Integrating the processes of enzymatic hydrolysis and PHB production has a great potential to avoid product inhibition, and simplify the conversion of cellulosic biomass into PHB. This study focused on PHB production using waste paper through SSF by C. necator, where both enzymatic hydrolysis and sugar fermentation coincide. C. necator accumulates high quantity of PHB inside the cell as a reserve material of carbon and energy. It synthesize the PHB by catalyzing the condensation of two molecules of acetyl CoA from the tricarboxylic acid cycle, into acetoacetyl CoA with the help of the enzyme β- ketothiolase encoded by the gene phbA. Then the reduction of acetoacetyl CoA into 3-hydroxybutyryl-CoA is mediated by acetoacetyl CoA dehydrogenase enzyme encoded by phbB gene. Finally, the PHB synthases (encoded by phbC) facilitated the polymerization of 3-hydroxybutyryl-CoA monomers into PHB homopolymer [26].
In the SHF study, the hydrolysis was conducted at 50 ℃, and pH 4.8, while the microbial fermentation was carried out at 30 ℃ and pH 7.2 [3, 13]. However, in the case of the SSF lower temperature and higher pH may affect the efficiency of the enzyme. Therefore, we tried to increase the temperature to a level that will not inhibit the growth of C. necator. Hence, in this study, the conditions for microbial growth were fixed as 35 ℃ and pH 7.0, which may ultimately affect the saccharification process of waste paper. Therefore, the strategy adopted to increase the sugar release was increasing the enzyme concentration by 1.5-fold in the SSF process and an extra-pretreatment using Triton X-100.
3.2. PHB production through batch and fed-batch SSF mode
Initially, OPH and OPHT treated paper were assessed at different substrate loadings (3, 5, and 10% paper) through batch SSF. The dry weight represents both residual paper and the microbial biomass (Figs. 2 and 3). They have an inverse relationship where the paper was decreasing, and the biomass was increasing during the course of SSF. In addition to the free cells in the liquid medium, a good amount of bacterial cells attached to the shredded paper makes the separation difficult. OPHT showed a higher reduction rate of the dry weight than OPH, particularly between 24 and 72 h, which was visually liquefied faster. At the last 24 h of the fermentation, the paper conversion rate decreased significantly to around 4% in both 5% OPH and 5% OPHT, and even less than 1% in 3% OPHT. Almost 7.03 and 5.77 g/L of dry weight remained in both 3% OPH and 3% OPHT (Fig. 2A and B) while 11.63 and 12.93 g/L in 5% OPH and OPHT (Fig. 2A and B), respectively. Paper conversion was evidenced by the residual glucose content that has reached the maximum at 48 h of incubation (Fig. 2A-D). However, more residual glucose was found in both OPHT loading paper, reaching a maximum of 3.07 and 6.19 g/L from 3 and 5%, respectively. At the end of the fermentation, glucose concentration in both treatments at 3% loading was almost utilized by C. necator, while 0.71 and 3.37 g/L remained in 5% OPH and OPHT, respectively. PHB content was higher in OPH than the OPHT medium in both paper loadings. After 24 h, 5% OPH reached the maximum PHB (0.46 g/L) (Fig. 2C) while 3% OPH reached the maximum PHB (0.31 g/L) at 48 h (Fig. 2A) due to the C/N ratio.
Similarly, higher paper loading (10%) using both OPH and OPHT was tested on their effect on PHB production, and the fermentation period was extended for seven days (168 h). At the beginning of the experiment, OPHT took 48 h to liquefy, and hence the first samples were taken; however, OPH was faster in liquefaction. The results were consistent with the previous solid loading experiments regarding residual glucose, dry weight, and PHB (Fig. 3). The general overview of the OPH experiment showed that the residual glucose and dry weight were almost constant between 48 to 144 h with an increment of PHB yield. This is due to the simultaneous increment of enzymatic hydrolysis and, consequently, bacterial biomass, which is acknowledged by PHB accumulation. The maximum PHB yield 4.27 g/L was achieved using OPH at 6 days of incubation. Nevertheless, there was a sudden decrease in the PHB yield at the end of the incubation. Unlike OPH, the OPHT experiment showed a delay in the hydrolysis at the beginning because OPHT was processed twice, which resulted in a semi-ground structure. This increased the paper surface area, and consequently, higher absorption of water. Despite the higher glucose in the medium, the PHB yield was almost nil till 144 h of incubation. After that, the PHB production slightly increased in coinciding with glucose utilization.
The higher sugar concentration in OPHT treated paper proves the efficiency of additional triton X-100 treatment in improving the enzymatic hydrolysis of paper fibers [37, 38]. Application of surfactant in pretreatment and enzymatic digestion favored enzymatic hydrolysis of various biomass through different ways; 1) surfactant reduces the irretrievable adsorption of the enzyme keeping it free with superior activity; 2) surfactant preserves the enzyme from thermal denaturation by reducing the surface tension; 3) surfactant intensifies the electrostatic interaction between the enzyme and the biomass; 4) surfactant increases the lignin and hemicellulose removal, the primary hurdle for effective saccharification, through emulsions process since both contain hydrophobic part and 5) surfactant causes an alteration in biomass structure and increases surface area makes it suitable for enzymatic hydrolysis [39–42, 15]. Despite the very close paper conversion rate in terms of dry weight (paper and bacterial culture) of both treatments at 10% solid loading, the PHB yield was low in the OPHT. This is due to the presence of residual amount of Triton X-100 remained after pretreatment, which affected the microbial growth. Qu et al. [38] found that Triton X-100 affected the transportation and utilization of reducing sugar, leading to a detrimental effect on the metabolism and growth of Clostridium thermocellum. Moreover, Triton X-100 boosts the enzymatic hydrolysis, led to the release of high sugars compared to OPH treatment affeced the growth of the bacteria by a very high C/N ratio in the production medium. Despite nitrogen limitation is a favorable condition for higher PHB content, a higher C/N ratio than the optimum condition minimized C. necator growth and hence, the PHB yield [3]. In the case of 10% OPH, the higher PHB yield was achieved due to the higher released-sugar, which increased the C/N ratio compared to the previous paper loadings. In contrast, the later reduction in the PHB after 144 h was due to the polymer degradation by bacteria for its growth or due to the concomitant occurrence of both saccharification and fermentation, causing a fluctuation in the C/N ratio during SSF mode of fermentation.
A fed-batch SSF was also tried to increase the yield of PHB production by the gradual increase of the paper load in the medium of both OPH and OPHT, starting with 3% (Fig. 4). The results showed that the residual glucose increased after the addition of the paper (36 and 84 h) and decreased later, apparently due to the bacterial growth. In both cases, the production of PHB was deficient at the first stage of the growth, like the batch SSF (3% paper of both OPH and OPHT) experiment. The first addition of paper (3%) in fed-batch experiment led to an increase in the PHB reaching the maximum (4.19 g/L) at 66 h of incubation in the OPH, but not in OPHT. Hereafter, PHB production decreased in the OPH experiment despite the second batch addition of the paper. The results showed that the second supplement of the substrate increases the residual sugar concentration but not PHB production. However, the PHB production started to increase at the end of the fermentation (after 115 h). OPHT showed almost half the PHB yield (2.05 g/L) of the OPH, only after 114 h of incubation. This indicates that double pretreatment, specifically Triton X-100, did not improve the PHB production; instead, it affects the growth of C. necator by interfering with sugar transport.
Dahman and Ugwn [24] used both SHF and SSF processes for PHB production using C. necator by utilizing wheat straw as a feedstock. The results showed that SSF is more influential than SHF and even pure sugar in terms of PHB yield and microbial growth. More recently, cereal mash was used as a substrate for PHB production through SSF using Halomonas boliviensis, resulted in a 60% higher PHB polymer than SHF mode with a 47% reduction in the overall processing time [25]. Although the PHB yield of our fed-batch mode (4.19 g/L) was almost closer to the 10% batch mode (4.27 g/L) experiment, its production was achieved within a shorter time almost half period (66 h) in fed-batch. However, unlike previous studies, the production of PHB from waste paper through SSF was still lower than that produced through SHF. The PHB yield of this study is higher than that obtained by B. sacchari (3.6 g/L) [13], but slightly lower than using C. necator (5.3 g/L) in shake flask experiments using SHF [3]. Table 1 represented the PHB yield of C. necator and Burkholderia sp. by utilizing various lignocellulosic biomass. The fluffy nature of waste paper materials interfere during the analysis of PHB in the samples and affected the final yield. Thus, SSF application simplified the processes while achieving the comparable PHB production. However, this result was achieved using 10% paper loading, unlike the SHF, which achieved using 3% paper loading only. Indeed, the coordination of waste paper enzymatic hydrolysis and sugars assimilation is essential for further improving the efficiency of the SSF approach. Further optimization studies on SSF conditions such as temperature, substrate loading, enzyme loading, and agitation will increase PHB production. Also, the fed-batch SSF process can be improved further by optimizing the quantity and time of paper loading.
Table 1
Cell dry weight (CDW) and PHB yield of C. necator and Burkholderia sp. by utilizing various lignocellulosic biomass (LCB) through SHF and SSF mode
PHB producer | LCB | Operation mode | CDW (g/l) | PHB (%) | PHB (g/l) | Yield (P/S) | Ref. |
C. necator | Sugarcane Bagasse | SHF, Batch | 6.0 | 65 | 3.9 | ND | [53] |
C. necator | Water hyacinth | SHF, Batch | 12.0 | 58 | 7.0 | 0.24 | [54] |
C. necator | Sunflower | SHF, Batch | 10.9 | 73 | 7.8 | 0.40 | [55] |
C. necator | Wheat bran | SHF, Batch | 24.5 | 63 | 14.8 | 0.32 | [56] |
C. necator | Sargassum | SHF, Batch | 5.3 | 74 | 3.9 | 0.47 | [49] |
B. sacchari | Waste OP | SHF, Flask, Batch | 3.6 | 44 | 1.6 | 0.15 | [13] |
C. necator | Waste OP | SHF, Flask, Batch | 7.7 | 57 | 4.5 | 0.21 | [3] |
C. necator | wheat straw | SHF, Flask, Batch | 12.2 | 58 | 7.1 | 0.13 | [24] |
C. necator | wheat straw | SSF, Flask, Batch | 15.3 | 65 | 10.0 | 0.16 | [24] |
C. necator | Waste OP | SSF, Flask, Batch | ND | ND | 4.27 | ND | This study |
| | SSF, Flask, Fed-batch | ND | ND | 4.19 | ND | This study |
*ND: Not Defined, *SHF: separate hydrolysis and fermentation, *SSF: simultaneous saccharification and fermentation |
The structural changes of the paper after the fermentation was observed through SEM. Figure 5A-F showed representative figures of 3 and 5% OPH and OPHT filtered residues, containing paper residues and bacterial culture, at different magnifications. Figure 5B and C illustrated the abundant growth of C. necator attached to the paper fibers. Paper fibers were particularly hydrolyzed into fermentable sugars and utilized by the microbes (Fig. 5D). At the end of the fermentation, the toner particles were visually noticed after the hydrolysis (Fig. 5E). Interestingly, it was observed that the tonner crystal did not affect the growth of C. necator. Instead, the bacterial cells used the tonner crystal as a surface for their growth (Fig. 5F).
FTIR spectra of both OPH and OPHT before and after SSF were carried out in the range of 750‒4000 cm‒1 (Fig. 6) to assess the nature of residues remained at the end of the experiment. Overall, peaks intensity decreased after hydrolysis compared to unhydrolyzed materials, specifically at 1000‒1770 cm‒1. Besides, the peak at 875 cm‒1, which represents C-H of syringl content in lignin is almost disappeared. Lignin and cellulose peaks are significantly declined. Cellulose was utilized by bacteria in the form of glucose, while lignin derivatives were definitely released in the medium, especially in OPH treatment. Although PHB is higher in the OPH experiments, lignin derivatives could have a side effect compared to the PHB yield of SHF [3].
3.3. PHB characterization
Thermal properties of PHB differs among PHB producing microbes [43]. The thermal strength of the PHB produced by C. necator (PHB/CN) was evaluated using thermogravimetric analysis (TGA) having the standard/pure PHB (PHB/STD) procured from Sigma (363502) as a reference (Fig. 7A). The TGA is used to analyse the changes in the mass of a sample with increased temperature in a time period. The dehydration of solvents in the PHB resulted in a weight loss from 130‒230°C. Further decrease in PHB weight occurred ~ 300°C led to the crotonic acid formation [44, 45]. From 300‒800°C the PHB/CN produced using waste paper substrate exhibited a high stability and residual weight. The delay in complete degradation compared to the PHB/STD showed the possible presence of some impurities. The differential thermogravimetric (DTG) curves deduced from TGA confirms the thermal stability of the produced PHB. The degradation temperature of the PHB/CN and the PHB/STD were in the same range, 293.39 and 301.50 respectively (Fig. 7B).
The molecular structure of the PHB was studied using 1H and 13C NMR spectra (Fig. 8). The presence of methyl group was represented by peaks near 1.3 ppm in 1H NMR. The methylene group was presented by two peaks at 2.4 and 2.6 ppm and the methine group by 5.2 5ppm. The 13C NMR spectrum showed the presence of CH3, CH2, CH and C = O groups at 19, 40, 67 and 169 ppm respectively. The above studies demonstrated that the PHB produced by SSF was a homoplomer and comparable to the previous studies [45, 46].
The FTIR spectrum results (Fig. 9) revealed the presence of C‒O stretching ester band at 1055.8 cm‒1 and C‒O‒C symmetric stretching amorphous band at 1183.2 cm‒1 [47]. The bands at 1277.8 cm‒1, 2930.5 cm‒1 and 1379.7 cm‒1 are assigned to methylene, methane andmethyl groups respectively. The strong band at 1721 cm‒1 denotes the crystalline carbonyl group in the ester group of PHB. The FTIR spectral study supported the NMR results and corroborates the earlier reports [48, 49, 47, 45].
The XRD diffractograms of the extracted PHB (PHB/CN) and standard (PHB/STD) are presented in Fig. 10. The diffractogram of PHB is predominated by five major peaks at the corresponding 2θ = (020) at 13.5°, (110) at 16.9°, (101) at 21.4°, (111) at 22.5°, and (121) at 25.5°. The crystallinity of the PHB revealed by the strong peak located at 2θ = 13.5o at 020 plane. The PHB produced through SSF, PHB/CN, is less crystalline or slightly amorphous when compared to standard PHB. The less intensed peak at 2θ of 020 in the extraxted PHB evidenced the low crystallinity [50–52]. The other peaks observed at various 2θ of both PHB/CN and PHB/STD are reflecting the same pattern and similar to the PHB produced from sugarcane bagasse (Salgaonkar and Bragança, 2017) and rice straw [50].