Bioplastic Production in Circular Economy Paths with Glycerol and Whey

1. Introduction

The annual production of plastics has doubled from 234 million tons (Mt) in 2000 to 460 Mt in 2019. Moreover, in the same period, plastic waste has more than doubled from 156 Mt in 2000 to 353 Mt in 2019 (1).

Some basic actions are needed to reduce the impact of plastic pollution. Some of the most important are:

1) To promote the circular economy, including the design of reusable and recycled products.

2) To increase bioplastics production from renewable feedstocks by utilizing residual byproducts as raw materials (2).

This work examines the use of whey and glycerol as substrates for bioplastic production. Whey is a byproduct of the dairy industry, and glycerol is the main byproduct of biodiesel manufacturing.

Biopolymers produced by microorganisms are easily biodegraded by them and within the digestive systems of higher animals, including humans. The PHAs, a type of biopolymer, are natural polyesters made up of various D-hydroxyalkanoic acids, starting with polyhydroxybutyrate (PHB). PHAs are generated by a range of prokaryotes and some eukaryotic organisms, such as plants, animals, and fungi.

Figure 1 displays the chemical structure diagram of PHAs. Depending on the bacterial strain, carbon source, and growth conditions, molecular weights can vary from tens of thousands to hundreds of thousands (2). All PHAs originate from renewable materials, are genuinely biodegradable, and exhibit high biocompatibility (3). They hold great promise for food packaging and compare favorably to plastics like polyethylene and polypropylene.

Some bacterial and fungal species store PHA as a carbon and energy source to survive during starvation conditions. PHA accumulates in cells when there is an excess of a carbon source in the culture medium, but bacterial growth is limited by another nutrient; these limiting nutrients can include nitrogen, sulfur, phosphate, iron, magnesium, potassium, or oxygen (4).

One of the most promising bioplastics is polyhydroxybutyrate (PHB), which belongs to the PHA family of bioplastics, where the molecular weight coefficient x (shown in Figure 1) equals 3. It is a non-toxic polymer produced and stored by certain bacterial and fungal genera, such as Alcaligenes, Azotobacter, Bacillus, Beijerinckia, Pseudomonas, Rhizobium, and Rhodospirillum (5). The PHB molecules in the cytoplasm are crystalline, forming granules about 0.5 µm in diameter. These granules can be isolated or extracted using solvents. Some researchers believe that PHB has significant potential to replace high-density polyethylene (PE) and polypropylene (PP) due to its chemical and mechanical properties (see Table 1) and with proper disposal practices to establish a fully circular plastic lifecycle (6). However, its current use is limited by high production costs, low yields, manufacturing technology, and downstream purification challenges. Therefore, it is necessary to study and optimize conditions that enhance its accumulation within cells, develop methods for cell disruption without damaging the PHB molecules, and improve purification from the culture media.

PHB biodegradation occurs within a reasonable timeframe when it contacts degrading microorganisms in biologically active environments, such as soils, freshwater, and aerobic and anaerobic composting (7).

Metabolic Pathways

The production of PHB using bacteria has been extensively documented in the literature. A variety of raw materials have been reported, including simple sugars like glucose and sucrose, vegetable oils, agro-industrial waste (molasses, whey, fruit peels), crude glycerol, and even methane and methanol.

Whey as Substrate

The main compound of whey (excluding water) is lactose. Lactose catabolism begins with the transport of lactose into the cell through the cell’s membrane via a lactose-specific phosphotransferase system (8,9). Figure 2 illustrates the steps of the lactose metabolic pathway leading to PHB production, including the enzymes that catalyze each reaction.

Glycerol as Substrate

This study examines the production process using crude glycerol, a byproduct of biodiesel manufacturing. The metabolic pathway that converts glycerol to PHB is illustrated in Figure 3 (10).

2. Results and Discussion

2.1. Experiments with Whey

The experimental tests were conducted in triplicate; the results shown in Figure 4 are the averages of the three measurements for each experiment. At a pH level of 8, the production of PHB was 2.35 times higher than at pH 3, 1.35 times higher than at pH 6, and 1.24 times higher than at pH 7. Therefore, a basic pH promotes PHB production because it stresses the cells of Bacillus megaterium.

That makes sense because B. megaterium is a neutrophilic bacterium, and its optimal growth pH ranges from 6.5 to 7.5. However, at acidic pH levels, these bacteria do not accumulate PHB in their cytoplasm. This occurs because low pH levels denature certain enzymes involved in the PHB production pathway, such as β-ketothiolase [β-ket], Acetoacetyl-CoA reductase [A-CoA-R], and β-PHB synthase [PHB-S].

Table 2 presents results from other studies reported in the literature on Bacillus megaterium and various substrates for PHB production. This table shows that one of the key issues researchers aim to address is finding low-cost raw materials or, ideally, residues from other industries such as biodiesel production (glycerol), cacao liquid wastes, and oil palm empty fruit bunches (waste from oil palm cultivation). The PHB concentration observed in this work falls within the same range as reported in other similar studies. The PHB concentration at each hour of the bioreaction is the most comparable variable across different studies.

2.2. Experiments with Glycerol

Figure 5 shows solid PHB obtained after the recovery process with B. subtilis [A] and B. megaterium [B]. It can be observed with the naked eye that the amount of PHB obtained with B. megaterium is greater than that obtained with B. subtilis.

Figure 6 and Figure 7 show the experimental results of glucose consumption, biomass growth, and PHB production with B. subtilis. Figure 6 shows the results of experiments carried out without trace elements, and Figure 7 shows the results with trace elements. As can be seen without trace elements (Figure 6), there are three phases of biomass growth. The first one [I] is the exponential phase from time 0 to 12 hours; the stationary phase begins at 12 hours and ends at 60 hours; then the death phase begins. Glucose is not totally consumed in these experiments; the percentage of glucose consumed at the end of the exponential growth phase [${{\mathit{C}}_{\mathit{S}}}^{\mathit{E}}]$was 16.4% [Eq. 1], and at the end of the experiment [${{\mathit{C}}_{\mathit{S}}}^{\mathit{F}}]$was 49.6%, calculated with Eq. 2, where ${\mathit{C}}_{\mathit{S}\mathit{i}}$ is the glucose initial concentration, and ${\mathit{C}}_{\mathit{S}\mathit{f}}$ is the glucose concentration at the end of the exponential growth phase.

The experimental yield coefficient biomass/substrate, calculated at the end of the exponential growth phase, was 0.38 grams of cell produced per liter per gram of glucose in the phase [${{\mathit{Y}}_{{\displaystyle \raisebox{1ex}{$\mathit{X}$}\!\left/ \!\raisebox{-1ex}{$\mathit{S}$}\right.}}}^{\mathit{E}}$], see Eq. 3. Where ${\mathit{C}}_{\mathit{S}\mathit{f}\mathit{e}}$ is the glucose concentration at the end of the exponential growth phase.

Part of the substrate consumed during the stationary growth phase is used to produce PHB, while the rest supports cell maintenance. That is because, even though biomass stopped increasing at hour 12 of the experiment, the PHB concentration continued to increase, quickly until the end of the stationary growth phase and slowly thereafter. The yield coefficient product / substrate calculated at the maximum PHB concentration [${{\mathit{Y}}_{{\displaystyle \raisebox{1ex}{$\mathit{P}$}\!\left/ \!\raisebox{-1ex}{$\mathit{S}$}\right.}}}^{\mathit{M}}$] obtained was: 0.45, see Eq. 5. Where ${\mathit{C}}_{\mathit{P}\mathit{f}\mathit{m}}$ is the maximum PHB concentration, and ${\mathit{C}}_{\mathit{S}\mathit{f}\mathit{m}}$ is the glucose concentration at time equal to maximum PHB concentration.

$$\% {C_S}^E={\displaystyle \frac{C_{Si}-C_{Se}}{C_{Si}}}100$$

(1)

$$\% {C_S}^F={\displaystyle \frac{C_{Si}-C_{Sf}}{C_{Si}}}100$$

(2)

$${Y_{{\displaystyle \raisebox{1ex}{$X$}\!\left/ \!\raisebox{-1ex}{$S$}\right.}}}^E={\displaystyle \frac{C_{Sfe}}{\left(C_{Si}-C_{Sfe}\right)}}$$

(3)

$${Y_{{\displaystyle \raisebox{1ex}{$P$}\!\left/ \!\raisebox{-1ex}{$S$}\right.}}}^E={\displaystyle \frac{C_{Pfe}}{\left(C_{Si}-C_{Sfe}\right)}}$$

(4)

$${Y_{{\displaystyle \raisebox{1ex}{$P$}\!\left/ \!\raisebox{-1ex}{$S$}\right.}}}^M={\displaystyle \frac{C_{Pfm}}{\left(C_{Si}-C_{Sfm}\right)}}$$

(5)

Figure 7 displays the experimental results for B. subtilis in a culture medium with trace elements. The same three growth phases are observed, but the durations of each phase differ from those in the previous case. The exponential growth phase lasts until hour 33 of the experiment. Then, a stationary phase occurs until hour 48, followed by the death phase.

Glucose is not completely consumed in these experiments, but the final concentration is lower than in the previous set. The percentage of glucose used up at the end of the exponential growth phase was 42.8%, and at the end of the experiment, it was 64.8%, as calculated using Equations 1 and 2, respectively.

The yield coefficient of biomass substrate during the exponential growth phase was 0.34, dropping to 0.14 by the end of the experiment. Conversely, the yield coefficient of product substrate was 0.07 at the end of the exponential growth phase and increased to 0.45 by the experiment’s conclusion. This is because a major portion of PHB is produced after the exponential growth phase has ended. The yield coefficient of PHB/substrate was 0.22 at the end of the exponential growth phase and rose to 0.61 by the end of the experiment, calculated using the same equations as before.

Figure 8 shows the experimental results for B. megaterium in a broth without trace elements. The same three growth phases were observed, with different durations for each. The exponential growth phase ended at hour 18, the stationary phase concluded at hour 40, and the death phase persisted until the end of the experiments.

The maximum biomass concentration was 1.9 g L^-1, and the maximum PHB concentration reached was 3.1 g L^-1. B. megaterium did not consume all the glucose in the broth; the glucose concentration at the end of the experiment was 11 g L^-1. The yield coefficient of biomass/substrate, at the end of the exponential growth phase, was 0.3, and at the end of the experiment was 0.026. The yield coefficient product/substrate at the end of the exponential growth phase was 0.2633, and the yield coefficient at the end of the experiment was 0.8.

Figure 9 displays the experimental results for B. megaterium in a culture medium with trace elements. In this case, glucose was also not fully consumed. The biomass/substrate yield coefficients at the end of the exponential growth phase and at the end of the experiment were 0.2 and 0.04, respectively. The yield coefficients of PHB substrate at the end of the exponential growth phase and at the end of the experiment were 0.18 and 0.42, respectively.

Figure 10 and Figure 11 display the comparison of yield coefficients for biomass/substrate and PHB/substrate, respectively, across the four experiments conducted. In Figure 10, the biomass substrate yield coefficients are depicted in red for the culture medium without trace elements and in blue for the medium with trace elements. As demonstrated, B. subtilis produced more cells per gram of substrate consumed in the medium lacking trace elements than in the one containing them, and it achieved the highest biomass-substrate yield coefficient among all the experiments.

The same trend was observed with B. megaterium: the biomass/substrate yield coefficient was higher in media without trace elements compared to those with trace elements. It is important to note that the C:N ratio used in all experiments was significantly higher than the optimal C:N ratio for growth. The optimal C:N ratio for B. subtilis ranges from 8:1 to 11.5:1 (19), (20), and for B. megaterium, it ranges from 10:1 to 20:1 (21). In these experiments, the C:N ratio exceeded 200.

Regarding PHB substrate yield coefficients, B. megaterium growing in a culture medium lacking trace elements produced the highest amount, as shown in Figure 11. In this experiment, the PHB production was 1.8 times greater than the production obtained in second place (B. subtilis without trace elements), 1.9 times greater than the production of B. megaterium with trace elements, and more than 6 times greater than the production with B. subtilis with trace elements.

In three of the experiments (1, 3, and 4), the amount of substrate used by cells to produce PHB was greater than the amount used for growth. Experiments conducted without TE increased PHB production in both strains. As previously reported by Gómez Cardozo et al. (22), B. megaterium is more efficient at producing PHB than B. subtilis.

To achieve short exponential growth phases and extended stationary phases, optimize PHB production. Trace element concentration has a greater impact on PHB production than the C:N ratio when the C:N ratio is too high.

The characterization of PHB was performed by Fourier-transform infrared spectroscopy (FTIR). The characteristic signals of PHB were identified in both Bacillus strains. One of the most important signals is the signal observed in the 1700–1750 cm^-1 region, characteristic of the carbonyl group (>C=O) present in PHB, as shown in Figure 12. This signal is very intense in the PHB spectrum produced by B. subtilis. Another characteristic signal was observed in the 1250-1150 cm^-1 range, corresponding to esters (C₂H₇C₆). In the 3000-2850 cm^-1 range, the characteristic signals of the CH₃ and CH₂CH₂ groups are visible in Figure 12 (23).

Figure 13 shows the SEM micrographs of the PHB produced, recovered, and purified from both strains, where homogeneous and amorphous masses with smooth textures are observed (23).

Image A displays PHB from B. megaterium observed at 1000x magnification. Amorphous structures with irregular surfaces and some aggregation are visible. The scale indicates a reference of 100 µm, suggesting the PHB aggregates are quite large.

Figure 13B displays the PHB produced by B. subtilis, visualized at a magnification over 2000X, showing a morphology with denser, rougher structures. The 20 µm scale indicates that the fragments observed are smaller than those in Figure 13A.

2.3. Material Balance

A carbon mass balance was conducted to determine the carbon dioxide produced by cell respiration and to investigate why glycerol was not consumed in the experiments. The initial carbon dioxide concentration in the culture media was set to 0.001 g L-1. This value represents the average CO2 concentration in water at 2,440 meters altitude, with an atmospheric pressure of 0.77 atm and a temperature of 25 °C, which are the conditions in Mexico City. Figure 14 illustrates the time-dependent carbon dioxide production for each experiment.

The yield coefficients of carbon dioxide to glucose obtained are shown in Table 3 for each experiment, compared with the coefficients reported in literature (18,24). All of these values are within the range reported in the literature. Therefore, glycerol was not consumed in the experiments.

2.4. Model

The growth kinetics of B. megaterium and B. subtilis were modeled with the logistic model. The production of PHB for both strains was simulated using the Luedeking-Piret model, with an initial product concentration [PHB] set to zero. Using the experimental results from all batch experiments in the exponential growth phase, kinetic parameters were estimated with the Marquardt non-linear estimation method (25).

Comparisons of model and experimental results for all cases are shown in Figure 14. Graph A shows the results for B. subtilis grown in culture media without trace elements. Graph B shows the results for B. megaterium with trace elements in the culture media. Symbols represent experimental data, and lines represent model results. As shown in Figure 14, the model fits the experimental data reasonably well, with parameters obtained using the Marquardt algorithm.

Table 4 shows the model results for parameters related to growth kinetics, PHB production, and substrate consumption using the models outlined in the Model section. As seen, among the maximum biomass concentration parameters, B. subtilis in a culture medium with trace elements had the highest x_max, followed by B. megaterium with TE, B. megaterium without TE, and B. subtilis without TE. Likewise, the yield coefficients for biomass production per gram of substrate were higher in experiments without TE, probably due to excess carbon relative to nitrogen.

Respect the comparison between the parameters α and β of the Ludeking-Piret model: in all cases, α is greater than β, because PHB is produced in the metabolic pathways of glycolysis and the Krebs cycle, which are part of the growth metabolic pathway.

The highest product yield coefficients were achieved without TE in the culture media, and B. megaterium produced more PHB per gram of substrate than B. subtilis.

The absence of trace elements in the culture media, specifically Fe, Mo, and Ni, inhibits the activity of nitrogenase and hydrogenase enzymes, redirecting reductive equivalents to the β-cetocetiolase → acetoacetyl CoA reductase → PHA polymerase pathway, which promotes PHB accumulation. The lack of Mg in the culture media has been shown to increase PHB production by up to 80%. Additionally, omitting trace elements from the culture medium reduces the cost of PHB production.

A light, basic pH level, between 7.5 and 8, enhances the activity of the PHA-polymerase enzyme because it influences the solubility of certain trace elements, decreasing the activity of some enzymes and increasing the activity of others involved in the PHB production pathway, as demonstrated by the experimental results with Whey.

3. Conclusion

This study shows the technical feasibility of using industrial by-products, especially residual glycerol and cheese whey, in circular economy pathways to produce polyhydroxybutyrate (PHB) through biochemical methods. Based on the experimental results, the following conclusions can be made:

• Effect of pH on Biosynthesis: The pH level of the culture medium was identified as a key factor directly affecting bioproduction yield. PHB production showed a positive correlation with increasing pH within the evaluated range, rising from 2.52 g/L at pH 3 to a maximum at pH 8.
• Effect of nitrogen and carbon sources: The lack of nitrogen and the excess of carbon sources promote PHB production with B. megaterium and B. subtilis.
• Effect of trace element deficiency: The absence of trace elements in the culture media increases PHB production and the product-to-substrate yield coefficient.
• PHB production after the exponential growth phase: In all experiments with glycerol, PHB production continued during the stationary and death phases. This is because trace elements like magnesium and calcium, which are involved in enzyme activity, return more slowly to the metabolic pathways responsible for growth and energy generation.
• Waste Valorization and Sustainability: Using glycerol and whey as sources of carbon and nitrogen not only cuts production costs related to pure substrates but also offers a sustainable method for managing agro-industrial waste. This strategy advances the shift toward a circular bioeconomy by converting polluting waste into high-value, biodegradable bioplastics.
• This work shows that using residual material, suppressing certain trace elements, maintaining a high C:N ratio, and controlling pH can create cost-effective and environmentally friendly processes to produce bioplastics like PHB.

4. Materials and Methods

In this part of the study, Bacillus megaterium was used to produce PHB in the laboratory with whey as the substrate, and both B. megaterium and B. subtilis were employed to produce PHB using glucose and glycerol as carbon sources. Glycerol is a primary byproduct of biodiesel production, while whey is a byproduct of the dairy industry. Some aspects considered were:

• Pyruvate produces ethanol or lactate under anaerobic conditions in a nutrient-balanced culture medium.
• Under aerobic conditions, pyruvate is converted into oxaloacetate and enters the Krebs cycle to produce energy and support cell growth, using a nutrient-balanced culture media.
• PHB production is increased in culture media with excess carbohydrates and a deficiency of nutrients such as nitrogen, phosphorus, oxygen, sulfur, or trace elements. When cells are cultured in media containing excess glucose, sucrose, lipids, glycerol, and other carbon sources, Acetyl CoA accumulates. The lack of nitrogen, phosphorus, oxygen, and sulfur halts the synthesis of nucleic acids and proteins, leading to a decrease in cell growth.
• The excess of NADPH and Acetyl Co-A increased the synthesis of 3-hydroxybutyryl-CoA, the monomer of PHB, because high levels of NADPH and NADH inhibit citrate synthase in the TCA cycle, which ensures the availability of acetyl CoA to connect with 3-Phosphoglycerate.
• Cells form PHB’s conglomerate in the cytoplasm as a future source of carbon and energy.

All experiments took place in the Process Analysis Laboratory at the Metropolitan Autonomous University campus Azcapotzalco. Two raw materials were tested: whey from a cheese factory and glycerol from biodiesel production.

Both Bacillus species were grown in flasks containing culture medium containing 15 g L^-1 of meat peptone, 1.5 g L^-1 of yeast extract, and 5 g L^-1 of NaCl at 30 °C and 150 rpm for 24 hours to obtain the inoculum.