Production of Poly(3-hydroxybutyrate) by Pseudomonas sp. phDV1 Strains Using Second Cheese Whey Effluent

TL;DR

This paper explores using cheese whey waste to produce a natural plastic-like polymer with Pseudomonas bacteria, offering a sustainable solution for waste management and resource recovery.

Contribution

The study demonstrates the use of second cheese whey as a low-cost substrate for PHB production by Pseudomonas sp. phDV1 strains.

Findings

Pseudomonas sp. phDV1 strains can grow and produce PHB in second cheese whey with or without enzymatic treatment.

Fluorescence microscopy and HPLC confirmed PHB formation and localization in bacterial cells.

The process offers a sustainable method for dairy wastewater treatment and biomass valorization.

Abstract

The aim of the circular economy for plastics is to replace some of them with bio-based polymers in the future. In this work, second cheese whey (SCW) was used as a low-cost substrate for the production of the natural polyester poly(3-hydroxybutyrate)-hydroxybutyrate (PHB) by three Pseudomonas sp. phDV1 strains, namely, the wild type, a depolymerase PhaZ and PhaR knockout mutants. SCW has high polluting loads, characterized by high levels of lactose, phosphorus, nitrogen and salinity, as well as high turbidity due to the presence of whey solids. Initially, SCW was evaluated as the sole carbon source for the growth of the bacterial strains and the production of PHB. Fermentation conditions were screened to maximize polymer synthesis. Small-scale experiments showed that the strains could grow and produce PHB in SCW with and without enzymatic treatment. The formation and intracellular localization of PHB were determined with fluorescence microscopy, using Nile Red staining. Analytical HPLC was also used to quantify the PHB content in the cells and to optimize production. This study demonstrates the efficacy of Pseudomonas sp. phDV1 in SCW treatment and biomass valorization, providing a sustainable solution for dairy wastewater management while producing valuable resources.

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Full text

1. Introduction

Plastics are synthetic polymer molecules whose properties make them suitable for a wide range of applications; however, the environmental pollution caused by them continues to be widespread [1]. Replacing synthetic plastics with bio-based, compostable and/or biodegradable alternatives is already a well-known and widely accepted solution to the environmental problems associated with plastic waste [2]. Among other bio-based polymers, polyhydroxyalkanoates (PHAs) are microbially synthesized polyesters formed intracellularly by numerous bacteria, fungi, yeasts, algae, plants, cyanobacteria and others [3]. Poly(3-hydroxybutyrate) is the best studied member of the PHA family. It forms intracellular biopolymer granules and acts as a carbon and energy store in response to stress conditions [4,5].

Over 300 distinct bacteria, including Pseudomonas sp., are known to produce and store PHAs intracellularly [6,7,8]. Metabolic engineering has been used to improve the production of PHAs through a variety of approaches, such as the over-expression of the PHA synthesis operon or the elimination of the ability to consume PHAs [9,10,11,12]. PHAs are an ideal replacement for fossil-based plastics because they are biodegradable and break down naturally into carbon dioxide and water [13]. However, due to their high production costs, research and development of new methods that aim at their biosynthesis is a key area of focus.

PHA-producing Pseudomonas strains harbor the pha gene cluster encoding the PHA synthase (PhaC), depolymerase (PhaZ), and transcriptional regulator (PhaD) [6,14]. Divergently transcribed phasin genes also contribute to regulatory and structural functions [15,16]. Metabolic engineering strategies commonly target PHA depolymerases, as PhaZ inactivation increases bioplastic accumulation. PHA metabolism is controlled by complex regulatory networks linking carbon and nitrogen assimilation [10,11]. The repressor PhaR regulates PHA synthesis by controlling PhaP expression, thereby influencing phasin production and PHA granule formation [12,16,17].

In recent years, microbial production of PHAs has shifted from pure substrates to renewable feedstocks and industrial wastes in the search for cost-effective and environmentally friendly resources [18]. The agri-food industry produces a wide range of waste products which, although often of varying quality or even containing harmful substances, can be re-used for the production of PHA. The use of agro-industrial wastes as carbon sources for PHA production has been proposed as a promising solution with the dual advantage of minimizing production costs and alleviating the economic burden of processing unwanted streams and residues [19]. Utilizing these wastes for PHA synthesis can yield high value-added products.

Cheese-making is one of the most important agro-industrial sectors in Europe [20]. The cheese industry is a very water-intensive sector and produces a significant number of wastewaters [21,22]. A by-product of cheese production is cheese whey (CW), which, after the addition of milk and salt, can be used to make whey cheese. The by-product of this stage is called second cheese whey (SCW), and contains important nutrients such as lactose, nitrogen, free amino acids, mineral salts, phosphorus and various other nutrients [23]. These nutrients make SCW a cost-effective substrate for bio-based production of lactic acid, bioethanol, environmentally friendly bioplastics, biofuels, beverages, bioactive peptides and microbial starters [23]. Although many studies have included dairy ingredients, only a few have focused on CW, while there are no known reports on SCW as a substrate for Pseudomonas [10,24,25,26,27].

In this context, the main objective of the present study is the utilization of SCW as the sole carbon source for PHB production. To achieve this, three different stains of Pseudomonas sp. phDV1 were used, the wild type, the phaZ and the phaR knockout mutants, in order to examine which strain can attain maximum bioplastic production. To the best of our knowledge, the valorization of highly saline SCW with Pseudomonas sp. has not been reported. Our approach highlights the bioremediation potential of Pseudomonas with the production of high-value bioplastics, contributing to a more holistic understanding of microbial dairy wastewater treatment.

2. Materials and Methods

2.1. Cultivation of Pseudomonas sp. phVD1 in High Salinity

First, experiments were carried out to assess the ability of Pseudomonas sp. phDV1 to grow under high salinity conditions, since SCW is characterized by high organic loads and high salt concentrations. The wild-type strain of Pseudomonas sp. phDV1 was cultivated in M9R minimal medium at 32 °C and 200 rpm for 225 h. Succinate served as the sole carbon source in the medium at concentrations of 1 M, 1.5 M and 2 M NaCl. All media were autoclaved at 120 °C and all cultures were performed in a sterile environment, in order to prevent contamination from other microorganisms. Growth was monitored by measuring optical density at 600 nm using a plate reader (Multiskan SkyHigh Microplate Spectrophotometer, ThermoFischer Scientific, Waltham, MA, USA). Culture purity was ensured through microscopic monitoring, using an oil-immersed lens on a Nikon ECLIPSE E800 microscope (Nikon Instruments Inc., Melville, NY, USA). All cultures were performed in triplicate, ensuring accurate and reliable results.

2.2. Second Cheese Whey as Carbon Source

The SCW was kindly supplied by a local cheese manufacturing industry, located in the prefecture of Rethymno, on the island of Crete (Greece). SCW was generated from the production of a salty whey cheese (anthotyros), which is a traditional Greek dairy product. The production of anthotyros involved the addition of salt to primary CW, which was mixed with a small amount of goat and sheep milk. SCW was stored in polyethylene bottles and the samples were kept frozen at −20 °C before they were used in the experiments. The SCW was extensively characterized for its physicochemical characteristics and nutrient content [28]. Throughout the entire experimental series, the SCW samples were autoclaved at 100 °C for 20 min to prevent contamination by indigenous microorganisms. After autoclaving, the solids were removed via centrifugation at 5000× g for 10 min. The supernatant (whey permeate) represents the SCW fraction used in the experiments. The pre-treated SCW was subjected to lactose hydrolysis using a commercial β-galactosidase (Lamberts Lactase Complex 350 mg (9000 FCC), Lamberts Healthcare, Tunbridge Wells, Kent, UK), added in 100 mL SCW (10 U/mL). After continuous shaking (4 h, pH = 4.5, T = 55 °C) the solution was autoclaved at 100 °C for 20 min to inactivate the β-galactosidase. The pH of the solution was adjusted to 7.4 and was centrifuged at 5000× g for 10 min in order to remove possible solids. The supernatant represents the pre-treated SCW. The process of hydrolysis of lactose was monitored by measuring the glucose produced using a commercial glucose meter.

2.3. Cultivation of Pseudomonas Strains in Second Cheese Whey

The three Pseudomonas strains (WT, ΔphaR, ΔphaZ) were cultured in three conditions, containing SCW and pre-treated SCW in different concentrations. In the first conditions tested, all Pseudomonas strains were cultured in M9R minimal medium, in the presence of SCW and pre-treated SCW in a quantity similar to that of succinate [29]. These cultures were carried out at 32 °C and 200 rpm for 72 h. In the second type of conditions, the M9 minimal medium water was replaced with SCW and pre-treated SCW, which also served as the provided carbon source, while in the third type of conditions pure SCW and pre-treated SCW were used, without any additives. These kinds of cultures were performed at 32 °C and 200 rpm for 144 h. All media, apart from SCW and pre-treated SCW, were autoclaved at 120 °C and all cultures were performed in sterile environment in order to prevent contamination from other microorganisms. Growth was monitored by measuring optical density at 600 nm using a plate reader (Multiskan SkyHigh Microplate Spectrophotometer, ThermoFischer Scientific), while culture purity was ensured through microscopic monitoring, using an oil-immersed lens on a Nikon ECLIPSE E800 microscope (Nikon Instruments Inc., Melville, NY, USA). All cultures were performed in triplicate, ensuring accurate and reliable results.

2.4. Nile Red Staining

In order to assess any potential PHB production, cells from all different conditions were stained, using Nile Red, a hydrophobic stain that binds to hydrophobic molecules through hydrophobic interactions and non-polar solvation [30]. To prepare the sample, 1–2 mL of cells was centrifuged at 13,000× g for 60 s and then resuspended in 50 μL of growth medium. In the reaction tube, 1 μL of a 250 μg/mL Nile Red solution in DMSO was added to 3 μL of cells. Agarose pads were prepared by pipetting 30 μL of hot (60 °C) 1% (w/v) agarose solution onto a slide. Immediately afterwards, 4 μL of the stained cell suspension was added to the agarose pad. After allowing it to dry for a few seconds, the coverslip was placed on top of the agarose. The cells were then observed using an oil-immersed lens on a Nikon ECLIPSE E800 microscope (Nikon Instruments Inc., Melville, NY, USA) with excitation and emission wavelengths of 562/40 and 594 nm, respectively.

2.5. Quantification of PHB with HPLC

PHB quantification was determined by measuring the absorbance of crotonic acid at 215 nm using HPLC. The pellet was harvested from 20 mL of cell culture by centrifugation (15,317× g, 10 min, 4 °C) and washed twice with equal volumes of acetone and ethanol. Conversion of the PHB to crotonic acid was achieved by digesting the pellet in 10 mL of concentrated sulfuric acid (Merck, Darmstadt, Germany) for 30 min at 105 °C. Following digestion, the samples were diluted with nanopure H_2_O at a volume ratio of 1:10 and filtered using 0.22 μm filters. The filtered samples were then analyzed using an Agilent 1260 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA). The samples were loaded onto a reversed-phase InfinityLab Poroshell 120 EC-C18 column (4 µm pore size, 4.6 × 150 mm, Agilent Technologies, Santa Clara, CA, USA) and eluted with 85% (v/v) of a 10 mM KH_2_PO_4_ (pH = 2.3) buffer and 15% (v/v) acetonitrile (Fisher Scientific, Portsmouth, NH, USA) at a flow rate of 0.5 mL/min and a temperature of 30 °C. Crotonic acid in the samples was detected by a diode array detector at 215 nm and quantified based on a standard curve.

2.6. Examination of the Utilization of Lactose by Pseudomonas sp. phVD1

First, 1 mL of the cell cultures that contained pure SCW and pre-treated SCW without additives was transferred to a 2 mL Eppendorf tube and centrifuged at 10,000× g for 10 min. Samples were collected at 0 h, 72 h and 144 h of growth. Then, 500 μL of the supernatants was mixed with 600 μL of deuterated water containing 0.5% trimethylsilylpropanic acid (TMSP) and sonicated for 10 min. After solubilization, the suspension was carefully transferred into 5 mm NMR tubes (Deutero GmbH, Kastellaun, Germany), where the analysis was performed directly in a 500 MHz NMR model Bruker DPX-500 (Bruker, Billerica, MA, USA) to avoid solid precipitation. The number of scans and dummy scans were 128 and 4 respectively, at a temperature of 298 K, using standard pulse programs from Bruker libraries, with pre-processing in the Topspin 4.0 (Bruker, Billerica, MA, USA) software. The results were then analyzed using the Chenomx NMR Suite version 10.1, where metabolites were automatically identified using the library of over 300 available metabolites. Finally, statistical analysis was performed in BioStatFlow 2.9.6. During processing in BioStatFlow 2.9.6., PCA and PLS-DA analysis and a Mean Comparison Test were performed.

2.7. Characterization of PHB by NMR Analysis

The PHB granules were isolated according to the protocol of Geladas et al. [29]. The PHB samples were dissolved in 600 mL of deuterated chloroform (CDCl_3_) and transferred into 5 mm NMR tubes (Deutero GmbH, Kastellaun, Germany) after dissolution via sonication. Experiments were performed on a Bruker DPX-300 spectrometer (Bruker, Billerica, MA, USA) at 298 K using standard Bruker pulse program libraries. Spectral processing and analysis were performed using TopSpin 4.0 software (Bruker, Billerica, MA, USA). All chemical shifts reported are referenced to the residual chloroform peak (δ 7.26 ppm).

3. Results and Discussion

3.1. Growth of Pseudomonas sp. phVD1 in High Salinity

The main by-products of the cheese making process are cheese whey (CW) (also called primary cheese whey) and SCW [31]. SCW is characterized by high concentrations of organic load and significant salinity and represents over 90% of the original cheese whey used in the process [31]. Due to the high salinity, SCW is more difficult to process and has higher disposal costs than CW [32]. According to the growth curves concerning the development of the WT in high salinity conditions (Figure 1), we observed a delay in growth compared to growth in the presence of succinate alone. This may be due to the longer adaptation time required by the bacterium due to salt stress [33]. As the strain was able to grow at NaCl concentrations of up to 2 M, which are substantially higher than the salinity of SCW, its growth had to be evaluated as well, using SCW as the carbon source. Pseudomonas sp. phVD1 has the highest tolerance to salt among the strains of Pseudomonas when compared to other strains of the genus Pseudomonas [34].

3.2. Growth of Pseudomonas sp. phVD1 in SCW

CW and SCW are complex, non-sterile and often variable by-products, with high microbial loads, and their direct use in both laboratory and industrial fermentations may therefore be challenging [35]. A number of studies reporting PHA production from CW or SCW have actually been carried out on their derivatives, which have undergone a series of pre-treatments, rather than CW and SCW themselves [35]. A widely used pre-treatment of liquid CW and SCW is the removal of most of its proteins and other solids by heat treatment, centrifugation and filtration, producing a supernatant that retains most of the CW and SCW lactose [36].

Lactose, at a concentration of approximately 3–6%, is the major carbohydrate in SCW [23]. Most Pseudomonas species are unable to metabolize lactose. To date, only a few strains of Pseudomonas have demonstrated the capacity to utilize lactose or whey waste as a carbon source for growth [24,37,38]. As mentioned in the case of the most widely studied strain, Pseudomonas putida KT2440, lactose can only be utilized as a carbon source through genetic modification. The aim of this is to expand the catabolic profile of the organism [39]. Pseudomonas sp. phVD1 was reported to use monocyclic aromatic compounds as carbon sources [40]. More recently, grape pomace, which is rich in sugars and polyphenolic compounds, has been biodegraded using this strain and two mutant strains [10]. No information on the potential direct lactose-converting ability of Pseudomonas sp. phVD1 is available to date. Similar to other Pseudomonas strains, the genome of Pseudomonas sp. phVD1 reveals the absence of the β-galactosidase gene [41]. For this reason, we proceeded with hydrolysis using β-galactosidase to use glucose and galactose derived from the enzymatic hydrolysis of SCW lactose (see Section 2).

We investigated three different conditions for both of the SCW supernatants. According to the growth curves, the wild type, ∆phaZ and ∆phaR showed a long adaptation time compared to other carbon sources and high growth rates when using SCW and hydrolyzed SCW as carbon sources (Figure 2 and Figure 3). Further, the results showed that individual or combined addition of phosphate buffer, ammonium chloride and trace elements did not lead to remarkable cell growth. Biomass production was observed in all cases, but the ability of the bacterial strains to grow without dilution and in the absence of nutrients represents the most promising aspect. Although the growth of the WT strain in pre-treated SCW is higher (Figure 2), the ability of the strain to grow in untreated SCW provides an opportunity for biotechnological applications using an inexpensive carbon source.

3.3. Utilization of Lactose by Pseudomonas sp. phVD1

The ability of the Pseudomonas sp. phVD1 strains to utilize the carbon source present in the both SCW supernatants was investigated by ^1^H NMR. Multivariate analysis was performed in order to investigate the overall metabolomic shifts of the three different timepoints in each type of samples. Principal Component Analysis (PCA), shown in Figure S1 (Supplementary Materials), revealed partial clustering of samples based on both treatment and incubation time. Some overlap was observed at 0 h for both types of samples but at 72 h and 144 h, clear trends of separations were shown, indicating time- and treatment-dependent metabolic changes. KODAMA-PLS-DA was also performed, in order to increase group separation and visualize distinct changes (Figure S1, Supplementary Materials). The results demonstrated enhanced separation between SCW and pre-treated SCW samples, in agreement with the suggestion that the pre-treatment of SCW leads to metabolic changes in Pseudomonas sp. phDV1 over time. More specifically, in the case of the SCW, we found a decreasing amount of six compounds during the growth period of the cultures (Figure 4). Three carbohydrates, namely, lactose, lactulose and arabinose, are metabolized by the strain. Three amino acids, hydroxlysine, proline and threonine, were also found to be metabolized by the strain. While lactose is milk sugar, lactulose is an isomerization product of lactose during the heat treatment of milk in pasteurization and sterilization [42].

In the case of pre-treated SCW, we found more metabolites with a statistically significant difference, because β-galactosidase hydrolyses, not only lactose but also other β-galactosides to monosaccharides [43]. We observed a reduction in both β-galactosidase products, glucose and galactose (Figure 5). While the strain is known to metabolize glucose, this is the first time that galactose metabolism has been demonstrated [44]. In addition, the catabolism of glucose, and of glucose derivates such as glucoronate and gluconate was also observed. The glucuronic acid pathway is a quantitatively minor pathway of glucose metabolism, providing biosynthetic precursors and interconverting some less common sugars to those that can be metabolized [45]. Further we observed degradation of arabinose, similar to untreated SCW. We also found differences in metabolites of energy and amino acid metabolism, namely, lactate, citrate, 3-amino isobutyrate, methyl glutarate and hydromethyl glutarate (Supplementary Materials, Figure S2).

Organic nitrogen, which makes up a high proportion of total nitrogen, and inorganic phosphorus in SCW increase the pollutant load of this industrial waste. In order to further assess the remediation potential of Pseudomonas sp. phDV1, the removal of protein and phosphorus from SCW was determined for the WT strain. The protein estimation over different time points of Pseudomonas sp. phDV1 grown in SCW and pre-treated SCW shows no changes in the protein content of the supernatants. These results are an indication that the strain is not able to metabolize whole proteins, although it can metabolize free amino acids, as shown by the NMR analysis. At the same time, the total inorganic phosphorus content of both untreated and pre-treated SCW was shown to significantly decrease over time, a very promising result concerning the bioremediation ability of Pseudomonas sp. phDV1 (Supplementary Materials, Table S1).

3.4. PHB Production from SCW

The ability of Pseudomonas sp. phDV1 wild type and the mutants ∆phaZ and ∆phaR to produce PHB from grape pomace was previously reported by Drakonaki et al. [10]. In this study, we are investigating the ability of the wild type and different mutants of this bacterium to use different waste materials from the cheese industry as cheap carbon sources to produce PHB. We tested SCW treated and untreated with β-galactosidase, without any supplements for growing. Figure 6 shows a microscope image of wild-type cells stained with Nile Red 72 h and 144 h after culture, showing the production of PHB within the cells. Both forms of SCW can be used by the strain to produce PHB (Figure 6). Although Nile Red staining is only a qualitative method, it allows the detection of PHB granules after 144 h cultivation of Pseudomonas sp. phDV1 in both forms of SCW. In addition, the light microscopy results indicate that the ∆phaZ and ∆phaR mutants were also able to produce PHB using SCW and pre-treated SCW as carbon sources (Supplementary Materials, Figures S3 and S4).

To confirm these qualitative findings, the actual concentration of PHB produced by the Pseudomonas sp. phDV1 strains grown in SCW and pre-treated SCW was quantified by HPLC. It is clear from the comparison of PHB production and the growth time of bacterial strains that its production increases with growth time in all carbon sources (Table 1). At the same time, the bacterial strains show the highest PHB yield when pre-treated SCW is used as a carbon source, rather than SCW. When SCW is used, all bacterial strains exhibit similarly low PHB production, ranging from 0.7 to 2.9 g/L. However, a significant change occurs when pre-treated SCW is used, with PHB production increasing over 144 h. The ∆phaZ mutant produced 0.83 g/L, the WT produced 3.6 g/L and the ∆phaR mutant produced 7.26 g/L.

According to Geladas et al., growth of Pseudomonas sp. phDV1 in M9R medium resulted in a PHB content of 0.03% of the total dry weight cells [29]. This condition was therefore used as the control for comparison with the results obtained under the tested conditions. Comparing the PHB production of the strains with the reported value of PHB production using grape pomace or different concentrations of phenol, we observed a higher PHB production using SCW and pre-treated SCW as a carbon source [10]. The ∆phaR mutant producing the most PHB in relation to other strains indicates the need to create genetically modified strains to improve PHB production [10,12,17]. When we compare the PHΒ production performance of Pseudomonas sp. phDV1 with that of the classic PHΒ producer Cupriavidus necator, we observe that it lags behind the latter but is much higher than that of other Pseudomonas strains [46,47,48]. Although the ∆phaR mutant produces most of the PHB, the wild type’s ability to produce around a third of the PHB directly from SCW reduces waste treatment and PHB production costs. The ability of Pseudomonas sp. phVD1 to grow in SCW and pre-treated SCW by metabolizing the carbohydrates to PHB makes it a promising candidate for SCW bioremediation.

3.5. Characterization of PHB by NMR Analysis

In this work, ^1^H-NMR analysis was carried out to characterize the isolated polymers. The PHB granules were isolated from cells grown under SCW as a carbon source. To overcome signals arising from impurities in the sample, we recorded a 2D heteronuclear ^1^H-^1^H gCOSY NMR experiment of the isolated PHA material, depicted in Figure 7, which shows correlations between neighboring protons connected via scalar J couplings. The gCOSY 2D NMR spectrum is similar to the reported 2D NMR spectrum of the isolated PHB granules and coincides exactly with the chemical shift reported for the methyl protons (1) of PHB [8,29]. To confirm the presence of PHB in the ΔphaR and ΔphaZ mutants, the isolated PHB granules were investigated using ^1^H-NMR. The presence of PHB was confirmed in both strains by gCOSY 2D NMR spectra (see Supplementary Materials, Figure S5). The ability of the Pseudomonas sp. phDV1 strain, as well as the ΔphaR and ΔphaZ mutants, to produce PHB has been confirmed previously [8,10,29]. This study investigates whether these three strains can use waste from the cheese industry as a carbon source to produce PHB. All strains were able to produce PHB from SCW without treatment, indicating their adaptability in the bioremediation of SCW.

4. Conclusions

SCW can be used as an alternative, sustainable medium for cultivating Pseudomonas sp. phDV1 strains. The suitability of this food waste has been tested in both hydrolyzed and non-hydrolyzed forms. The biomass obtained from the hydrolyzed SCW was higher than that obtained from the other formulated media. The carbon hydrate profile was affected in both SCW media, with a lower profile obtained after treatment of the SCW with the strains. Furthermore, the Pseudomonas sp. phDV1 strains were shown to be capable of producing PHB using both SCW media. We obtained the highest yield after cultivating for 144 h. Future research should focus on optimizing culture conditions and process parameters to enhance biomass formation and PHB accumulation by Pseudomonas sp. phDV1 when grown on SCW-based media. Particular attention should be given to cultivation modes (e.g., fed-batch or continuous systems) to assess productivity improvement and process robustness. Scale-up studies would also be necessary to evaluate the feasibility of large-scale PHB production using dairy waste substrates. This study demonstrates, for the first time, the feasibility of using SCW as a low-cost and sustainable cultivation medium for Pseudomonas sp. phDV1 strains in PHB production. Unlike conventional approaches relying on refined substrates, this work evaluates both pre-treated and untreated SCW, highlighting the effect of waste pre-treatment on biomass formation, carbohydrate utilization, and PHB yield. The findings provide new insights into the direct valorization of food waste streams for biopolymer production and contribute to the development of economically and environmentally sustainable PHB production processes.