganism. Dual production of PHA and pigments using microbes is reported (97).

Kumar et al., reported the use of the halophile Paracoccus sp.LL1 in the conversion of glycerol and waste cooking oil to value added products PHA and carotenoids. The biotransformation demonstrated the inherent capacity of the Paracoccus sp. LL1 to produce the value added products. This can be further improvised by use of other cosubstrates and fermentation strategies (98,99).

Corrêa, P. S., & Teixeira studied the effect of pure and crude glycerol on total carotenoids, phycocyanin and PHA content in Arthrospira platensis culture. The highest final biomass concentrations, 1.68 and 1.40 g L⁻¹, and PHAs contents, 1.1 and 0.5% were achieved by supplying a Zarrouk medium with 6.14 g L⁻¹ of pure or crude glycerol, respectively. crude glycerol at 3.07 g.L⁻¹ attained the highest content (10.8%) of phycocyanin. other characteristics such as easy harvesting operation, fast growth and high phycobiliproteins content represent advantages of using the Arthrospira genus (100).

Violacein pigment is the secondary metabolites produced by bacteria such as Chromobacterium violaceum, Duganella sp., Janthinobacterium lividum. This constitutes of violacein and deoxyviolacein. It possesses various biological activities and has commercial applications. Statistical optimization and bench-scale bioprocesses in 22.0 L fermentor for the co-production of PHB and violacein pigment were investigated using Iodobacter sp. PCH 194 (101). The two important biomolecules quantified was PHB (11.0 ± 1.0 g/L, 58% of dry cell mass) and violacein pigment (1.5 ± 0.08 g/L).

Park et al., reported production of PHB with pyomelanin, a black colored pyoPHB in a single fermentation broth with 0.52 g/L of PHA and pyomelanin using engineered E.coli culture. The composite possess both physical properties of PHB and pyomelanin with increased thermostability thereby enhancing commercial applications (102)

4. Future scope and limitations of the study

The dual production of PHA and another high value-added product is still in the inchoate stage, but the future looks promising. The review is restricted to reporting the organism, the experimental studies conducted for dual production, and the PHA content analysis. Exploration of different carbon sources and the possibility of another product was studied. A clear understanding of the mechanism for the partition of the nutrients in the formation of two products, separation of dual products, and process optimization promises to be the key to the progress of the formation of dual products. Optimization of the nutrients and bioprocess parameters for the yield of the two products provides ample scope. Therefore, the identification and production of the two products remain a challenge. Future experiments need to focus on the increase of the intracellular PHA content. Additional features such as biodegradation, eco-toxicity, and cytotoxicity tests of the material have to be performed according to valid norms and standards to receive admission of PHA obtained to find its applications. The literature data on these aspects are fragmentary and incomplete, thus providing a fertile ground for researchers in the field of biotechnology and advancing the frontiers of knowledge by combining metabolic engineering, genetic engineering, and bioprocess engineering.

5. Conclusion

The various studies are proof of the concept of the simultaneous production of two products. A more rigorous approach is required to identify the right organism, manipulation of the media components, and search for the right substrate, or agro-industry residue. The dual production of PHA and PGA, biosurfactants, Xanthan gum, enzymes, and biofuels are proof of simultaneous production. The usage of feedstocks for a useful product also tackles the issue of waste management. The SWOT analysis of feedstocks, the genetics of the organism, and the co-production strategy reveals a systematic and logical approach to sustainable PHA production and paves a strong way for commercial PHA production. For this to be carried out and for the successful viable production of PHA, a committed and unified approach of microbiologists, biochemists, system biologists, bioprocess engineers, industrialists, and economists is the need of the hour. Identifying the valuable products and coupling them to the PHA producer and substrate with the optimization of media components holds a promising alternative to sustainable PHA production in the future.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by The Department of Biotechnology (DBT), Government of India (PR 18430/BIC/101/703/2016) and grateful to The Department of Biotechnology, Manipal Institute of Technology, Manipal Academy of Higher Education (MAHE), India; for providing the facilities to carry out the research work.