The microorganism-substrate combination plays an important role in the global process effectiveness. PHBV producing bacteria can utilize a wide variety of organic molecules as substrates, principally sugars, alcohols and organic acids. As previously said, to enhance the process productivity and/or increase the 3HV fraction, the addition of co-substrates (precursors) is a strategy of main relevance. Many studies investigating the effect of different types of precursors as well as the required precursor dosage in the medium, have been performed, although, as expected, the precursors utilization results in a considerable increase of the production costs. Therefore, the use of waste organic material is reasonably more advisable, as it allows moving the process towards a biorefinery scenario fed with abundant and inexpensive materials (i.e. organic wastes). Moreover, the utilization of pre-treatments which generate precursors from wastes has been also proposed as a promising production strategy.
Addition of 3HV fraction precursors
As mentioned above, the majority of bacteria are capable to produce PHBV instead of PHB only if specific precursors are available. The presence of precursors is also fundamental to adjust the 3HV monomer fraction in PBHV and, consequently, modify the polymer properties. Therefore, several studies have been addressed to verify the effect of a large number of synthetic carbon sources (e.g. methane, glucose, fructose), linked to these precursors (Table 2). These studies have confirmed that methanotrophic bacteria, Ralstonia eutropha, Pseudomonas species and Hydrogenophaga pseudoflava were capable to produce only PHB when fed solely with the main substrate (Kim et al. 1994; Inn et al. 2003; Myung et al. 2015; Cal et al. 2016; Martla et al. 2018) while the specie Ralstonia eutropha DSM 545 was capable to produce PH3HB4HB when fed with sole glycerol (Cavalheiro et al. 2012). The addition of precursors led, to PHBV production in all cases investigated.
Valeric acid and propionic acid have been the most studied precursors. Valeric acid is clearly a precursor of the 3HV monomer as it leads to the formation of the 3HV-CoA enzyme, which is successively polymerized (Han et al. 2015). Moreover, valerate concentration in the culture medium strongly affects the 3HV fraction of the PHBV biopolymer. Myung et al. (2015) tested various combination of CH4 and valerate using a methanotrophic consortium. The authors observed that the 3HV fraction increased when the valerate concentration as well as the fraction of the oxidized methane were increased. Inn et al. (2003) reached similar results using the bacterium Ralstonia eutropha and butyrate as principal carbon source: a maximum 3HV fraction of 62% was reached when a valerate fraction of 100% was used in the culture medium. Moreover, Sheu et al. (2009) showed that the modification of the valerate concentration in a sugar rich medium could be used to produce the desired 3HV fraction (from 10 to 90%) using the thermophilic bacterium Caldimonas taiwanensis.
The addition of valerate and, therefore, the increase of the 3HV fraction is relevant as it enhances the quality of the final product. For instance, Koller et al. (2008) observed that the polymer produced through valerate addition presented superior thermal properties compared to the polymer obtained without the precursor addition. According to their analysis, the product quality was appropriate for melt extrusion and film blowing technologies. In the study conducted by Inn et al. (2003), the analysis of the characteristics of the produced polymers showed that an increase of the 3HV fraction led to a decrease of the melting and glass transition temperatures while the polymer composition did not substantially influence the molecular weight distribution. It is worth noticing that the accumulation of high concentrations of acids in the culture medium can result in bacteria inhibition. To induce the reduction of free protons generation in the cell cytoplasm and avoid the acid accumulation in the medium, Loo and Sudesh (2007) converted the valeric acid into the relative salt prior to feeding cells, thus obtaining a reduced inhibitory effect.
Concerning the use of propionic acid as precursor, it was used in 1970 by Imperial Chemical Industries Ltd. to produce PHBV for the first time. In that case, the 3HV-CoA was obtained from condensation of acetyl-CoA and propionyl-CoA to 3-ketovaleryl-CoA and the subsequent reduction of the condensation product to 3HV-CoA. These two reactions were catalysed by β-ketothiolases and acetoacetyl-CoA reductases, respectively (Steinbüchel and Lütke-Eversloh 2003). In the following years, various authors tested different propionate concentrations dissolved in the culture medium. For instance, Yu et al. (2005) used culture media containing glucose and three propionate concentrations (5, 7 and 15 g/L) with the strain Ralstonia eutropha. The tested media led to increase 3HV fractions in PBHV (respectively 30%, 40% and 60%). Similarly, Doi et al. (1987) showed that an increase of the propionate concentration increased the 3HV fraction in the produced biopolymer (from 22 to 45%) using the bacteria Ralstonia eutropha H16.
Kim et al. (1994) studied the effect of three different propionic acid to glucose mole ratios (0.17, 0.35, and 0.52) using Ralstonia eutropha NCIMB 11,599. The final PBHV concentrations of 117, 74, and 64 g /L with 3HV fractions of 74%, 57%, and 56.5% respectively, were obtained. Propionate concentration in a glucose medium was also investigated by Park et al. (1997) in presence of Bacillus thuringiensis R-510. The 3HV fraction increased from 0 to 85%, by increasing propionate concentration from 0 to 0.8% (w/v). A minimum melting point of 65 °C was measured when the polymer contained 35% 3HV fraction.
Other authors tested propionic acid addition to culture media and observed that without the addition of the precursor, only PHB was produced. Conversely, the use of propionic acid as co-substrate led to the production of a PHBV polymer with superior thermal and mechanical properties. In particular, the melting temperature, thermal stability, tensile strength and elongation at break were found to be, respectively, 90 °C, 220 °C, 10.3 MPa and 13.3% (Balakrishna Pillai et al. 2020). On the other hand, the same side effect responsible for culture inhibition produced by high valerate concentrations occurs with high propionate concentrations. In particular, propionic acid was even found to be more toxic compared to valeric acid. Indeed, Loo and Sudesh (2007) observed that the inhibitory effects of the 3HV precursors increased in the following order: valerate salt < valeric acid < propionate salt. The formation of Acetyl-Coa from propionic acid was found to be the rate-limiting step in HVCoA formation, thus reducing the substrate consumption rate, when propionate was tested as single substrate. Dionisi et al. (2004) tested lactate, acetate and propionate as single substrates and their mixture. The authors found that when Acetyl-CoA was formed from acetic or lactic acid instead of being formed from sole propionate, higher fractions of the 3HV monomer were achieved. Moreover, the uptake rate of the propionic acid increased. The importance of using propionic acid as co-substrate rather than as sole carbon source was also underlined by Grousseau et al. (2014). They showed that the simultaneous availability of a second carbon source (butyric acid) led to high conversion rate of propionic acid into 3HV, in presence of Ralstonia eutropha.
Due to the high costs of both valeric and propionic acids, during the last few years, alternative less costly compounds have been tested. For instance, pentanol, which can be oxidized via valeraldehyde to valeric acid and then converted to the 3HV monomer. It has been demonstrated that increasing pentanol fraction by 20% in a methanol-pentanol medium resulted in a valerate increase by 50% and in the stimulation of the PHBV production. Conversely, higher pentanol concentration resulted to be toxic for microorganisms (Ezhov et al. 2013). Despite the reduced costs of the process, pentanol is less effective for PHBV production compared to valerate. Indeed, Cal et al. (2016) tested methanotrophic bacteria fed with sole methane, a mixture of methane with valerate and a mixture of methane with pentanol, by changing the co-substrates concentration. The authors found that the 3HB/3HV molar ratio in PBHV was directly related to the valerate concentration in the culture medium. The same strain (i.e. Methylocystis WRRC1) produced pure PHB when the process was fed with sole methane and 50% lower amount of PHBV when it was fed with a mixture of methane with pentanol rather than valerate.
In addition, levulinic acid, the most inexpensive precursor among all those considered, has been tested to increase the 3HV fraction in PBHV. However, mechanisms leading to PHBV production from levulinic acid have not been clarified yet. Novackova et al. (2019) studied the adaptation of the bacteria Ralstonia eutropha to levulinic acid: the analysis of the PHBV into cells showed a high content of 3HV when the mentioned precursor was used. The influence of levulinic acid on PHBV production by Ralstonia eutropha was also investigated by Chung et al. (2001): the precursor addition greatly increased the molar fraction of 3HV from 7 to 75.l% by increasing the levulinic acid concentration from 0.5 to 4.0 g/L in a culture medium containing fructose as main carbon source.
A comparison between the utilization of propionic acid, valeric acid and levulinic acid was performed by Choi et al. (2003). They added precursors to a glucose medium using the bacterium Alcaligens SH 69. Precursors greatly increased the molar fraction of 3HV to 38–77%. The highest 3HV fraction of 77% was reached by adding levulinic acid.
Also, a few studies reported that PHBV could be synthesized through the propionate pathway when some amino acids like threonine, valine and isoleucine act as precursors for propionyl CoA (Steinbüchel and Pieper 1992; Yoon et al. 1995).
Finally, a very interesting and convenient option is neither the use of waste substrates containing precursors or the adoption of waste pre-treatments generating precursors. Indeed, the use of waste and wastewater is a strategy of main relevance for the reduction of process costs.
Waste substrates and pre-treatments used to enhance the productivity
Over the last few years, various organic wastes and wastewaters have been used as feedstock for PHBV production.
One of the most widely used waste has been the crude glycerol, which is the main by-product of biodiesel industry (Cavalheiro et al. 2012; Hermann-Krauss et al. 2013; Van-Thuoc et al. 2015; Martla et al. 2018). Crude glycerol is particularly suitable for PHAs accumulating species. Indeed, carbon atoms are reduced in glycerol stronger than in any other molecule (e.g. carbohydrates). Consequently, cells using glycerol are in a more evident reduced physiological state, which favours intracellular polymer synthesis (Hermann-Krauss et al. 2013).
It is worth to underline that pure glycerol is actually an expensive material. However, the biodiesel industry residues approximatively 10 kg of crude glycerol per 100 L of produced biodiesel. Yield of biodiesel and related by-products are growing annually, thus causing a sharp decrease of crude glycerol cost (Ghosh et al. 2012; Hermann-Krauss et al. 2013).
Van-Thuoc et al. (2015) tested, comparatively, glucose, maltose, xylose, sucrose, fructose, dextrin and glycerol as substrates for PHBV production, thus obtaining the best results with glucose and glycerol in terms of PHA content and with maltose and glycerol in terms of 3HV fraction. Hermann-Krauss et al. (2013) compared the utilization of crude glycerol and pure glycerol to feed Haloferax mediterranei. The authors pointed out that the amount of the polymer produced and its characteristics were almost the same in the two investigated cases. Therefore, due to the abundance of crude glycerol and the limited costs of its production, its use resulted more convenient than that of the pure glycerol.
By-products from the ethanol industry have been tested as well. Smith et al. (2008) used a condensed corn soluble (CCS) medium to feed Rhodospirillum Rhubum. CCS is a coproduct of corn ethanol production and contains organic acids (lactic acid, succinic acid and acetic acid), glycerol, glucose, maltose, dextrins, microelements, phosphorus and a small amount of free nitrogen. Therefore it represents a suitable source of nutrients for different species of bacteria. Bhattacharyya et al. (2012, 2014) tested vinasse and stillage, highly polluting wastes of the ethanol industry. Results showed that both substrates were effective for PHBV production, and they could be degraded easily during the process, thus obtaining an important lowering of the organic load at the end of the processes.
Agricultural wastes also represent abundant and inexpensive organic sources. Due to the high carbohydrates content in their hemicellulose and cellulose structures, they can be used for PHBV production.
Chen et al. (2006) showed that corn starch, which is rich in sugars, can be successfully used. Poplar hydrolysate has been positively used as well (Yin et al. 2019), and the use of madhuca flowers from India, which contain a large quantity of sugars, proteins, mineral nutrients and organic acids, has been proposed which success (Anil Kumar et al. 2007; Kerketta and Vasanth 2019).
Among others, rice straw is worldwide the most abundant agricultural waste (approximately 700–800 million tons generated every year). Therefore it can be a potential candidate for the industrial PHA production (Ahn et al. 2016b, a). Nagamani and Mahmood (2013) used rice straw to feed Ralstonia eutropha, thus obtaining better results in terms of PHBV productivity and 3HV fraction compared to pure glucose, whey, starch and bagasse. Rice bran was compared with wheat bran to replace part of the pure starch in the culture broth by Huang et al. (2006). Both waste substrates increased both the cell concentration and the PHBV accumulation. However, the maximum cell concentration, PHBV concentration and its content were achieved when rice bran was used as co-substrate with starch, setting a waste to starch mass ratio of 1:8 (w/w).
Due to their high organic load, organic wastes and wastewaters from food industry represent further potentially effective substrates for PHAs accumulating bacteria. A widely utilized waste has been cheese whey, the major by-product from the cheese factories (Nagamani and Mahmood 2013; Pais et al. 2016; Suhazsini et al. 2020). The use of cheese whey for PHBV production in presence of Haloferax mediterranei has revealed to be interesting due to the high salinity requirement of the mentioned bacteria. Indeed, as various types of cheese require along the production process the addition of large quantities of salt, the obtained waste is a high saline cheese whey which already contains the quantity of salt required by Haloferax mediterranei (Pais et al. 2016). Fruit and vegetable wastes represent, certainly, inexpensive and abundant substrates, rich in sugars and nutrients. Vegetable waste has been used as sole carbon source by Ganzeveld et al. (1999). Du & Yu (2002), instead, coupled anaerobic digestion of food scraps with PHB and PHBV production using the digested food waste as substrate for the PHAs production step. Alsafadi et al. (2020) tested date palm, one of the most successful and vital crops in Middle East region as well as in other arid and semiarid regions, to feed Haloferax mediterranei.
Finally, wastes from vegetable oils production, such as OMW as well as jatropha, sunflower, palm and coconut oils have revealed to be effective for PHBV production (Alsafadi and Al-Mashaqbeh 2017; Lee et al. 2008; Mumtaz et al. 2009; Ng et al. 2011). Results obtained from the jatropha oil conversion to PHBV revealed that the quality of the produced copolymer was essentially the same as that produced from other pure carbon sources, such as sugars (Ng et al. 2011).
It has to be highlighted that the use of waste substrates usually requires appropriate pre-treatments, aimed at neither reducing the size and/or the molecular complexity of the organic waste or eliminating toxic compounds.
As it can be noticed from Table 2, physical, chemical or biological pre-treatments can be successfully used. The physical pre-treatment, based on thermal or mechanical processes, are aimed at reducing the solid waste size or extracting simpler molecules. Kerketta & Vasanth (2019) dried, boiled and filtered madhuca flowers to extract macro and micronutrients. In the study of Alsafadi et al. (2020), both mechanical and thermal pre-treatments were applied to date palm fruit waste: fruit seeds were firstly removed manually, then dates were sliced to small pieces (1 cm × 1 cm × 0.5 cm). Successively, carbohydrates extraction was performed using a thermal pre-treatment. The thermal extraction was investigated by testing different conditions (e.g. temperature and extraction times). The maximum carbohydrates concentration (210 g/L) was obtained using 6 h extraction time and 40 °C temperature. Another widely used pre-treatment is the extrusion. Extruders are used to mix and considerably reduce the waste size, in order to facilitate the metabolic activity of bacteria. This treatment was applied to corn starch by Chen et al. (2006) and rice bran by Huang et al. (2006). Both studies compared the utilization of extruded waste and raw waste obtaining better results using the extrudates as carbon source.
Thermal pre-treatments have been also coupled with hydrolysis. Yin et al. (2019) studied hot compressed water method for delignification and promoting the successive enzymatic saccharification of poplar wood. Hot water pre-treatment increased the efficiency of cellulase enzymatic hydrolysis and the yield of reducing sugars. The optimized pre-treatment conditions resulted in being the use of hot water at 200 °C for 30 min, and the enzymatic hydrolysis at 45 °C for 3 days. In addition, the conversion of enzymatically hydrolysed cheese whey into PHBV by Haloferax mediterranei was investigated by Martin Koller et al. (2008), while Pais et al. (2016) performed the cheese whey chemical hydrolysis using the same microbial strain. Results obtained using the enzymatic hydrolysis were better in terms of PHBV productivity. However, the study was conducted with precursors addition.
Chemical hydrolysis has been reported to be less expensive than that enzymatic. Moreover, chemical hydrolysis of cheese whey, requiring alkali addition for hydrolysate neutralization, results in a saline substrate, which is an advantage whenever Haloferax mediterranei is utilized as microbial strain. Pais et al. (2016) tested different HCl concentrations (0.4, 0.7 and 1.0 M) and different reaction times (30, 60, 90 min). The most efficient lactose hydrolysis (96%) with no appreciable degradation of glucose (3.6%) and galactose (0.9%) was obtained using 1.0 M HCl and reaction time of 90 min (Pais et al. 2016).
Kim et al. (2016) studied the effect of thermal pre-treatment and chemical hydrolysis on PHBV production, using rice straw. The pre-treatment conditions strongly affected the substrate composition and the process productivity. The increasing sulfuric acid concentration from 2 to 6% generated a larger PHBV production while the 3HV fraction decreased. To obtain a higher 3HV fraction, an additional heating process of 60 min was conducted following 2% sulfuric acid digestion. In such a condition the highest 3HV mole fraction (22.9%) was achieved. On the other hand, shorter or longer thermal pre-treatment time resulted in a lower 3HV fraction. The obtained results were attributed to the generation of both sugars and levulinic acid, which are precursors of the 3HV fraction.
PHAs production can be coupled with biofuels generation if a biological anaerobic process is conducted as pre-treatment. Such process is inexpensive and environmental-friendly and leads to organic acids generation as soluble products (Pagliano et al. 2017).
Du & Yu (2002) developed a new technology to couple anaerobic digestion of food scraps with PHBV production. The food wastes were digested in an anaerobic reactor producing acetic, propionic, butyric, and lactic acid. The produced acids were successively transferred through membranes via molecule diffusion into an air-bubbling reactor and utilized to produce PHBV. On the other hand, Mumtaz et al. (2009) used anaerobic fermentation as pre-treatment to obtain a mixture of acetic, butyric and propionic acid, which was successively used for PHBV production by Comamonas.
Bhattacharyya et al. (2012) used, instead, a different process, i.e. adsorption on activate carbon, to pre-treat vinasse. This pre-treatment was aimed at removing polyphenolic compounds that are toxic to microorganisms.
Obviously, the convenience of any pre-treatment has to be evaluated by considering the costs/benefits balance. Generally, the use of a substrate rich in simple macro and micro nutrients as well as free of toxic compounds is advisable. For instance, Bhattacharyya et al. (2014) compared the use of stillage with the use of vinasse. The authors reported an increase in the 3HV fraction using stillage. The improvement was possibly due to the higher amount of available organic acids in stillage, including 3HV precursors. Moreover, stillage did not require any pre-treatment, while vinasse has to be treated through adsorption, as previously mentioned. Consequently, stillage was more cost effective than vinasse for PHBV production.