Production of Polyhydroxybutyrate from Lignocellulosic Hydrolysates Using Mixed Microbial Cultures

Abstract

This study investigated the production of poly(3-hydroxybutyrate) (PHB) using mixed microbial cultures (MMCs) with lignocellulosic hydrolysates as a carbon source. Single-factor experiments were conducted to examine the effects of substrate concentration, C/N ratio, and pH on PHB synthesis. The highest PHB yield (612.35 mg/L) was achieved at a substrate concentration of 1700 mg/L (R1700), with an effective C/N ratio of approximately 31 and pH 7.0. Nitrogen limitation and neutral pH were favorable for PHB production. Microbial community analysis via 16S rDNA sequencing revealed Chryseobacterium as the dominant genus in all reactors. These findings provide insights into the efficient conversion of waste lignocellulose into biodegradable PHB using MMCs.

1. Introduction

The overuse of petroleum-based plastics has caused serious environmental problems because of their very slow rate of degradation under natural conditions [1,2]. Therefore, the development of bio-based and biodegradable plastics as suitable substitutes for petroleum-based plastics has attracted great interest. Polyhydroxyalkanoates (PHAs), the only class of biopolymer that can be completely synthesized and degraded by living cells, are produced by various microorganisms as intracellular carbon and energy reserves under nutritionally limited conditions [3,4,5]. PHAs have physicochemical characteristics and thermoplastic properties similar to those of petroleum-based plastics, with additional advantages such as non-toxicity, biocompatibility, biodegradability, piezoelectricity, antioxidant activity, gas-barrier properties, optical activity, and ultraviolet resistance [6,7]. Because of these excellent properties, PHA is considered one of the most promising alternatives to petroleum-based plastics and can be widely applied in biodegradable packaging materials, tissue engineering, conductive materials, carrier materials, fuel and spices [8]. At present, the industrial production of PHA is mainly carried out by fermentation using pure cultures, which requires sterile conditions and expensive pure organic substrates such as glucose, fatty acids, or vegetable oils [9]. High raw material costs and stringent sterilization requirements make PHA production 2–5 times more expensive than petroleum-based plastics, thereby hindering its large-scale commercial development [10]. Consequently, the exploitation of low-cost and abundant carbon sources has become an urgent demand for PHA biosynthesis [11]. Reducing the cost of carbon substrates and simplifying the fermentation process are therefore key strategies for improving the economic feasibility of PHA production.

Lignocellulosic biomass, such as agricultural straw waste and forestry processing residues, is one of the most abundant, inedible, and renewable feedstocks on the planet [12]. It has the characteristics of low commercial value, year-round availability, and does not compete with food and feed resources [13]. Therefore, it can be considered as a potential raw material for PHA synthesis, because its enzymatic hydrolysis can release the fermentable sugars such as glucose and xylose, which are the building blocks for the production of PHA [14,15]. More importantly, by regulating the fermentable sugar composition generated during lignocellulose hydrolysis and the conditions of microbial culture, the synthesis of PHA with different monomer compositions can be controlled [16,17]. Studies on PHA synthesis from lignocellulose-derived fermentable sugars can not only improve the efficient and high-value utilization of lignocellulosic resources but also enable the preparation of wood-derived bioplastics with diverse performance characteristics, thereby expanding the application range of bioplastics. However, lignocellulosic hydrolysates are complex substrates containing a mixture of fermentable sugars, organic acids, and other diverse compounds that can vary depending on the biomass source and pretreatment method [18]. Efficient utilization of such heterogeneous substrates for PHA production requires microbial systems capable of metabolizing multiple carbon sources simultaneously and adapting to fluctuating substrate composition. Therefore, the development of mixed microbial cultures with high metabolic versatility is essential for efficient PHA production from lignocellulosic resources [19].

The use of mixed microbial cultures, such as activated sludge, instead of pure cultures has also been proposed as an effective alternative to potentially reduce the current cost of PHA production [20]. The advantage of using this technology is that sterilization is not required, and the structure of bacteria genus is relatively stable, because the selection of mixed microorganisms is based on ecological principles, thereby ensuring stable long-term operation of the reactor [21]. In addition, the control process is simpler because the operating conditions can be adjusted more easily, allowing the use of semi-continuous systems [22,23]. During PHA production by MMCs, operational parameters such as substrate concentration, carbon-to-nitrogen ratio, and pH strongly influence the balance between biomass growth and intracellular polymer accumulation [24].

Therefore, the purpose of this study was to synthesize biodegradable plastic polyhydroxybutyrate instead of petroleum-based plastics using waste lignocellulosic hydrolysates and activated sludge as mixed microbial cultures (MMCs). To determine the maximum yield of PHB, the effects of the carbon-to-nitrogen ratio (C/N ratio), pH value, and concentration of lignocellulose hydrolysate under controlled pH conditions on PHA production were studied. Finally, the species composition and abundance of the microbial community were investigated using 16S rDNA sequencing.

2. Materials and Methods

2.1. Materials

30 g of poplar wood powder (Total cellulose content: 75.18%) dried at 103 °C for 48 h was mixed evenly with ultra-pure water in a mass ratio of 1:4, and then reacted in a 0.5 L high-temperature and high-pressure reactor (Dalian Tongda, Dalian, China) at 200 °C for 30 min. The pretreatment solution was transferred to a beaker and made up to 1 L with ultrapure water, and the pH value was adjusted to 4.8. Then, 3 mL of cellulase (CTec2, Novozymes Biotechnology Co., Ltd., Bagsværd, Denmark, cellulase enzyme blend) was added and the mixture was reacted for 72 h at 45 °C under magnetic stirring at 330 rpm. Cellulase was a standardized liquid enzyme mixture rather than a single purified cellulase, containing cellulases, β-glucosidases and hemicellulases. Its declared activity was approximately 1000 BHU-2/g, with a density of approximately 1.15 g/mL. Finally, the product was placed in a 90 °C water bath for 20 min and filtered to obtain the supernatant. The process was based on previous studies [7]. The obtained enzymatic hydrolysate of poplar wood contained reducing sugar, furfural and 5-hydroxymethylfurfural, with concentrations of 15.91 g/L, 512.61 mg/L and 239.34 mg/L respectively.

The activated sludge used in the experiment was taken from the secondary sedimentation tank of Jinnan Sewage Treatment Plant in Tianjin, and the contents of total solids (TS) and volatile solids (VS) in the activated sludge were 1.48 g/L and 1.03 g/L, respectively. TS and VS were determined according to standard methods (APHA, 2017). In short, TS was measured by drying the sample to constant weight at 105 °C, while VS was determined by igniting the dried residue in a muffle furnace at 550 °C.

2.2. Use of Lignocellulose Hydrolysate for PHA Production by Activated Sludge

Aerobic dynamic feeding technology was used for the domestication and enrichment of PHA synthesis mixed bacteria and the synthesis of PHA. A 2 L glass beaker was used as an activated sludge reactor (sealed) for PHA domestication and synthesis experiments. Throughout the entire study period, the reactor was kept on a 28 °C constant temperature water bath, mechanically stirred to ensure uniform mixing, and supplied with oxygen through an air pump to achieve a dissolved oxygen concentration of at least 2 mg/L during each aerobic stage of the cycle. Each operational cycle consisted of feeding → aeration and mixing → settling → discharge, with a total duration of 12 h, including a 1 h settling phase. The dissolved oxygen (DO) concentration in the reactor was measured using a dissolved oxygen meter during the reaction process. The cycle of the sequencing batch reactor (SBR) system was 12 h, and the operating cycle included initial feeding phase (15 min), reaction phase (645 min), sedimentation phase (50 min), and effluent withdrawal phase (10 min). The carbon source of the influent substrate in the system was lignocellulose hydrolysate, the nitrogen source was NH₄Cl, and the phosphorus source was KH₂PO₄. Each liter of system influent substrate also contained 100 mg of MgSO₄ and 1 mL of mineral salts. The composition of the mineral salts used for culture was (mg/L): FeCl₃·3H₂O, 3; CaCl₂, 40; CoCl₂·6H₂O, 0.3; ZnSO₄·7H₂O, 0.24; H₃BO₃, 0.3; MnCl₂·4H₂O, 0.3; KI, 0.06; CuSO₄·5H₂O, 0.06. When PHA was synthesized, the initial mixed sludge suspension (MLSS) of activated sludge was 1500 mg/L. In order to investigate the effects of carbon source concentration, C/N ratio, and pH value on PHA synthesis, a single-factor experiment was designed (

Table 1. Single-factor experimental design.

Experiment No.	Substrate (Lignocellulose Hydrolysate mg/L)	Nitrogen Source (NH4Cl mg/L)	pH Value	C/N Ratio
R500	500	55	7	9
R800	800	55	7	15
R1100 (N55/P7)	1100	55	7	20
R1400	1400	55	7	26
R1700	1700	55	7	31
N33	1100	33	7	33
N44	1100	44	7	25
N66	1100	66	7	17
N77	1100	77	7	14
P6	1100	55	6	20
P6.5	1100	55	6.5	20
P7.5	1100	55	7.5	20
P8	1100	55	8	20

Note: The concentrations of KH₂PO₄ and MgSO₄ were fixed at 11 mg/L and 100 mg/L, respectively, in all experiments. The Trace element solution was also added at a constant concentration of 1 mL/L for all experimental groups. Bold values indicate the factor level that was varied in each single-factor experiment.

). Activated sludge samples were collected at the end of each cycle and at fixed times during the last cycle to detect MLSS, chemical oxygen demand (COD), pH value, and PHA content in microbial cells, thereby evaluating the PHA accumulation potential of mixed microorganisms.

2.3. Analytical Methods

In the PHA accumulation experiment, samples were collected from the mixed solution in the reactor after each cycle and at fixed times during the last cycle and centrifuged at approximately 6790× g for 10 min. The obtained supernatant was filtered through a 0.45 μm inorganic filter membrane and stored at 4 °C, while the precipitate was stored at −26 °C for subsequent analysis of substrate concentration (COD) and PHA. COD was determined using the dichromate-based rapid digestion method. Briefly, the sample was mixed with the COD digestion reagent in a digestion tube and heated in a multifunctional intelligent digestion instrument (EF-X16L, Hefei Enfan Instrument Equipment Co., Ltd., Hefei, China). After digestion and cooling to room temperature, the COD concentration was measured using a multiparameter water quality analyzer (ITS2004, Shanghai Yoke Instrument Co., Ltd., Shanghai, China). Freeze-dried particles were used for PHA quantitative analysis and monomer composition detection by gas chromatograph (Agilent 7890A, Agilent Technologies, Santa Clara, (CA), USA) equipped with HP innovatx capillary column and flame ionization detector (FID), following the method described by Yin et al. [22]. For PHA quantification by GC, the freeze-dried biomass was suspended in 1 mL of chloroform, and 2 mL of esterification solution (prepared by dissolving 1.0 g of benzoic acid in 110 mL of acid methanol, composed of 100 mL chromatographic methanol and 10 mL concentrated sulfuric acid, followed by ultrasonic treatment for 10 min) was added. The mixture was incubated in a constant temperature oven at 103 °C for 4 h. After cooling, 1.0 mL of high-purity water (conductivity = 2.4 μS·cm⁻¹) was used for extraction over 12 h. The resulting esterified PHA was then analyzed by GC. MLSS was measured using the standard reference method (APHA, 1998). Due to budget constraints, each experimental data point in this study was obtained from a single independent experiment without repeated measurements. Therefore, no statistical analysis or error bars were applied to the experimental results.

2.4. Microbial Community Analysis Methods

During the PHA accumulation process, 16S rDNA genes were analyzed in samples collected under different carbon source concentrations (R500, R800, R1100, R1400, and R1700) to characterize the microbial community. 10 mL biomass samples were collected from each reactor at the PHA accumulation stage. Before DNA extraction, the samples were centrifuged at approximately 6790× g, and the obtained pellets were stored under −80 °C to minimize DNA degradation. Total genomic DNA was extracted using Soil DNA Kit according to manufacturer’s protocols. DNA extraction, library preparation, 16S V3–4 rDNA gene amplicon sequencing on an Illumina Miseq PE250/300 platform (Illumina, San Diego, CA, USA), and bioinformatics processing were performed by Suzhou jinweizhi Technology Co., Ltd. (Suzhou, China). The V3–V4 region of the 16S rRNA gene was amplified using forward primers (CCTACGGRRBGCASCAGKVRVGAAT) and reverse primers (GGACTACNVGGGTWTCTAATCC). After sequencing, low-quality reads, ambiguous bases, and chimeric sequences were removed to obtain high-quality effective sequences. The sequences were then clustered into operational taxonomic units or amplicon sequence variants, and taxonomic annotation was performed using a reference ribosomal RNA database. Finally, the relative abundance of microbial taxa at different classification levels was calculated to compare the effects of carbon source concentration on microbial community composition during PHA accumulation.

3. Results and Discussion

3.1. Effects of Substrate Concentration on PHB Production

The effect of different initial substrate (lignocellulose hydrolysate) concentrations (R500:500 mg/L; R800:800 mg/L; R1100:1100 mg/L; R1400:1400 mg/L; R1700:1700 mg/L) on PHB production in a batch reactor was evaluated. The yield of PHB was generally similar throughout the seven cultivation cycles Figure 1(a₁–e₁) and the single cycle Figure 1(a₂–e₂). Figure 1(a₁–e₁) shows the changes in MLSS and PHB at different substrate concentrations over seven cultivation cycles. In the R500, R800, and R1100 reactors, MLSS and PHB first increased and then decreased, reaching their maximum values in the fifth cycle, possibly because more carbon sources were used for cell growth rather than PHB synthesis after five cycles [25]. In the R1400 and R1700 reactors, as cultivation time increased, MLSS gradually increased, while PHB showed a trend of first increasing and then decreasing. When the initial concentration was 1700 mg/L, PHB reached its maximum value in the third cycle (0.6124 g/L), which was related to the unbalanced state created by the high carbon source concentrations during the first three cycles, thus favoring the accumulation of PHB [26]. The PHB production observed in this study was higher than that reported in some previous studies using lignocellulose hydrolysate as a carbon source, but lower than that obtained with volatile fatty acids as the carbon source, likely because different substrates involve different microbial metabolic conversion pathways [27,28,29]. Zeng et al. investigated the effects of varying carbon-to-nitrogen (C/N) ratios ranging from 5 to 80 on PHA production under a constant carbon input. The results showed that, at a COD concentration of 4000 mg/L, a high C/N ratio favored PHA accumulation, whereas low C/N ratios of 5–20 promoted biomass growth. The maximum PHA production, 412.88 mg/L, was achieved at a C/N ratio of 80 [30]. Verdini et al. investigated PHA production from corn stover hydrolysate using mixed microbial cultures. The results showed that corn straw hydrolysate could be effectively converted into PHA, with the maximum PHA concentration reaching 0.42 g/L when the biomass-derived monosaccharides were supplied at 20 g/L at 30 °C for 96 h [31].

The single operational cycle results (Figure 1(a₂–e₂)) showed the changes in COD and PHB at different initial substrate concentrations in the eighth operational cycle. For all tested concentrations, the substrate was rapidly consumed during the first 2 h of incubation, and the maximum PHB content increased linearly during the eighth operational cycle. There was no clear pattern in the synthesis of PHB in the R500, R800, and R1100 reactors, and the PHB content fluctuated within a small range at different time points, indicating that the microorganisms in these reactors may have become less active during this operational cycle. In the R1400 and R1700 reactors, PHB showed a trend of first increasing and then decreasing. In this cycle, the increase in PHB within 1 h after carbon source addition was much smaller than the decrease after 1 h. This result was consistent with the gradual decrease in PHB synthesis observed in the later cultivation cycles.

3.2. Effects of C/N Ratio on PHB Production

The C/N ratio critically affects the growth of PHA-producing strains and the composition of PHA products [32]. As a key operational parameter, the C/N ratio determines the availability of nitrogen relative to carbon, thereby regulating microbial metabolic pathways, enzyme synthesis, and intracellular carbon flux distribution. In mixed microbial cultures (MMCs), this parameter is particularly important because it not only influences individual cell metabolism but also shapes the competitive interactions among different microbial populations. Figure 2 shows the experimental results obtained under different C/N ratios (N33: 33; N44: 25; N55 (R1100): 20; N66: 17; N77: 14) at the same lignocellulose hydrolysate concentration and pH value. The changes in MLSS and PHB under different C/N ratios are shown in Figure 2(a₁–e₁). As reactor operation proceeded and the C/N ratio decreased, the maximum value of MLSS showed a trend of first decreasing and then increasing, whereas PHB content generally showed a trend of first increasing and then decreasing. Specifically, the highest accumulation of PHB (0.4445 g/L) and the lowest maximum MLSS value (3.66 g/L) were obtained when the C/N ratio was 20. This result indicated that nitrogen deficiency caused by a higher carbon-to-nitrogen ratio did not necessarily direct carbon flux toward PHB storage; instead, part of the carbon may still have been used for cell growth, which may be related to the relatively low concentration of carbon sources applied during the cultivation process [30]. In addition, under moderate nitrogen limitation, microorganisms tend to reduce protein synthesis and cell division, while redirecting excess carbon toward storage compounds such as PHB as a survival strategy under nutrient imbalance conditions [33]. When the C/N ratio was 14, the maximum accumulation of PHB was the lowest (0.1367 g/L), and the maximum value of MLSS was the highest (3.66 g/L). This is because sufficient nitrogen supply increases the activity of citrate synthase, inducing acetyl-CoA to enter the TCA cycle and thereby shifting microbial carbon allocation toward growth and metabolism [34]. Under such nitrogen-rich conditions, the synthesis of nucleic acids and proteins is enhanced, promoting rapid biomass growth and reducing the accumulation of storage polymers. Overall, the carbon-to-nitrogen ratio played a crucial regulatory role in the allocation of carbon between growth and storage pathways in mixed microbial communities.

The single operational cycle results (Figure 2(a₂–e₂)) showed the changes in COD and PHB at all C/N ratios during the eighth operational cycle. When the carbon-to-nitrogen ratio decreased from 33 to 14, the maximum PHB accumulation under different conditions was 456.28 mg/L, 316.29 mg/L, 264.42 mg/L, 211.07 mg/L, and 171.94 mg/L, respectively. These results indicated that a higher C/N ratio was more conducive to PHB accumulation than to biomass growth, thereby helping to avoid endogenous respiration during nutrient deprivation [30]. This is consistent with the findings of other researchers, indicating that a high C/N ratio promotes PHB accumulation [35]. However, this trend is inconsistent with the results shown in Figure 2(a₁–e₁), which may be attributed to differences between short-term and long-term system behavior. During repeated operation, microbial community adaptation, limited carbon availability under low substrate loading, and endogenous consumption of stored PHA may collectively reduce net PHA production [36].

3.3. Effects of pH on PHA Production

Microbial activity and metabolic pathways can be influenced by pH value, thereby indirectly affecting the enrichment of PHA-producing bacteria and the synthesis of PHA [35]. As an important environmental factor, pH can regulate nutrient transport, intracellular enzyme activity, membrane stability, and the competitive relationships among different microbial populations in mixed microbial cultures (MMCs) [37]. Therefore, maintaining a suitable pH range is essential for improving substrate utilization efficiency and promoting intracellular carbon storage. The experimental data in Figure 3(a₁–e₁) show the changes in MLSS and PHB during batch culture at different pH values (P6: 6; P6.5: 6.5; P7: 7; P7.5: 7.5; P8: 8) over seven culture cycles. As illustrated in Figure 3(a₁–e₁), the MLSS values varied with pH, with the highest MLSS at pH 8 (4.18 g/L), followed by a slight reduction at pH 7 (3.66 g/L), indicating that microbial growth at alkaline pH was usually a more favorable environment for cell proliferation. Slightly alkaline conditions may enhance the physiological activity of the mixed culture and support biomass formation, possibly by improving cell viability and reducing acid stress during repeated batch operation. However, a higher MLSS value does not necessarily correspond to higher PHB accumulation, because part of the consumed carbon may be directed toward cell growth and maintenance rather than polymer storage [37]. The highest PHB yield (0.4446 g/L) was observed at pH 7. This trend may be explained by the optimal activity of enzymes involved in the sugar metabolism and PHA biosynthesis, such as acetyl-CoA synthetase and polyhydroxybutyrate synthase, under neutral conditions [38]. Under excessively acidic or alkaline conditions, enzyme conformation and catalytic efficiency may be affected, leading to reduced substrate assimilation and weakened PHB biosynthetic capacity. In addition, unsuitable pH may alter the composition of the microbial community, thereby reducing the relative abundance or activity of PHB-accumulating microorganisms.

The single operational cycle results (Figure 3(a₂–e₂)) showed the changes in COD and PHB at different pH values during the seventh cycle. The single operational cycle data confirmed that at pH 7, microorganisms rapidly absorbed carbon source within the first hour after substrate addition, resulting in optimal PHB accumulation. Subsequently, the consumption rate gradually slowed, possibly because of substrate limitation during microbial growth. These findings are consistent with previous studies that a neutral pH environment promotes the highest production of PHB by mixed microorganisms. For example, Tripathi reported that operation at neutral pH in a microaerobic environment led to higher substrate degradation rates, dehydrogenase activity, and reduction current than acidic or alkaline conditions, thereby enhancing PHA production [39].

Potential Interactions Among Key Operational Parameters: Although our study employed a single-factor experimental design, potential interactions among substrate concentration, C/N ratio, and pH can be inferred from the observed trends. For example, pH may influence the assimilation efficiency of specific carbon and nitrogen sources, thereby affecting microbial growth and PHB accumulation. Similarly, high substrate concentrations may impose osmotic stress that could alter the optimal C/N ratio for polymer synthesis. Such interactions suggest the presence of synergistic or antagonistic effects among these key parameters, which should be further explored in future multivariate optimization studies, such as Response Surface Methodology.

3.4. Microbial Community Analysis

To understand the microbial dynamics during PHA production, we analyzed the microbial community composition using 16S rDNA sequencing. Figure 4 presents the microbial community structure at the genus and phylum levels. At the genus level in Figure 4a, Chryseobacterium showed an increasing trend in relative abundance from 9.63% in the R500 reactor to 28.37% in the R1700 reactor, becoming one of the dominant genera at higher substrate concentrations. In contrast, Nakamurella was the predominant genus in the R500 reactor. The increase in Chryseobacterium abundance with higher substrate concentration may be attributed to the greater availability of fermentable sugars (such as glucose and xylose) in the lignocellulose hydrolysate. In addition, previous studies have shown that Chryseobacterium species can produce extracellular enzymes that facilitate the breakdown of complex organic compounds, further enhancing their ability in lignocellulosic environments [40,41]. This result also indicated that Chryseobacterium had a higher tolerance to osmotic stress and could effectively utilize lignocellulosic hydrolysates as carbon sources [42]. Such osmotic tolerance is particularly important in high-strength wastewater or concentrated hydrolysate systems, where microbial survival depends on robust stress response mechanisms [43,44]. Importantly, although Chryseobacterium enhances the availability of monomers such as sugars and organic acids, there is limited evidence that this genus itself accumulates PHB in significant amounts. Instead, low-abundance genera such as Paracoccus and other known PHA producers likely utilize the monomers released by Chryseobacterium for PHB synthesis [42]. This suggests a synergistic metabolic division of labor within the mixed microbial culture, where Chryseobacterium acts primarily as a degrader, while other PHA-producing genera carry out PHB accumulation. Other genera including Paracoccus, Chitinophagaceae, Nakamurella, and Spirosoma were also identified in each reactor, but at lower relative abundances (typically >1%), further demonstrating the adaptability of mixed microorganisms to various carbon sources, including lignocellulose hydrolysate. These genera are often associated with nutrient cycling, organic matter degradation, and biopolymer synthesis, indicating that even low-abundance taxa may play supportive or complementary roles in the overall PHA production process.

Figure 4b presents the microbial community structure at the phylum level across all samples. The dominant phyla in all reactors were Bacteroidetes, Proteobacteria, Actinobacteria, and Patescibacteria. Among them, the abundance of Bacteroidetes changed the most with increasing substrate concentration, rising from 28.31% in R500 reactor to 54.65% in R1700 reactor. This indicated that Bacteroidetes mainly absorbed fermentable sugars from lignocellulose hydrolysate for growth, proliferation and PHB accumulation [45]. Moreover, members of Bacteroidetes are known to possess diverse carbohydrate-active enzymes (CAZymes), which enable them to efficiently degrade polysaccharides into utilizable monomers, thereby supporting both cell growth and intracellular polymer synthesis. As the substrate concentration increased, the relative abundance of Proteobacteria, Actinobacteria, and Patescibacteria decreased. This change may be due to the osmotic pressure generated by high substrate concentrations, which limited the ability of these bacteria to absorb substrates effectively, thereby reducing their abundance in the reactor [42]. Additionally, competition for readily available carbon sources may further suppress the growth of less competitive microbial groups under high substrate conditions. These findings are consistent with previous research findings showing that high substrate concentrations can exert selective pressure on microbial communities, favoring species that can tolerate such conditions. The results revealed that different substrate concentrations had no significant effect on microbial community type but had a greater impact on relative abundance. Overall, this suggests that the core microbial consortium remained stable, while functional dominance shifted toward species better adapted to high substrate and osmotic stress environments, ultimately influencing PHA production efficiency.

4. Conclusions

This study investigated the use of pretreated lignocellulosic hydrolysate as a carbon source for the production of polyhydroxybutyrate (PHB) by mixed microbial cultures (MMCs). The results demonstrated that a nitrogen concentration of 55 mg/L and a pH of 7 led to the highest PHB production (612.35 mg/L) when the substrate concentration was 1700 mg/L. Microbial community analysis revealed Chryseobacterium as the dominant genus, which played a key role in the efficient conversion of lignocellulosic hydrolysate into PHB. These findings provide valuable insights for optimizing the production of PHB from lignocellulosic waste and for advancing the industrial-scale application of MMCs-based PHA production. This study is limited by the use of a single-factor experimental design, which does not account for potential interactions among key parameters such as substrate concentration, C/N ratio, and pH. Future work should employ multivariate optimization approaches (e.g., Response Surface Methodology) to better elucidate the synergistic and antagonistic effects among these factors and achieve more comprehensive process optimization.