Screening of PHA-producing strains and characterization of PHA
Out of the total Bacillus sp., one of them demonstrated to produce PHA using Nile red and Sudan Black B staining procedure on Saline-Alkali LB agar (pH 8.0). It can be observed that the PHA in the B. cereus HBL-AI specifically binds to Nile red and emits red fluorescence under the excitation of ultraviolet light (Fig. 1a). Under the microscope, black particles strained with Sudan black inside the strain were observed (Fig. 1b). To further determine the accumulation of PHA, the strains fermented for 72 h were observed by TEM (Fig. 1c). It can be seen that PHA granules exist in the cells with a diameter of 200-1000 nm.
The PHA produced by this strain was extracted. The infrared spectrum of the product is shown in Fig. 1d. 1724 cm-1 is the C=O stretching vibration absorption peak, which is the characteristic absorption peak of PHA. It can be confirmed that the extracted polymer is PHA. The monomer types of the PHA polymers were analyzed by GC-MS. The GC-MS spectrum (Fig. 1e) shows that the main peak with a retention time of 4.20 min is methyl 3-hydroxybutyrate, indicating that the intracellular polymer is Poly-β-3-hydroxybutyrate (PHB). The structure of the product is similar to the previous results by mostly isolated Bacillus strains (Zhang et al. 2013).
Fed‑batch production of PHB
B. cereus HBL-AI was open cultured in a 5 L fermentor, and the Fed‑batch production of PHB was tested. In this batch culture, the supply of glucose was particularly important for the sustained production of PHB by this strain, so it was added continuously to ensure that its content in the fermentor was kept at a concentration of 20 g/L. Its sufficient supply ensured the accumulation of PHB. With the growth of the strain, the concentration of PHB was enhanced. The accumulation concentration of PHB reached a maximum value of 12.8 g/L in 48 h and then decreased slightly (Fig. 2). A maximal CDW of 20.1 was also obtained. It means that HBL-AI accumulated 63.7% w/w P(3HB) during fermentation. The fermentation performance was higher than that of recently reported B. cereus FA11 (1.42 g/L) (Evangeline et al. 2019), B. thuringiensis B417-5 (2.768 g/L) (Thammasittirong et al. 2017) and B. megaterium CAM12 (8.31 g/L) (Silambarasan et al. 2021). In the end, the total consumption of glucose was 100 g/L. The final product was extracted and further identified by NMR. It also proved that the intracellular polymer obtained fromHBL-AI was PHB, and its monomer was 3-hydroxybutyric acid (3HB) (Fig. 1f, g). This open fermentation form of Bacillus requires no sterilization of the culture medium, which not only shortens the fermentation cycle, but also saves costs.
Construction of the expression system and purification of PHA surface binding protein
The PHB synthesis gene cluster of B. cereus HBL-AI was found according to the genome-wide functional annotation of protein-coding genes (Wang et al. 2021). HBL-AI has a pha locus that consists of phaR-phaB-phaC operon and phaJ-phaP-phaQ operon in the opposite direction (Fig.3a). The phaR (366 bp) and phaC (1086 bp) genes encode PHA synthase subunits, whereas phaB (744 bp) encodes an NADPH dependent acetoacetyl-CoA reductase (PhaB), which plays a role in the supply of (R)-3HB-CoA monomer for PHA polymerization (Kumar et al. 2020). The phaP gene (525 bp) encodes PHA granule-associated protein (PhaP). PhaP is a non-enzymatic protein localized on the surface of PHA granules in the cells and functions to block the binding of unnecessary proteins. The phaQ gene (453 bp) encodes a P(3HB)-responsive transcriptional regulator (PhaQ) that negatively controls the expression of both phaQ and phaP (McCool et al. 2001). The phaJ gene (540 bp) encodes R-specific enoyl-CoA hydratase (PhaJ), which is a member of the MaoC-like protein family. PhaJ is a monomer supplying enzyme from fatty acid β-oxidation (Kihara et al. 2017).
The whole genome of HBL-AI was used as a template, and the PHA surface binding proteins genes of phaR, phaP, phaQ, and phaC were successfully cloned by PCR amplification (Fig. 3b). These gene fragments were inserted into plasmids pET-28a and were transformed into E. coli BL21(DE3)plysS separately. Protein expression and purification of these recombinant strains were conducted, and the purified PhaR, PhaP and PhaQ were obtained except for PhaC that is an inclusion body. As shown in SDS-PAGE (Fig 3c), those protein bands are single, the size of the protein is the same as the predicted molecular weight, which are 17.3 kDa, 23.9 kDa and 20.7 kDa respectively for PhaR, PhaP and PhaQ.
Emulsion function of PHA surface binding protein
Diesel oil, vegetable oil and lubricating oil were selected as the oil phase to test the emulsifying properties of solutions of PhaR, PhaP, PhaQ and BSA compared with the traditional chemical emulsifiers SDS, Tween-20 and sodium oleate. As shown in Fig. 4a, PhaR, PhaP and PhaQ are of good emulsifying effects on diesel oil, with an obvious emulsifying layer. Tween-20 and sodium oleate can emulsify diesel oil to form an emulsifying layer, but the emulsifying layer is small, indicating that their emulsifying ability of diesel oil is weak. SDS produce slight emulsification at the water/oil interface. BSA fail to emulsify diesel oil. It can be seen from Fig. 4b that the emulsification values of PhaR, PhaP and PhaQ do not change significantly after 30 days of sample standing, which indicates that the emulsion system formed is stable and the three proteins have good emulsion layer stability.
PhaR, PhaP and PhaQ also have a good emulsifying effect on lubricating oil, and an obvious emulsion layer can be observed. Other surfactants have no significant effect on the formation of the emulsifying system. PhaP is slightly better than PhaR and PhaQ in emulsifying effect in the lubricating oil (Fig. 4c). The emulsification values of PhaR, PhaP and PhaQ did not change significantly after the samples were kept for 30 days (Fig. 4d), indicating that the emulsification system formed by these three proteins had good stability.
As shown in Fig. 4e, PhaR, PhaP, PhaQ, BSA and SDS had good emulsifying effects on vegetable oil, and an obvious emulsifying layer can be observed; Tween 20 can emulsify vegetable oil, but its emulsifying effect is not obvious; sodium oleate cannot emulsify vegetable oil. It can be seen that PhaP is slightly better than PhaR and PhaQ in emulsifying vegetable oil, and the emulsifying properties of the three proteins to vegetable oil are better than other surfactants. After the sample was left standing for 30 days, the emulsification values of PhaR, PhaP and PhaQ did not change significantly, but the emulsification values of SDS, Tween-20 and BSA all decreased significantly (Fig. 4f). It is indicated that the stability of the emulsification system formed by emulsifying vegetable oil with three protein solutions is better than that formed by other surfactants, and all three proteins have good emulsion layer stability.
Effect of surface binding protein concentration on emulsion stability
Diesel oil, vegetable oil and lubricating oil emulsions with different concentrations of surface binding protein (PhaR, PhaP and PhaQ) were prepared under the same vortex intensity. After 48 h of still-standing, various emulsion stabilities were observed. It can be concluded that the higher concentration of the PhaR, PhaP and PhaQ, the more stable the emulsion (Fig. 5a). Whereas the emulsifying effect of protein solutions with different concentrations on the oil phase is not the same. In the case of the diesel oil, emulsion layers started to form stably when the concentration of PhaR, PhaP and PhaQ exceeded 500 μg/mL. Stable emulsions could be observed in the presence of 200 μg/mL of PhaR and PhaP in the case of vegetable and lubricating oil. However, the concentration of PhaQ in vegetable oil to form a stable emulsion layer was as low as 50 μg/mL, while in lubricating oil, the emulsion layer was formed at a high concentration of 500 μg/mL. PhaR and PhaP from B. cereus have the same emulsion layers and emulsion indexes as that of PhaR and PhaP from A. hydrophila 4AK4 in the three oil phases at the same protein concentration (Wei et al. 2011; Ma et al. 2013). However, the minimum concentration of PhaQ (50 μg/mL) from Bacillus that achieves the same emulsification efficiency in vegetable oil is significantly lower than that of PhaR and PhaP, which also shows that PhaQ can reduce the amount of protein used in emulsification and indirectly save costs.
Stability test of PhaR, PhaP and PhaQ
The capability of inversion and thermal stability of PhaR, PhaP and PhaQ in emulsion was studied. Firstly, the emulsion systems of PhaR, PhaP and PhaQ solutions with diesel oil, vegetable oil and lubricating oil were turned upside down, and whether flow changes in the emulsion layer were observed. It can be seen from Fig. 5b that all water layers stably stayed on the upper part of the vial without leaking no matter which emulsion system it is. These also suggested that PhaR, PhaP and PhaQ could form a fairly stable emulsion structure with the oil phases at a protein concentration of 1000 μg/mL.
Secondly, the thermal stabilities of PhaR, PhaP and PhaQ were investigated at 60 ℃ and 90 ℃ for 30 min. Similar to the emulsification of unheated proteins in oil, it was observed that when the three proteins were heated to 60 ℃, a good emulsified layer was formed in the three oil phases (data not shown). When the temperature rose to 90 ℃, the three proteins could only keep good emulsification in vegetable oil, and the emulsification values of PhaR, PhaP and PhaQ were 80%, 86.7%, and 66.7%, respectively. But the emulsifying ability of the three proteins in lubricating oil was obviously reduced and that was completely lost in diesel oil (Fig. 5c). The poor performance of the three proteins in diesel oil is in sharp contrast to the good thermal stability of PhaR from A. hydrophila 4AK4 [8], which might relate to the source of diesel oil. The structural stability of PhaR, PhaP and PhaQ in vegetable oil after heating is also conducive to their application in food or cosmetics, because vegetable oils such as soybean oil and related derivatives are usually used as additives (Sarubbo et al. 2022). Amphiphilic proteins as surfactants have better biocompatibility and safety than chemical surfactants.