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Article

Synthesis and Properties of Polyhydroxyalkanoates on Waste Fish Oil from the Production of Canned Sprats

by
Natalia O. Zhila
1,2,*,
Kristina Yu. Sapozhnikova
1,2,
Evgeniy G. Kiselev
1,2,
Ekaterina I. Shishatskaya
1,2 and
Tatiana G. Volova
1,2
1
Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, 50/50 Akademgorodok, Krasnoyarsk 660036, Russia
2
Basic Department of Biotechnology, School of Fundamental Biology and Biotechnology, Siberian Federal University, 79 Svobodnyi Av., Krasnoyarsk 660041, Russia
*
Author to whom correspondence should be addressed.
Processes 2023, 11(7), 2113; https://doi.org/10.3390/pr11072113
Submission received: 14 June 2023 / Revised: 6 July 2023 / Accepted: 13 July 2023 / Published: 15 July 2023
(This article belongs to the Special Issue Production, Extraction, Analysis and Degradation of Bioplastics)
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Abstract

:
The waste fish oil obtained from Baltic sprat waste in the production of canned sprats was studied as a sole carbon substrate for PHA synthesis by the wild-type strain Cupriavidus necator B-10646. Sprat oil contained a set of fatty acids with a chain length from C14 to C24, saturation factor 0.63, and provided bacterial growth and PHA synthesis. Bacteria metabolized fatty acids unevenly utilizing polyenoic acids and not using monoenoic and saturated acids. The bacterial biomass yield and the intracellular polymer concentration were 6.5 ± 0.5 g/L and 65 ± 5% by fed-batch culture in flasks. The synthesized PHAs were three-component copolymers with a predominance (97–98 mol.%) of 3-hydroxybutyrate monomers and small inclusions of 3-hydroxyvalerate and 3-hydroxyhexanoate; the ratio of monomers changed slightly depending on the sprat oil concentration. The series of samples had a temperature (Tmelt) of 158–165 °C, a molecular weight (Mw) of 540–760 kDa, and a degree of crystallinity (Cx) of 66–72%. For the first time, the waste fish oil from the production of sprats studied as a carbon substrate is a promising, affordable, and renewable substrate for PHA biosynthesis.

Graphical Abstract

1. Introduction

Degradable polyhydroxyalkanoates (PHAs) are viable candidates for the gradual replacement of petroleum-derived synthetic plastics, the widespread use of which is a global environmental problem [1]. The combination of useful properties makes PHA one of the most promising materials of the 21st century for applications in various fields, from utilities and agriculture to pharmacology and biomedicine [2]. The key and acute problem of success in conquering the market with degradable PHAs is to reduce their cost by attracting available carbon raw materials, whose share in the cost structure can be up to 45–50%.
Potentially, various substrates can serve as raw materials for PHA synthesis. Among them are individual compounds (sugars, organic acids, alcohols, methane, carbon dioxide and its mixtures with hydrogen, glycerol, etc.) [2,3,4,5] and also, which is especially significant, waste from the technosphere (hydrolysates of plant raw materials of various origins, waste from the food, pharmaceutical, alcohol, pulp, and paper industries, etc.) [5]. The possibility of attracting waste for the production of target products, including PHA, is the contribution of biotechnology to solving the problem of biosphere clutter and increasing the efficiency of industrial production. Therefore, PHA has an undoubted potential for implementing the principles of “The Circular Economy” [6].
Large-tonnage fat-containing wastes of the food industry, which generate up to 29 million tons of waste fat annually, the disposal of which is expensive, are considered as a promising substrate for obtaining target biotechnology products [7]. Waste fish oil (WFO) has found application in the production of biodiesel, and the synthesis of degradable plastics may become a new area of application. Interest in lipid substrates for PHA synthesis has been formed relatively recently. In research, animal fat by-products, including used cooking oil, processed low-grade animal fats [3,8,9], and various vegetable oils [10].
The problem of utilization of fat-containing waste is also relevant for the fish-processing industry, where up to 50% or more of the fish raw material goes to waste during the processing, a significant part of which is carried away with wash water and creates environmental problems [11]. Relevant is the search for new waste-processing technologies not only for the routine production of fishmeal and industrial fat products [12] but also for more demanded products with high added value (fish oil, ω3-fatty acids, protein products, etc.).
The study of WFO as a substrate for the synthesis of PHA has begun relatively recently. A small number of research papers have been published, including the results of a study of PHA synthesis using hydrolyzed pollock oil [13], fatty acid extracts from fish-processing waste [14], spent fats from canned tuna [15,16], and fish cannery wastewater [17]. A series of works was published in Vietnam, in which the synthesis of PHA by various producers was studied: the halophilic bacterium Salinivibrio sp. M318 [18], isolates of the genus Ralstonia [19], and wild-type strain Cupriavidus necator H16 [20]. Using waste fish oil from various sources as a substrate, biomass yields and PHA yields were obtained from 5–10 g/L and 50–70% in flasks to 50–117 g/L and 82.9% in the fermenter. It is important to note that the theoretical yield of PHA on lipid substrates could be 0.7–0.8 g/g compared to 0.3–0.4 g/g on sugars [21]. Waste fish oil is a potentially new but little explored source of carbon feedstock that can become a large-scale and renewable substrate for biotechnology processes, including the production of PHA.
Large-tonnage fat-containing wastes are formed in the production of canned sprats with preliminary hot smoking of small herring fish. The main raw material for producing canned sprats is small sprat, the global catch of which is about 500,000 tons per year [22]. Removed smoked sprat heads contain up to 15% fat mass and 22% protein, as well as smoke components (mainly phenolic compounds and polycyclic aromatic hydrocarbons). Therefore, this source is not suitable for use in the composition of animal feed and aquaculture; it is taken to solid-waste landfills or burned, which makes it expedient to find solutions for the use of this waste fish oil.
This determined the purpose of this work: to assess the potential of waste fish oil in the production of canned sprats for the biosynthesis of polyhydroxyalkanoates.

2. Materials and Methods

2.1. Obtaining and Characterization of Sprat Oil

Sprat oil is obtained from waste products from the production of canned food “Sprats in oil” from the heads of smoked Baltic sprat (Sprattus sprattus balticus) in accordance with the state standard of the Russian Federation [23]. We obtain raw materials from the RosKon fish cannery (Kaliningrad region, Kaliningrad, Russia). The technological scheme is as follows: after washing the chilled or thawed fish, the carcasses are pierced onto metal rods and subjected to hot smoking in a tunnel oven at a temperature of 110–140 °C for 15–20 min. Smoke formation during smoking is carried out with sawdust of hardwood (alder, oak, birch without bark, etc.) in exothermic-type smoke generators, which provide sawdust pyrolysis at a temperature not exceeding 400 °C. After cooling the smoked fish, the heads are separated, crushed, and mixed with water in the ratio 1:1; heated to 90 °C and kept under stirring for 15–20 min; then centrifuged at 3000 g (Megafuge 1.0R, Thermo Fisher Scientific, Waltham, MA, USA). the fat fraction of the supernatant is separated from the non-fat fraction by decanting.

2.2. Analysis of the Chemical Composition of Sprat Oil

The total content of lipids, protein, and carbohydrates in sprat oil was determined by conventional methods [24]. The fatty acid composition and FA dynamics during oil utilization by bacteria were analyzed. After methyl esterification with H2SO4:methanol (1:20) solution that lasted 2 h at 80 °C, fatty acid methyl esters (FAMEs) were analyzed using GC-MS (7890/5975C, Agilent Technologies, Santa Clara, CA, USA).
Acid number—the method is based on titration with a solution of sodium hydroxide [25]—and peroxide value—the method is based on titration with a solution of sodium thiosulfate of iodine, which is released during the interaction of fat peroxides with a solution of potassium iodide in the presence of glacial acetic acid [25]—were determined as indicators of the quality of fat.

2.3. PHA Producer Strain and Cultivation Technique

Polymers were synthesized using the wild-type Cupriavidus necator B-10646 strain, registered in the Russian National Collection of Industrial Microorganisms (VKPM) [26]. The strain is capable of synthesizing PHA from various carbon substrates; as a nitrogen source, the strain uses nitrates, ammonium salts, urea, and amino acids. The bacteria were grown in the mineral Schlegel medium [27], a strong phosphate-buffered solution. To prepare oil medium, distilled water containing Na2HPO4 (9.1 g/L) and KH2PO4 (1.5 g/L), and sprat oil were combined. Then, the medium was autoclaved, and MgSO4 (0.2 g/L), C6H5O7Fe (0.025 g/L), and trace elements were added from sterile stocks (3 mL per 1 L of the medium). The stock solution of trace elements was of the following composition (g/L): H3BO3—0.288; CoCl2·6H2O—0.030; CuSO4·5H2O—0.08; MnCl2·4H2O—0.008; ZnSO4·7H2O—0.176; NaMoO4·2H2O—0.050; NiCl2—0.008. Sprat oil served as the sole source of carbon; its concentration in the process of research and development of the regime of polymer synthesis varied from 10 to 40 g/L. NH4Cl was used as a nitrogen source.
Bacteria were cultivated in the mode of reserve PHA synthesis in a two-stage batch culture. At the end of the first stage (25–35 h), the nitrogen source concentration in the medium (initially 0.5 g/L) became a limiting factor limiting cell growth and synthesis of the main cellular macromolecules and stimulating the accumulation of PHA; at the second stage (after 35–40 h), the bacteria were in a nitrogen-free medium due to its exhaustion.
The cultivation of bacteria was carried out in glass flasks with a volume of 0.5–2.0 L, with a filling of 50% using thermostatically controlled shaker-incubators “Incubator Shaker Innova®” series 44 “New Brunswick Scientific” (New Brunswick Scientific, Edison, NJ, USA) at 200 rpm and 30 °C. Bacteria were cultured in a batch mode, observing the conditions developed earlier for PHA biosynthesis. The inoculum was prepared by resuspending the stock culture maintained on Schlegel agar medium at a temperature of 5 °C. The stock culture was grown in 0.5 L glass flasks under strictly sterile conditions. The inoculum was used to seed larger flasks. The starting concentration of cells in the medium was 0.1–0.2 g/L.
During the cultivation of bacteria in flasks, culture samples were periodically taken every 24 h for analysis. The concentration of ammonium nitrogen in the culture was measured by photometry of the supernatant of the culture medium with Nessler’s reagent, followed by quantitative determination of the nitrogen content according to the calibration curve. The concentration of sprat oil in media was measured as described previously [28]. The dynamics of cell growth in culture was detected by the optical density of the bacterial suspension at a wavelength of λ = 440 nm (UNICO 2100 photoelectrocalorimeter, Dayton, NJ, USA). The cell concentration (yield of bacterial biomass, X, g/L) was determined by centrifuging culture medium at 6000g (AvantyJ-HC centrifuge, BeckmanCoulter, Indianapolis, IN, USA) for 10 min and washing it twice with a mixture of distilled water and cold hexane. The bacterial biomass was dried at 105 °C for 24 h. Lipolytic enzyme activity was measured as described by Takaç and Marul [29].

2.4. Analysis and Properties of PHA

Intracellular PHA content was determined by analyzing samples of dry cell biomass and PHA samples isolated from cell biomass and purified to a homogeneous state. The purity of the polymer and copolymers was determined by chromatography of methyl esters of fatty acids after methanolysis of purified polymer samples using a 7890A chromatograph-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a 5975C mass detector (Agilent Technologies, Santa Clara, CA, USA).
The polymer was extracted from the cell biomass with dichloromethane; the resulting extract was concentrated on an R/210V rotary evaporator (Büchi, Flawil, Switzerland) and then precipitated with ethanol. Repeating the procedures of polymer dissolution and reprecipitation ensured the removal of impurities and obtaining homogeneous samples. The polymer samples were dried in a fume hood at room temperature for 72 h.
Molecular weight and molecular weight distribution of PHAs were examined using a size-exclusion chromatography (Agilent Technologies 1260 Infinity, Waldbronn, Germany) with a refractive index detector, using an Agilent PL gel Mixed-C column (weight average, Mw, and number average, Mn), and polydispersity (Ð = Mw/Mn) was determined. Thermal properties of the polymer were analyzed using a DSC-1 differential scanning calorimeter (Mettler Toledo, Schwerzenbac, Switzerland). Melting points were determined from exothermal peaks in thermograms using the STARe software. Thermal degradation of the samples was investigated using a TGA2 thermal analysis system (Mettler Toledo, Schwerzenbac, Switzerland). XRD structure analysis and determination of crystallinity of the samples were performed employing a D8 ADVANCE X-ray powder diffractometer (Bruker AXS, Karlsruhe, Germany). Methods for analyzing the physico-chemical properties of PHA have been previously described in detail [30].

2.5. Statistics

Statistical analysis of the results was performed by conventional methods, using the standard software package of Microsoft Excel v14.0. Each experiment was performed in triplicate. Arithmetic means and standard deviations were found.

3. Results and Discussion

For the first time, sprat oil, a fat-containing waste from the production of canned sprats, was studied as a carbon substrate for the synthesis of PHA. The choice of this raw material is due to the fact that canned sprats are produced in large volumes everywhere—in the Baltic, Black Sea, Caspian countries, the Faroe Islands, Norway, as well as in Russia, in which the production volume is more than 20,000 per year (RBC (https://www.rbc.ru/business/01/12/2020/5fc4ecea9a79471b9e84966f, accessed on 9 June 2023)).
At the same time, about 10–12 tons of fish waste per day is generated in the Kaliningrad region alone; up to 8–10 tons fall on sprat heads of smoked sprat and herring [31,32]. These wastes are practically not recycled, which led to their evaluation as a substrate for PHA synthesis.

3.1. Characteristics of Sprat Oil

Vegetable and animal oils derived from natural raw materials are mainly composed of triacylglycerols (TAGs) in which FAs are attached to the glycerol backbone. The composition of oils and their FAs depends on the source of production and the technology of extraction and purification [33]. The composition of fat from fish raw materials is determined by the type of fish, the specifics of trophism, and habitat. Sprat oil contains lipids (95%), proteins (4%), and carbohydrates (1%). An assessment of the quality of fat in terms of acid (AN) and peroxide numbers (PN) showed that the AN is 3.26 mg KOH/g, and the peroxide value is 6.63 mmol of active oxygen/kg. This meets the requirements of the Technical Regulations of the Eurasian Economic Union (TR EAEU 040/2016). The fat isolated from the heads of smoked sprat had a dark brown color and had a specific pronounced aroma characteristic of smoked fish, without negative organoleptic shades and signs of oxidative damage.
Quantitative data on the composition of fatty acids in sprat oil are presented in Table 1. In sprat oil, the amount of saturated and unsaturated fatty acids is 38.5% and 61.5%, respectively, with a saturation factor of 0.63. In the composition of fatty acids of sprat oil lipids (Table 1), 20 FAs with chain lengths from 14 to 24 carbon atoms were identified. Among the dominant FAs, palmitic (28.0%), oleic (25.3%), docosahexaenoic (16.7%), and eicosapentanoic (8.7%) acids were identified. The content of other C18 fatty acids (stearic, linoleic, and linolenic), as well as myristic, is much lower, at the level of 2.5–4.5%. In small amounts (1.1–1.5%), long-chain monoenoic fatty acids C20 and C24 are present. Minor FAs (less than 1% present in the oil) are mainly represented by saturated fatty acids C15, C20, and C22, as well as monoenoic C16:1 and C17:1.
Comparison of the FA composition of sprat oil with published data showed that hydrolysates of crude fat from pollock waste contained FAs with a C-chain length of 14 to 22 carbon atoms, a saturation factor of 0.35 [13] with a predominance of monounsaturated FA (28.0%) and FA with a length of C20 atoms (23.0%). Fats from fish solid waste, including scales and intestines collected from the local fish market of Bhubaneswar, Odisha (India), consists of proteins (58.0%), lipids and minerals (19.0%), growth factors, palmitic acid, and oleic acid (22.0%). The waste fish oil (basa fish) contained 45.3% saturated fatty acids. Among the dominant FAs were palmitic acid (30.6%) and oleic acid (38.6%). The unsaturated fatty acids accounted for over 55%. The major fatty acids were oleic acid (38.6%), palmitic acid (30.6%), linoleic acid (9.0%), stearic acid (8.2%), myristic acid (4.2%), and palmitoleic acid (2.5%). Other FAs were present in minor amounts. The saturation ratio of this waste fish oil was 0.84 [18,19,20].
Thus, according to the FA composition, sprat oil is close to oil from pollock; however, it is characterized by a higher content of docosahexaenoic acid and palmitic acid. In addition, the presence of linolenic acid and the absence of eicosatetraenoic acid were noted in sprat oil. Differences in the composition of sprat oil from basa oil are more significant and fundamental, consisting in the minor content in basa oil of polyunsaturated long-chain fatty acids, a new substrate for the synthesis of PHA.

3.2. Growth and Synthesis of PHA by C. necator B-10646 from of Sprat Oil

The wild-type strain C. necator B-10646 has a broad organotrophic potential: it can utilize such carbon sources as CO2 [34], sugars, variously refined glycerol [35,36], vegetable oils [37], as well as hydrolysates of molasses [38], tubers, and vegetative biomass of Jerusalem artichoke [39].
The wide organotrophic potential of microorganisms and the ability to transform various compounds into cell biomass is determined by the richness of the range of constitutive enzymes in them and the ability to synthesize enzymes in response to the appearance of new substrates. The activity of microbial metabolism of lipid substrates depends on the activity of lipolytic enzymes, which hydrolyze lipids extracellularly, making them available to microorganisms. During the metabolism of lipid substrates, lipases catalyze the hydrolysis of triacylglycerols (TAGs) of lipids into diacylglycerols (DAGs), monoglycerols (MAGs), glycerol, and free fatty acids (FFAs) at the interface between lipids and water. Fatty acids are freely transported into cells in an undissociated form by nonionic diffusion [40]. In the cytoplasm of C. necator and many other PHA producing bacteria cells, fatty acids are metabolized by the β-oxidation pathway during which (R)-3-hydroxyacyl-CoA is formed. It is the building block for PHA monomers [41] (Figure 1).
In the work [42] in the culture of Alcaligenes eutrophus (later taxonomic names Ralstonia, Wautersia, and Cupriavidus) on a medium with olive oil, it was found that the bacteria have a lipase secreted by cells into the culture medium. The authors suggested that the enzyme hydrolyzes triglycerols (TAGs) with the formation of fatty acids, which, when entering the cells, are metabolized into acetyl-CoA along the FA β-oxidation pathway. In a series of subsequent studies, this assumption was confirmed, and it was shown that, under the action of extracellular lipases, TAG hydrolysis products form an emulsion in the culture medium, and the initially complex lipid substrate becomes available to cells, enters them, and is metabolized [43]. The patterns of manifestation of lipase activity in the culture of PHA producers when grown on vegetable and animal fats are described by Riedel et al. [8,44].
It was previously shown that the C. necator B-10646 strain, having lipase activity, ensures the growth and synthesis of PHA on palm, sunflower, and Siberian oilseed oils [37]. The manifestation of enzyme activity was recorded 2–4 h after inoculation of the medium with vegetable oils, followed by an increase in activity for 24 h as bacteria grew up to 11.5 U/mL. Further, as the culture aged, the lipase activity somewhat decreased. A similar decrease in lipase activity with the development of the Ralstonia eutropha microbial culture on a medium with palm oil was noted by Riedel et al. [44].
A study of the lipase activity of C. necator B-10646 showed the presence of the enzyme in cells grown on a medium with sugars in the absence of fat (0.4 U/mL). When sugars were replaced with sprat oil, the lipase activity of cells increased to 8.3 U/mL. This indicates the constitutive nature of the enzyme and the ability to metabolize fatty acids in sprat oil.
In connection with the known data that the components of the substrate have boundaries of physiological action specific for each specific producer, we studied the effect of various concentrations of sprat oil (from 10.0 to 40.0 g/L) on the growth and synthesis of PHA by the C. necator B-10646 (Figure 2).
The highest yields of cell biomass and polymer were obtained in the range of sprat oil concentrations from 15.0 to 25.0 g/L, 4.3–4.7 g/L and 58–62%, respectively. A change in the concentration in the direction of decreasing or increasing these values was accompanied by a decrease in both indicators, especially significantly in the area of limiting the growth of bacteria by a carbon substrate. A similar effect of the concentration of waste fish oil obtained from basa fish (Pangasius bocourti) in the range of 10–30 g/L on PHA production in C. necator culture was shown by Loan et al. [20], where the best results were obtained at a concentration of fish waste fat of 20–25 g/L.
The kinetic parameters of the bacterial culture were evaluated during the first 24 h of the experiment, which corresponded to the exponential growth phase (Table 2).
The highest specific growth rates were recorded for waste oil concentrations of 15, 20, and 25 g/L and amounted to 0.139–0.140 h−1. At the concentration of 10 g/L, bacteria were limited by a carbon substrate. As a result, the specific growth rate was only 0.116 h−1. A further increase of the substrate concentration to 30 g/L or more led to a decrease of the specific growth rate to 0.120 h−1, which indicates the inhibition of bacteria by substrate excess. The PHA specific synthesis rates for the substrate concentration range from 15 to 40 g/L were from 0.047 to 0.059 h−1. The low values of that parameter were due to the complete nutrient medium and the short-term experiment. The highest values of the biomass and PHA productivity were recorded for a waste oil concentration of 20 g/L and amounted to 0.098 and 0.061 g/L·h, respectively. For carbon substrate concentrations of 15 and 25 g/L, the productivity values were slightly lower. The lowest productivity values were obtained at an oil concentration of 10 g/L due to an insufficient amount of substrate.
The results of growing C. necator B-10646 on sprat oil (20.0 g/L) in batch culture in the PHA synthesis mode during the first half of the process (30–35 h) on a nitrogen-containing medium and the subsequent stage in a nitrogen-free medium are shown in Figure 3a.
The polymer concentration in 48 h was 60% with a biomass yield of 4.1 g/L. Continuing the process up to 72 h increased the polymer content to 70% and a biomass concentration of 4.6 g/L. The result is inferior to the results in a similar mode in flasks on sugars (80–85% and 8–9 g/L) when glucose and fructose were used at the concentrations of 10.0–15.0 g/L [45] and comparable to the results in the culture of this strain on molasses hydrolyzate (75%), which contained a mixture of fructose and glucose (total sugar concentration was 18.0 g/L) [38] and glycerol (at the concentrations 5.0–10.0 g/L) [35]. It should be noted that scaling the process on glycerol in fermenters with a volume of 30 to 150 L gave a higher result, comparable to sugars [36].
The obtained indicators, apparently, are associated with the studied carbon source in the accepted periodic mode of bacterial cultivation. A well-known disadvantage of periodic cultures is the cessation of growth and synthesis of target products as a result of the accumulation of cell metabolism products in the culture medium and the exhaustion of the substrate. When using sprat oil containing a set of various fatty acids, uneven consumption of fatty acids probably took place, with rapid consumption and exhaustion of some and accumulation in culture of others that are not utilized by bacteria.
The effect of uneven consumption of fatty acids from three vegetable oils with different compositions was described by us earlier [37]. Comparison of the composition of FAs in the initial oils and the residual non-utilizable substrate showed that in the C. necator B-10646 culture, when grown on palm oil, FAs were utilized evenly, which is consistent with the data of works [28,44]. However, when growing on sunflower and Siberian oilseed oils, the cells primarily used polyenoic fatty acids. The uneven utilization of FA by bacteria was reflected in the saturation coefficient of the residual substrate, which decreased when palm oil was used and increased when bacteria grew on Siberian oilseed and sunflower oils. The completeness of the use of oils by bacteria varied and was maximum (80%) for palm oil and significantly lower (50%) for sunflower oil. This also affected the productivity of the process in terms of biomass and polymer yield [37].
This prompted us to analyze the initial composition of FAs in sprat oil and changes during the development of the bacterial culture (Figure 3c). The residual concentration of sprat oil was 10.2 g/L (Figure 3a), that is, almost half remained unused. This corresponds to the economic coefficients for biomass and polymer equal to YX/FAs = 0.46 and YPHA/FAs = 0.33 g/g, respectively, and is inferior to the known data on the efficiency of PHA production on fatty substrates since the theoretical yield of PHA from fatty acids is 0.65 g/g [46].
The dynamics of the composition of fatty acids, reflecting the uneven consumption of bacteria, is shown in Figure 3c. In the original sprat oil, the content of saturated, monoenoic, and polyenoic fatty acids was, respectively, 38.5, 28.1, and 32.6% of the total FA. In the course of cultivation, the bacteria utilized polyenoic fatty acids, the content of which by the end of the experiment decreased by almost eight times to 0–2% of the total FA, while the content of monoenoic and saturated fatty acids increased. The ratio of residual concentrations of saturated, monoenoic, and polyenoic FAs in non-utilized fat at the end of the experiment was, respectively, 59.7, 36.4, and 3.9% of the total FA amount, which differs significantly from the initial content and ratio. The predominant consumption of linolenic (18:3), eicosapentaenoic (20:5), and docosahexaenoic (22:6) acids by bacteria was registered, the concentration of which by the end of the process (72 h) fell many times, by 10 or more times. Uneven consumption of FAs by bacteria led to the fact that polyenoic FAs (linoleic, linolenic, eicosapentaenoic, and docosahexaenoic) were utilized by bacteria almost completely. Myristic, palmitic, stearic, and oleic fatty acids were practically not utilized, so their content increased by more than 1.4 times.
There are not many data on the regularities of the assimilation of fatty acids from the fatty waste of fish processing with PHA producers. It should be noted the work [13] which studied the consumption of FAs from hydrolyzed pollock fat and, using the example of six strains of Pseudomonas, not only showed uneven consumption of fatty acids by bacteria but also found that, depending on which pathway and which composition, PHAs were synthesized, and the utilization of fatty acids occurred in different ways. In the case of scl-P(3HB) synthesis, two strains utilized up to 42–46% of fatty acids from the initial content, and in the case of mcl-PHA synthesis, the other four strains utilized up to 61–69% of fatty acids. During the synthesis of mcl-PHA in the residual oil, a decrease in the level of myristic (C14:0), palmitic (C16:0) and palmitoleic acids (C16:1) was noted, and during the synthesis of scl-P(3HB), a decrease in the concentration of EPA and DHA was noted, which shows a lower efficiency of the short-chain PHA synthesis pathway.
The work of a team of researchers from Italy [47] should be noted, which presents a large array of results of the process of unicellular protein synthesis on food waste (fruits and fish) in the culture of Saccharomyces cerevisiae. The authors studied the simultaneous biovalorization of food wastes with an assessment of the dynamics of yeast utilization of the components of this complex substrate, including protein, crude lipid, fatty acids, lignin, and mineral elements at different fermentation times. Starting levels of the saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) were 24.9, 35.5, and 38.3, respectively. The uneven assimilation of fatty acids by cells was shown. The content of SFAs did not change during the fermentation process; conversely, MUFAs and PUFAs showed an opposite trend during all the fermentation process. MUFAs increased significantly, up to 47.3%, while PUFAs decreased down to 26.3%. A decrease in the polyunsaturation of the residual lipid substrate, according to some authors, is a positive fact due to the lengthening of its shelf life [48,49]. The uneven consumption of fatty acids is associated with the specifics of yeast metabolism, which, during growth and metabolism, break down lipids to obtain energy, followed by intracellular beta-oxidation, which leads to polyunsaturated reduction [50,51].
The revealed uneven consumption of FAs from sprat oil and the active utilization of polyunsaturated and monoenoic long-chain fatty acids by C. necator B-10646 bacteria and their sharp reduction in culture as part of the residual substrate against the background of accumulation of saturated FAs did not ensure continued cell growth and accumulation of the polymer in them. Therefore, the continuation of the process of cultivating bacteria, apparently, is possible with the implementation of a periodic mode with replenishment of the substrate with the additional introduction of new portions of sprat oil into the culture. Figure 4 illustrates the results of this regime. The input concentration of sprat oil was reduced to 15.0 g/L.
The use of fed-batch cultivation had a positive effect only on the biomass yield of C. necator B-10646, which increased to 7.0 g/L, but did not affect the yield of the polymer (63%) and the completeness of the use of the lipid substrate (about 60%). The YX and YP were 0.48 and 0.30 g/g, respectively, which is comparable to the experiment in batch culture. Reducing the concentration of sprat oil to 10.0 g/L in a similar fed-batch mode slightly reduced the biomass yield (to 6.1 g/L) and polymer (58.0%) and did not increase the completeness of fat utilization, which was close to batch cultivation at the initial concentration oils 20.0 g/L. Consequently, during the synthesis of PHA on sprat oil, the problem of the substrate reutilization arises, the use of which is possible, for example, to obtain industrial fats.
The dynamics of the composition of fatty acids indicating the uneven consumption of bacteria, is shown in Figure 4c. During cultivation, the bacteria utilized polyenoic fatty acids (linoleic and linolenic fatty acids), the content of which was recorded only in trace amounts to the end of the experiment. There was also a decrease in the content of polyenoic fatty acids (20:5ɷ3 and 22:6ɷ3), whereas the content of saturated fatty acids and oleic acid is slightly increased.
The results obtained are generally comparable with those published under similar conditions for growing PHA producers in flasks. Thus, depending on the strain specificity, Pseudomonas representatives, growing on hydrolyzed pollock waste fat, synthesized from 42 to 69% of the polymer at a biomass yield of 1.7 to 4.8 g/L [13]. The Ralstonia M91 strain synthesized up to 3.93 g/L of biomass and 2.43 g/L of polymer on a medium with 15 g/L of fat in flasks; in the 10-L fermenter, the values were slightly higher, respectively, 5.32 g/L and 2.73 g/L (61%) [19]. In the culture of Bacillus subtilis (KP172548), using an extract of fatty acids from fish-processing waste, the yield of biomass and polymer for 72 h was 2.3 and 1.62 g/L. Up to 22–37% PHA was synthesized by mixed cultures of activated sludge from fish-canning plants [15,17]. Bacteria Cupriavidus necator TISTR 1095 had higher rates and synthesized up to 7.5 g/L of biomass and 3.8 g/L of P(3HB-co-3HV) copolymer using condensate of fat waste from tuna processing [16].
Higher rates were obtained only when implementing processes in fermenters, from 20–30 to 117 g/L and from 20–50 to 82–85% [20]. For the synthesis of PHA, the authors of these works used microbial cultures belonging to different taxa, which were cultivated in different modes, and sources of fat, which were obtained from the waste of various fish species and by various methods, were used as a carbon substrate.

3.3. Composition and Properties of PHA Synthesized on Sprat Oil

The PHA samples were purified to a homogeneous state, and the chemical composition and physicochemical properties were studied. C. necator B-10646 bacteria synthesized 3-component copolymers on sprat oil. The dominant monomer was 3-hydroxybutyrate (3HB) (97–98 mol.%), and there were monomers of 3-hydroxyvalerate (3HB) (1.3–1.7 mol.%) and minor inclusions of monomers of medium-chain 3-hydroxyhexanoate (3HHx) (0.3–0.7 mol.%). Regardless of the concentration of sprat oil in the medium, which influenced the intracellular concentration of PHA, the basic properties (molecular weight and temperature characteristics, and degree of crystallinity) were similar (Table 3).
With an increase in the sprat oil concentration in the medium, a slight increase in 3HV monomers and a decrease in 3HHx monomers were noted. The number average molecular weight (Mn) of the samples practically did not depend on its concentration and amounted to 116–133 kDa. At the same time, an increase in the sprat oil concentration in the medium was accompanied by the synthesis of a polymer with a higher weight average molecular weight (Mw), which led to an increase in the polydispersity value (Đ).
X-ray studies did not reveal significant changes in the degree of crystallinity; Cx values were in a fairly narrow range (66–72%), regardless of the concentration of the C-substrate. The melting point (Tmelt) and thermal degradation temperature (Tdegr) for all the studied samples had similar values, not beyond the significant differences. Thus, the Tmelt values were in the range of 158–160 °C and had double peaks; the thermal degradation temperature (Tdegr) was in the range of 287–293 °C. In general, the properties of PHA synthesized on sprat oil with low inclusions of 3-hydroxyvalerate monomers and minor 3-hydroxyhexanoate monomers are close to those of poly(3-hydroxybutyrate) homopolymer.
Synthesis of three component copolymers P(3HB-co-3HV-co-3HHx) was shown in cultures of wild-type strains grown on vegetable oils as a sole substrate [37] or with additions of precursors into the medium for the corresponding monomers synthesis [52]. When C. necator B-10646 was cultivated on palm, vegetable, and Siberian oilseed oils, the bacteria synthesized an analogous copolymer with a similar monomer content (3HV up to 1.9 mol.%, 3HHx up to 1.0 mol.%); however, the Mn values were slightly higher (130–190 kDa) at Đ from 4.1 to 5.2. The Tmelt of the obtained copolymers was also slightly higher, 170–171 °C, with lower Tdegr values (271–281 °C) [37]. When the same strain was grown on glucose as the main carbon source and various precursors (potassium valerate + potassium hexanoate), a similar copolymer was obtained, but with higher inclusions of 3HV monomers (up to 26.1 mol.%) and 3HHx (up to 13.6 mol.%), which led to higher Mn (147–225 kDa) and Tmelt (172–175 °C); however, Tdegr and Cx in this case were lower (262–270 °C and 53–63%, respectively) [52].
The synthesis of P(3HB-co-3HV-co-3HHx) is also shown for recombinant strains. Thus, C. necator transformant harboring phaC of Chromobacterium sp. USM2 growing on CPKO and sodium valerate or propionate synthesized a copolymer with a 3HV content of 3–85 mol.% and 3HHx content of 1–7 mol.% had Mn 130–210 kDa and Tmelt 89–148 °C [53]. In another work, the synthesis of P(3HB-co-3HV-co-3HHx) by a recombinant strain of Aeromonas hydrophila 4AK4 harboring β-ketothiolase gene phaA and NADPH-dependent acetoacetyl-CoA reductase gene phaB cultivated on lauric acid with the addition of sodium valerate was studied. The copolymer contained 3HB, 3HV (0.0–23.8 mol.%), and 3HHx (0.0–28.3 mol.%) monomers having a wide range of properties with respect to Mn (210–551 kDa), Tmelt (54.4–170.1 °C), and Tdegr (231.8–258.1 °C) [54].
An analysis of publications on the synthesis of PHA on the fatty waste of fish processing showed that the chemical composition of polymers, depending on the source of raw materials and the species specificity of producers, differs significantly. Representatives of Pseudomonas, when growing on a substrate from crude pollock oil, depending on the strain, synthesized polymers of various compositions, among which, in addition to P(3HB), were scl-PHA with a low content of 3-hydroxyhexanoate monomers, and the dominance of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acids [13]. It was shown that P(3HB) had weight average molecular weight (Mw) values of 206,000–195,000 g/mol, polydispersity 2.0–2.2, and copolymers had lower Mn values (from 84,000 to 153,000 g/mol) but close polydispersity values (2.08–2.61). Fatty wastes from effluents from the tuna processing plant ensured the synthesis of Cupriavidus necator TISTR 1095 of P(3HB-co-3HV) copolymer containing up to 20 mol.% of 3HV monomers. The Mn values of the copolymer and polydispersity were 2000 kDa and 2.5, respectively [16]. The bacteria B. subtilis, when grown on an FSW extract from fish waste, synthesized a highly crystalline P(3HB) homopolymer. The thermal degradation temperatures of the polymer remained almost constant (150–466 °C) [14]. Using the spent fish oil basa (Pangasius bocourti) in the Cupriavidus necator H16 culture and strain M91 isolated from Ralstonia, a P(3HB) homopolymer was synthesized [19,20]. The molecular weight, number average molecular weight and polydispersity for P(3HB) produced by Ralstonia sp. M91 from crude fish oil was 670 kDa, 230 kDa, and 2.83, respectively. The moderately halophilic bacterium Salinivibrio sp. M318 (VTCC 910086), when grown on fish sauce from spent fish oil and glycerol, synthesized poly(3-hydroxybutyrate), and with the addition of precursor substrates, copolymers hydroxyvalerate P(3HB-co-3HV) were synthesized [18]. Depending on the composition of the P(3HB-co-3HV) copolymers and the proportion of 3HV, the molecular weight characteristics varied: from 120 to 320 kDa for Mn and from 200 to 630 kDa for Mw with polydispersity 1.4–2.0. In the melting region, two peaks were recorded depending on the inclusion of 3HV at 141 and 160 °C and at 132 and 150 °C (content of 3HV monomers, respectively, 13.0 and 17.0 mol.%). At a higher inclusion of 3HV (24.7 mol.%), one melting peak was recorded at 139 °C.
Thus, the use of fat waste from the production of sprats as a carbon substrate makes it possible to synthesize not only the P(3HB) homopolymer but also more technologically advanced PHA copolymers.

4. Conclusions

The PHA synthesis by the wild-type strain C. necator B-10646 on sprat oil was studied for the first time. The highest biomass concentration (up to 7.0 g/L) was obtained in the mode with C-substrate feeding at PHA content up to 63%, and the completeness of substrate utilization was 45–50%. It is comparable with most of the results obtained with similar modes of bacterial cultivation on waste fish oil. The synthesized polymers were poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) and had similar temperature, molecular weight characteristics and crystallinity. In general, it can be concluded that the studied sprat oil is a promising substrate for PHA synthesis.

Author Contributions

N.O.Z.—conceptualization, investigation, and writing; K.Y.S.—investigation and visualization; E.G.K.—investigation, visualization, and formal analysis; E.I.S.—formal analysis and writing; T.G.V.—conceptualization, writing, review and editing, supervision, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Russian Science Foundation (grant N 23-64-10007).

Data Availability Statement

All data are available in the paper.

Acknowledgments

The authors would like to express their special thanks to the Krasnoyarsk Regional Center of Research Equipment of Federal Research Center “Krasnoyarsk Science Center SB RAS” for providing equipment to ensure the accomplishment of this project.

Conflicts of Interest

The authors declare no conflict of interest.

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Processes 11 02113 g001 550
Figure 1. Overall metabolic steps from fatty acids to PHA.
Figure 1. Overall metabolic steps from fatty acids to PHA.
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Figure 2. Effect of sprat oil concentration on bacterial biomass concentration of C. necator B-10646 and PHA content. The letters indicate the significance of differences when comparing groups according to the Mann–Whitney test at the level of p < 0.05; identical letters indicate no significant differences.
Figure 2. Effect of sprat oil concentration on bacterial biomass concentration of C. necator B-10646 and PHA content. The letters indicate the significance of differences when comparing groups according to the Mann–Whitney test at the level of p < 0.05; identical letters indicate no significant differences.
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Figure 3. Sprat oil concentration dynamics (a), biomass concentration and polymer content of C. necator B-10646 (b), and FA profiles in the sprat oil (c) during batch cultivation in flasks.
Figure 3. Sprat oil concentration dynamics (a), biomass concentration and polymer content of C. necator B-10646 (b), and FA profiles in the sprat oil (c) during batch cultivation in flasks.
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Figure 4. Sprat oil concentration dynamics (a), biomass concentration and polymer content of C. necator B-10646 (b), and FA profiles in the sprat oil (c) during fed-batch cultivation in flasks. The arrows show the time of adding the substrate (sprat oil).
Figure 4. Sprat oil concentration dynamics (a), biomass concentration and polymer content of C. necator B-10646 (b), and FA profiles in the sprat oil (c) during fed-batch cultivation in flasks. The arrows show the time of adding the substrate (sprat oil).
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Table
Table 1. Fatty acid composition of sprat oil.
Table 1. Fatty acid composition of sprat oil.
Fatty Acid IndexFatty Acid TypeContent, % of Sum of Fatty Acids
14:0Myristic3.5 ± 0.1
15:0Pentadecanoic0.5 ± 0.0
16:0Palmitic28.0 ± 1.2
16:1ω7Palmitoleic0.3 ± 0.0
18:0Stearic4.5 ± 0.2
18:1ω9Oleic25.3 ± 1.3
18:1ω6Linoleic2.5 ± 0.1
18:1ω3Linolenic4.3 ± 0.1
20:0Arachidic0.3 ± 0.0
20:1Eicosenoic1.1 ± 0.0
20:2Eicosadienoic0.4 ± 0.0
20:5ω3Eicosapentaenoic8.7 ± 0.3
22:0Docosanoic0.5 ± 0.0
22:6ω3Docosahexaenoic16.7 ± 0.2
24:1Tetracosenoic1.5 ± 0.1
Other * 1.3 ± 0.1
∑saturated FAs/∑unsaturated FAs 0.63
* i-14:0, i-14:0, i-16:0, ai-16:0, 17:1.
Table
Table 2. Kinetic and production parameters of C. necator B-10646.
Table 2. Kinetic and production parameters of C. necator B-10646.
Waste Fish Oil Concentration, g/LSpecific Growth Rate, h−1PHA Specific Synthesis Rate, h−1 Biomass Productivity, g/L·hPHA Productivity, g/L·h
10.00.1160.0200.0460.011
15.00.1390.0560.0900.054
20.00.1400.0590.0980.061
25.00.1390.0540.0940.054
30.00.1230.0520.0560.022
35.00.1180.0470.0580.020
40.00.1200.0510.0630.023
Table
Table 3. Physicochemical properties of PHA samples synthesized by C. necator B-10646 on sprat oil.
Table 3. Physicochemical properties of PHA samples synthesized by C. necator B-10646 on sprat oil.
Concentration of Sprat Oil in the Medium, g/LComposition of PHA, mol.%Mn,
kDa		Mw,
kDa		ĐCx,
%		Tmelt,
°C		Tdegr.,
°C
3HB3HV3HHx
1598.01.30.71165404.667158/163287
2097.81.70.51176695.766159/165292
2598.11.60.31337625.772160/165293
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Zhila, N.O.; Sapozhnikova, K.Y.; Kiselev, E.G.; Shishatskaya, E.I.; Volova, T.G. Synthesis and Properties of Polyhydroxyalkanoates on Waste Fish Oil from the Production of Canned Sprats. Processes 2023, 11, 2113. https://doi.org/10.3390/pr11072113

AMA Style

Zhila NO, Sapozhnikova KY, Kiselev EG, Shishatskaya EI, Volova TG. Synthesis and Properties of Polyhydroxyalkanoates on Waste Fish Oil from the Production of Canned Sprats. Processes. 2023; 11(7):2113. https://doi.org/10.3390/pr11072113

Chicago/Turabian Style

Zhila, Natalia O., Kristina Yu. Sapozhnikova, Evgeniy G. Kiselev, Ekaterina I. Shishatskaya, and Tatiana G. Volova. 2023. "Synthesis and Properties of Polyhydroxyalkanoates on Waste Fish Oil from the Production of Canned Sprats" Processes 11, no. 7: 2113. https://doi.org/10.3390/pr11072113

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