Composition of corn stover. The composition of native bagasse used in this study was determined to be 35.8 ± 0.51% cellulose, 24.2 ± 0.23% hemicellulose, 28.5 ± 0.41% Klason lignin, 5.3 ± 0.21% ash, and 6.24 ± 0.13% others on a dry basis. The carbohydrate content of native bagasse accounted for 60.0% (as cellulose and hemicellulose). The other components that are chemically bound are water- and ethanol-soluble materials and proteins.
Effects of different pretreatment of bagasse. The pretreatment of bagasse with various methods was first investigated (Table 1). The LHW pretreatment condition was tested at 180°C for 30 min without catalyst. In LHW, water is the main solvent at high temperatures under pressurized conditions. Under these conditions, hydronium ions are generated in situ by the ionization of water, leading to the release of acetic acid from hemicelluloses, which in turn autocatalyses the solubilization of hemicelluloses in an acidic environment and leads to the degradation of carbohydrates15. The presence of autocatalysts in LHW pretreatment could clearly enhance the glucose yield and remove hemicellulose and lignin from bagasse. The use of liquid hot water significantly improved the glucose yield by 50.2% compared to native bagasse (35.8%). Alkaline pretreatment of bagasse under conditions of 90°C for 30 min at a NaOH concentration of 5% w/v resulted in a higher cellulose content after delignification. The cellulose content in native bagasse increased from 35.79% (native) to 67.61% (pretreated). Alkaline pretreatment refers to the removal of lignin and part of hemicellulose so that it can enhance the accessibility of enzymes to cellulose. The saccharification effect can be significantly enhanced by alkaline pretreatment16. The hydrogen link between cellulose and hemicellulose and the ester bond between saponified hemicellulose and lignin molecules can both be weakened by OH during alkali treatment17. NaOH pretreatment can also cause raw wood fibers to swell, which damages the structure of the lignin and results in broken chemical connections between the lignin and the carbohydrates as well as increased internal surface area and decreased degree of polymerization and crystallinity18. Xu et al.19used beanstalk as a raw material and subjected it to a 24-hour period with 10% ammonia. The results showed that the lignin and hemicellulose yields decreased by 30.61% and 41.45%, respectively. Zhao et al.20 used Crofton weed stem as a raw material and treated it with various pretreatment methods for enzymatic hydrolysis. The results showed that the glucose yield from enzymatic hydrolysis from NaOH pretreatment was higher than the glucose yield obtained from H2SO4. Compared with LHW, alkaline pretreatment is more destructive to the ester bonds among lignin, hemicellulose, and cellulose and can avoid the breakage of hemicellulose polymers21. Previous studies have shown that using alkaline pretreatment on lignocellulosic biomass mainly depends on the lignin and hemicellulose contents in the raw materials22.
Table 1
Bagasse composition pretreated under various LHW pretreatment conditions
Sample | Cellulose | Hemicellulose | AIL | ASL | Ash | Other (Extractive) | Pulp yield |
Raw bagasse | 35.79 | 24.23 | 23.72 | 4.77 | 5.25 | 6.24 | - |
Remaining solid after liquid hot water pretreatment 180ºC 30 min | 50.18 | 7.83 | 25.21 | 4.57 | 6.69 | 5.53 | 58.91 |
Remaining solid after alkaline pretreatment (5%w/v NaOH) 90ºC 30 min | 67.61 | 22.36 | 4.43 | 2.04 | 2.35 | 1.21 | 48.04 |
Physiochemical characterization of solid fraction. The morphology and surfaces were characterized to comprehend the change in LHW pretreatment of bagasse. The morphological characteristics of the local and pretreated samples are depicted in Fig. 1. The untreated bagasse exhibited a smooth, compact, and fibrous appearance, attributed to the presence of recalcitrant structures (Fig. 1A). In contrast, the pretreated bagasse displayed noticeable surface disruption, leading to the exposure of internal structures (Fig. 1B). SEM micrographs confirmed that the cell wall of pretreated bagasse was broken down, which exhibited loosened fibrous networks and irregular, rough, microporous, and cracked surfaces23. These observations can be attributed to the removal of hemicellulose and lignin, as well as the effects of LHW and alkaline pretreatment. Further insight into the structural changes was gained through X-ray diffraction (XRD) analysis, as shown in Fig. 2. The results demonstrated that the crystallinity index increased during the LHW pretreatment from 48.3–54.2%, as during the alkaline pretreatment from 48.3–55.4%. This result revealed that amorphous structures (e.g., hemicellulose and lignin) were removed from the cellulose content as the main remaining solid fraction24. Furthermore, the physiochemical changes in bagasse during the pretreatment processes are s summarized in Table 2 in terms of surface area. The table provides an overview of the correlation between the surface area and pore volume of native and pretreated bagasse under optimized conditions. The surface area of the pretreated bagasse increased from 5.7 to 13.6 m2/g, which was 2.4-fold greater than that of the native bagasse. Additionally, the pore volume increased from 0.07 cm3/g to 0.21 cm3/g. A significant surface area corresponded to the surface change caused by LHW and alkaline pretreatment, as determined by SEM analysis. The crystallinity index of pretreated bagasse was observed due to the elimination of the amorphous structure of hemicellulose and lignin; nevertheless, the crystalline structure of cellulose was relatively unaffected. Previously, increases in lignocellulose crystallinity were observed in various lignocelluloses pretreated with LHW, for instance, rice straw pretreated with LHW25 and other pretreatment methods, e.g., dilute acid pretreatment26 and alkaline pretreatment27. On the other hand, certain pretreatment techniques, such as ionic liquids, have been reported to cause complete destruction of the crystalline cellulose structure28. This evidence supports the notion that the pretreatment of bagasse with either LHW or alkaline methodologies could facilitate further applications.
Table 2
Surface area and pore volume of native and pretreated bagasse under optimized conditions.
order | Native Bagasse | Pretreated Bagasse |
Pore volume (cm3/g) | 0.07 | 0.21 |
Surface area (m2/g) | 5.7 | 13.6 |
Enzymatic hydrolysis of pretreated bagasse. The effect of enzyme loading on the hydrolysis of bagasse was studied. A maximum glucose yield of 1.53 g/g cellulose was obtained when the LHW-pretreated sugarcane bagasse was hydrolyzed for 48 h at an enzyme loading of 25 FPU/g cellulose, whereas that of glucose from alkaline-pretreated hydrolysate was clearly lower (0.99 g/g cellulose) under the same conditions (Fig. 3).
Screening and identification of actinomycete for IAA production and medium selection. Thirty-four actinomycete isolates obtained from rhizosphere soil samples showed valuable IAA production in the range of 3.32–13.08 µg/ml. These isolates were grown in three different media (ISP No. 2, YM and GMP) supplemented with 5 mg/mL L-tryptophan. It was found that isolate BS50-1 produced the highest IAA (37.49 µg/mL) from ISP No. 2 medium supplemented with L-tryptophan. This isolate was selected for further study.
The isolate BS50-1 was identified based on the 16 s rRNA gene sequence, consisting of 1,420 nucleotides, and showed that isolate BS50-1 was closely related to Streptomyces lavenduligriseus species. The 16S rRNA gene sequences of strain BS50-1 shared 99.93% similarity with S. lavenduligriseus species. BS50-1 isolates were identified as S. lavenduligriseus, and their 16S rRNA gene sequences were deposited in the GenBank database with accession number OQ135193.
Optimization of IAA production. The effect of two independent variables, glucose (X1) and L-tryptophan (X2) on IAA production was determined using response surface methodology (RSM) based on central composite design (CCD) with 11 experimental runs, as shown in Table 3.
Table 3
The effects of process factors, including glucose and L-tryptophan, on IAA production obtained by experimental design
Run no. | Level | Actual level | IAA (µg/mL) |
X1 | X2 | X1 (Glucose, g/L) | X2 (L-tryptophan, g/L) | Observed | Predicted |
1 | -1 | -1 | 2 | 1 | 18.31 | 13.35 |
2 | 1 | -1 | 6 | 1 | 46.01 | 48.83 |
3 | -1 | 1 | 2 | 5 | 139.54 | 138.46 |
4 | 1 | 1 | 6 | 5 | 100.42 | 107.11 |
5 | -1.68 | 0 | 1.17 | 3 | 42.49 | 47.04 |
6 | 1.68 | 0 | 6.83 | 3 | 56.41 | 49.97 |
7 | 0 | -1.68 | 4 | 0.17 | 38.58 | 40.40 |
8 | 0 | 1.68 | 4 | 5.83 | 173.74 | 170.15 |
9 | 0 | 0 | 4 | 3 | 115.18 | 118.43 |
10 | 0 | 0 | 4 | 3 | 110.63 | 118.43 |
11 | 0 | 0 | 4 | 3 | 129.48 | 118.43 |
The regression-based determination coefficient, R2, was evaluated to test the model equation's fit. The value of the determination coefficient (R2 = 0.986) explained 98.6% of the fitness between the observed and predicted values, and the other 1.4% was affected by other variables. Fisher’s F test (71.09) and a very low probability value (p-model = 0.0001), which was indicative of the fit of the model demonstrated that the model was statistically significant (Table 4). The 0.70 lack-of-fit F value means that the statistical value relative to the pure error is not significant, which is good for the model. This result indicated that the response equation provided a suitable model of the relationship between the independent variables and the response. IAA production can be predicted by Eq. 2,
Table 4
Analysis of variance (ANOVA) for optimization of IAA production
Source | Sum of Squares | DF | Mean Square | F-value | p-value | |
Model | 24913.21 | 5 | 4982.64 | 71.09 | 0.0001* | |
X1 | 8.54 | 1 | 8.54 | 0.12 | 0.7412 | |
X2 | 16816.41 | 1 | 16816.41 | 239.95 | < 0.0001* | |
X1X2 | 1116.23 | 1 | 1116.23 | 15.93 | 0.0104* | |
X12 | 6887.24 | 1 | 6887.24 | 98.27 | 0.0002* | |
X22 | 243.61 | 1 | 243.61 | 3.48 | 0.1213 | |
Residual | 350.42 | 5 | 70.08 | | | |
Lack of Fit | 157.02 | 3 | 52.34 | 0.54 | 0.7001 | |
Cor Total | 25263.63 | 10 | | | | |
*Significance level = 95%, R2 = 0.9861, Adjusted-R2 = 0.9723, C.V. %= 9.49 |
$$Y=-156.99511+82.89134{X}_{1}+49.48105{X}_{2}-4.17625{X}_{1}{X}_{2}\pm 8.73075{X}_{1}^{2}-1.642{X}_{2}^{2}$$
2
where Y is IAA concentration (g/L), X1 is glucose (g/L), and X2 is L-tryptophan (g/L),
The results of the regression model revealed that only L-tryptophan (X2) had a significant effect on IAA production, whereas the effect of glucose concentration was not statistically significant. The interaction between glucose and L-tryptophan was significant. The quadratic terms of glucose (X12) were significant, while those of L-tryptophan concentration (X22) were not significant.
The interaction relationship and the optimal values of the variables were determined by the response surface plots (Fig. 4). Figures 4A and 4B indicate that there was a significant interaction between glucose and L-tryptophan for IAA production, as shown by the low p-value (0.0104 < 0.05) in Table 4. IAA production increased with increasing L-tryptophan and glucose concentrations. It was evident that when glucose increased from 2.0 g/L to 3.5 g/L, IAA production increased, but an excessive increase in glucose did not increase the yield of IAA any further. When L-tryptophan concentrations were increased from 1.0 g/L to 5.0 g/L, IAA production increased. In this case, the highest IAA concentration was obtained with glucose within the range of 3.40 to 3.60 g/L and concentrations of L-tryptophan at 5.0 g/L.
The optimized values of the variables were obtained from the regression equation and from the response surface contour plots using Design Expert software. The model predicted that the maximum IAA concentration of 159.47 µg/mL occurred at 3.55 g/L glucose and 5.0 g/L L-tryptophan. Validation of the experimental model, repeated three times under the optimum conditions, showed that the observed value of 159.25 µg/mL was close to the predicted value of 159.47 µg/mL. A yield and productivity of 0.07 g/g and 0.055 g/L/h, respectively, were obtained with an IAA concentration of 159.25 µg/mL, approximately 4.25-fold higher than that from the control medium.
The optimum conditions obtained from the preliminary experiment were further studied for different time intervals (24, 48, 72, 96, 120, 144, and 168 h) to determine the effect of incubation time on IAA production. The optimum time with the maximum IAA concentration at 159.25 was observed after 168 h of incubation. The obtained results are consistent with this work. Benadjila et al.29 optimized the biotechnological fermentation of IAA by strains of actinomycetes from agricultural waste (roots and leaves of wheat) using RSM. The results showed that Saccharothrix texasensis MB15 is the best IAA-producing strain on a medium based on the roots and leaves of wheat only. Factors including L-tryptophan, leaf extract, and inoculum quantity showed a significant effect on IAA production. After 96 h of incubation, the test result of 148 µg/mL IAA production agreed with the prediction and increased by 2.65-fold compared to the basal medium29.
In this study, the optimization of IAA production from rhizospheric actinomycetes using a low-cost medium based on pretreated bagasse was investigated. Thirty-four actinomycete strains with IAA biosynthesis efficiency were screened using different media. The results indicated that among these actinomycetes, the strain S. lavenduligriseus BS50-1 showed the highest IAA production on ISP No. 2 medium supplemented with L-tryptophan. The concentration of IAA production depends on the actinomycete strains and fermentation conditions. Several actinomycete genera, including Actinomadura, Actinoplanes, Frankia, Microbispora, Micromonospora, Mycobacterium, Nocardia, Nonomurea, Saccharopolyspora, Streptomyces, and Verrucosispora, can produce PGRs in various plants, e.g., beans, peas, rice, tomato, and wheat30,31,32. Statistical analysis of the significant model factors suggested that L-tryptophan had a significant effect on IAA production by S. lavenduligriseus BS50-1, which is consistent with previous works on IAA production by actinobacterial strains29,33. This finding suggests that L-tryptophan is believed to be a primary precursor for IAA biosynthesis by microorganisms34,35. According to the literature, there is no research on the use of bagasse waste for the biosynthesis of IAA from S. lavenduligriseus BS50-1. The statistical RSM based on the CCD approach was used to optimize the fermentation conditions of S. lavenduligriseus BS50-1. The highest amount of IAA found in this study was higher than that found in other studies for some plant growth-promoting microorganisms, such as Saccharothrix texasensis MB15 (148 µg/mL)29, Streptosporangium becharense MB29 (141.00 µg/ml)36, Streptomyces sp. PT2 (127 µg/ml)37, S. viridis CMU-H009 (143.95 µg/mL)38, Streptomyces sp VSMGT1014 (26.63 µg/mL)39, Enterobacter ludwigii BNM 0357 (30 µg/ml)40 and Pseudomonas aeruginosa (32 µg/ml)41.