Optimization of solid-state fermentation with recombinant T. reesei

Lignocellulolytic enzymes are essential to the complete decomposition of lignocellulosic biomass. Therefore, in this part, culture conditions for the yield of cellulase, laccase, and xylanase produced by recombinant T. reesei ZJ-09 during solid-state fermentation of lignocellulosic wastes were investigated using one-factor-at-a-time (OFAT) experiments.

Crop type

During SSF, solid substrate is critical to microbial growth and enzyme activity, since it not only supplies the culture nutrients, but also serves as an anchor for the microbial cells (Wong et al. 2017). In this study, enzyme production by T. reesei ZJ-09 under SSF was evaluated using several types of agricultural wastes (wheat straw (WS), rice straw (RS), corn straw (CS), and sugar cane bagasse (SCB)) as carbon sources.

Among these carbon sources, CS could induce the highest total cellulase activity (FPA) of 112.26 FPU/g, laccase activity of 20.50 IU/g, and xylanase activity of 5401.23 IU/g, respectively (Fig. 1). An explanation for this result is that CS had higher cellulose content compared to other lignocellulosic wastes (Additional file 1: Table S2) which led to the upregulation of genes associated with cellulase, hemicellulase, and laccase synthesis given that cellulose is a strong inducer for gene expression in Trichoderma reesei (Bischof et al. 2013). Besides CS, recombinant T. reesei also exhibited strong capability of cellulase, xylanase, and laccase production using RS and SCB as the substrate, indicating T. reesei ZJ-09 has great potential for degradation of various agricultural residues.

Fig. 1
figure 1

Effect of crop type on enzyme production by T. reesei ZJ-09 under SSF. All samples are collected on day 10 to assay the FPA, xylanase activity, and laccase activity. Data were average values of triplicate samples and error bars indicate standard deviations

Rice straw, an economically feasible carbon source commonly used as the substrate for SSF, is the most abundant lignocellulosic biomass in southern China (Abraham et al. 2016). It is rich in several nutrients, which could explain its better performance as it can enhance cell growth as well as metabolism. Considering the nature of the substrate, cost, and availability, rice straw was chosen as the carbon source for further experiments.

Bran content

It has been reported that the addition of wheat bran into SSF medium could provide adequate essential nutrients as well as inducers for microbial growth and enzyme secretion (Farinas 2015). Meanwhile, bran was discovered to have suitable particle sizes, good porosity, and offer fungi the anchorage to grow on during SSF process (Sun et al. 2008). Besides, its texture was kept loose even in moist conditions, thus providing a large surface area by holding water (Kar et al. 2013).

In this study, the optimal bran content for enzyme production from recombinant T. reesei through solid-state fermentation using rice straw as substrate was investigated at 30 °C, initial pH 5.0, 70% water content with 10% (v/w) initial inoculum over a bran content from 0 to 40%. As presented in Fig. 2a, FPA, laccase activity, and xylanase activity increased with bran content, and 30% bran content led to the maximum enzyme activity. After that, further bran content increase brought slight decreases in enzyme activities.

Fig. 2
figure 2

Optimization of fermentation conditions, including the effect of bran content (a), temperature (b), water content (c), pH (d), and inoculum size (e) on enzyme production by T. reesei ZJ-09 under SSF. All samples are collected on day 10 to assay the FPA, xylanase activity, and laccase activity. Data were average values of triplicate samples and error bars indicate standard deviations

Wheat bran is a cheap carbon source rich in several nutrients including cellulose, hemicellulose, protein, and essential minerals. It can also increase the surface area of mixed substrates, which may provide optimum support for enzymes production and cell growth of T. reesei ZJ-09.

Temperature

Temperature is an important indicator during SSF, which can affect enzyme production efficiency. The effect of temperature on enzyme production during SSF was evaluated from 24 °C to 34 °C. As presented in Fig. 2b, enzyme activities did not exhibit huge differences during the measured temperature range. Three enzyme activities reached maximum values when the temperature was set to 30 °C. A slight decrease in laccase activity occurred at temperatures higher than 32 °C. Laccase activity exhibited a sharp decrease (accounting for only 68.15% of the maximal activity) when the temperature was up to 34 °C.

Water content

Sufficient moisture is crucial to microbial growth and metabolism. It is previously reported that initial water content had a major impact on the cellulases production. In this study, different moisture contents (50—80%) were evaluated for cultures of T. reesei ZJ-09 under SSF using rice straw as substrate. As presented in Fig. 2c, the ideal water content was observed between 60 and 75%. FPA, laccase activity, and xylanase activity maintained above 80% of the highest enzymatic activities when moisture content varied in this range, indicating recombinant T. reesei ZJ-09 could thrive on a comparatively wide range of water content. 70% was proven to be optimal for enzyme production. When moisture was maintained at 70%, FPA, laccase activity, and xylanase activity reached 124.49 IU/g, 24.60 IU/g, and 5763.34 IU/g, respectively (data not shown). Further increase in moisture level had a negative impact on enzyme production.

pH

The optimal pH for enzyme production by T. reesei ZJ-09 under SSF was studied over a pH range of 3.5–7.0. The FPA, laccase activity, and xylanase activity were measured on the 10th day of fermentation. Enzyme activities from the recombinant T. reesei did not vary considerably at pH varying from 4.5 to 7.0 (Fig. 2d). Notably, though the laccase activity decreased when pH was lower than 4.0, the recombinant T. reesei still presented laccase production capacity, suggesting comparatively wide pH adaptability of T. reesei ZJ-09. Taken together, the pH of SSF for T. reesei ZJ-09 was set at 7.0 (natural pH) to simplify pH adjustment process.

Inoculum size

The effect of inoculum size on T. reesei SSF was studied over the range of 5–15%. As shown in Fig. 2e, the increase in inoculum size within the range of 5% to 10% resulted in increased enzyme activities which could be due to enhanced growth rates achieved in the initial phase. There is a trade-off between strain growth and enzyme production, if increase the inoculum size, the fungal growth rate can be accelerated, but at the same time nutrient depletion can be also aggravated. Fungal growth will be affected via nutrient depletion, which would do harm to improving the yield of enzymes, thereby the suitable inoculum size was set to 10% in this study.

Enzyme production by recombinant T. reesei on rice straw

The changes of enzyme activity of cellulase, laccase, and xylanase during the SSF process are given in Fig. 3. Similar to the original strain, recombinant T. reesei ZJ-09 had strong cellulase and xylanase producing capability. FPAs of T. reesei ZJ-09 and the host strain T. reesei ZU-02 increased rapidly from day 2 to day 7, peaking at 110.47 FPU/g and 126.27 FPU/g, respectively, on day 12. Taking other components of cellulase complex into consideration, ZJ-09 exhibited slightly lower β-glucosidase activity, cellobiohydrolase activity, and endoglucanase activity (Additional file 1: Table S1). Significant increases in xylanase activities were found after 4 days of fermentation, reaching 5723.39 IU/g and 5225.21 IU/g, respectively, on day 10.

Fig. 3
figure 3

Cellulase, xylanase, and laccase production by recombinant T. reesei ZJ-09 and original strain under optimized SSF conditions using rice straw as substrate. Error bars represent the standard deviation of three independent repeats

Notably, compared to the host strain, T. reesei transformant ZJ-09 could persistently secrete laccase from day 4 to day 10 with the activity of 24.45 IU/g on the 10th day of fermentation.

Taken together, recombinant T. reesei kept the abilities of efficient production of cellulase and xylanase from the original strain. Meanwhile, T. reesei ZJ-09 could use rice straw as a substrate to produce high activity laccase.

It was known that the complete degradation of lignocellulosic biomass needs a battery of enzymes targeting cellulose, hemicellulose, and lignin. Because of its complexity and rigid structure, lignin is hard to degrade. Despite the noted catalytic activity of laccases towards phenolic groups of lignin, laccase cannot oxidize lignin completely. Consequently, mediators, acting as electron carriers, are needed to assist laccase in depolymerizing lignin and are considered a crucial factor in efficient and complete lignocellulosic residues degradation. In this study, the early degradation of cellulose and hemicellulose catalyzed by the cellulase and xylanase from T. reesei ZJ-09 could release various phenolic compounds which could act as natural mediators for laccase. In this way, the formed laccase–mediator systems (LMS) could expand the oxidation ability of laccase, which made the recombinant T. reesei have greater potential to effectively degrade lignocellulosic wastes as well as organic pollutants.

Biodegradation of rice straw by T. reesei

Changes in the biomass compositions under SSF process are shown in Fig. 4. As it was expected, recombinant T. reesei ZJ-09 retained the strong cellulose and hemicellulose degradation ability of the original strain. The degrading ratio of cellulose and hemicellulose increased immediately after inoculation, reaching 21.41% and 23.64% on day 2, respectively, and then topped 59.12% and 52.61% on day 12, respectively.

Fig. 4
figure 4

Degradation of rice straw by T. reesei ZJ-09 (a) and original strain (b) under optimized SSF conditions. Error bars represent the standard deviation of three independent repeats

Additionally, it is noteworthy that T. reesei ZJ-09 was able to degrade lignin effectively (Fig. 4a). Despite the lower removal rate during the initial phase of SSF, the lignin degradation ratio for T. reesei ZJ-09 had been growing with the laccase activity since day 4. The lignin degradation ratio was up to 38.05% on day 12. By comparison, the lignin degradation ratio of the original strain was undetectable.

The lower percentage of lignin makes cellulose and hemicellulose more available to the microorganism, which leads to higher biomass loss. As a result, the final mass loss of ZJ-09 was 1.4-fold higher to 51.16% compared to that of the original strain.

POPs degradation by recombinant T. reesei koji

Target organic contaminants (nonylphenol, 2,4,5-trichlorophenol, and oxytetracycline) selected in this study are the three most recalcitrant and prevalent in the environment, which pose significant health risks to humans. The laccase-mediated removal rates of the three contaminants are lower than 15% when no mediator is added.

As expected, the commonly used synthetic mediator ABTS could facilitate laccase-catalyzed degradation of inert chemicals. After reacting for 4 h, ABTS-mediated treatments achieved the removal rates of 94.28%, 52.01%, and 46.73% for nonylphenol (NP), 2,4,5-trichlorophenol (TCP), and oxytetracycline (OTC), respectively (Fig. 5). Regardless of the proven efficiency of the laccase/ABTS system, artificial mediators lead to additional costs, exhibit potential toxicity (Becker et al. 2016), and can cause laccase inactivation (Fillat et al. 2012). In comparison, natural mediators, considered as the true mediators for fungal laccases, are good alternatives to artificial ones given that they are more environmentally friendly and economically feasible.

Fig. 5
figure 5

Degradation of POPs by different laccase–mediator systems. Individual POP on 50 mg/L was treated with 0.2 IU/mL laccase at pH 4.0, 50 ℃ for 4 h in the presence or absence of 0.3 mM individual mediator or Syr/Van complex with a ratio of 4:6. Control was performed on 50 mg/L individual POP with 0.2 IU/mL laccase without mediators. Residual POP concentrations were determined by HPLC. Error bars represent the standard deviation of three independent repeats. *p < 0.05, **p < 0.01 compared with control

As shown in Fig. 5, the removal rates were 94.55%, 86.42%, and 62.98% for NP, TCP, and OTC, respectively, by syringaldehyde/laccase system. Interestingly, an improved removal rate was observed when syringaldehyde and vanillin were present in combination, indicating the coexistence of various mediators could bring a synergistic effect in laccase–mediator systems. This was probably due to that not only the primary radical species, but also the secondary species produced by the chain reactions among primary radicals could act on the recalcitrant chemicals, resulting in the enhancement of the oxidation efficiency.

A similar result was found by POPs degradation by koji from SSF by recombinant T. reesei ZJ-09 (LKPB). Degradation reactions using supernatant from submerged fermentation of T. reesei ZJ-09 (control group) were also run along with the test. When no mediator was added, the 4 h removal rates mediated by LKPB (92.45% for NP, 87.21% for TCP, and 90.73% for OTC) were comparable to those achieved by syringaldehyde/vanillin co-mediator system, while those of the control group were all lower than 15%.

Previous studies showed that the combination of laccase and mediators can efficiently transform phenolic xenobiotics into less toxic compounds (Su et al. 2018). For example, the degradation of OTC by laccase–mediator system (LMS) showed a reduction in toxicity (Mir-Tutusaus et al. 2014) and antimicrobial activity (Yang et al. 2017). As for TCP, the toxicity, persistence, and biodegradability depend on the number and position of chlorine substituents in the phenolic ring (Rubilar et al. 2008). Çabuk et al. revealed that TCP was dechlorinated by laccase (Çabuk et al. 2012) and Liu et al. demonstrated that laccase could achieve a good detoxification effect while degrading chlorophenols (Liu et al. 2021). Also, the acute toxicity test pointed out that the detected metabolites of NP were less toxic than the parent compound after laccase treatment (Mtibaà et al. 2020).

However, the application of laccase is limited because it is hard to react with pollutants with high redox potentials alone (Su et al. 2018). It was found that natural phenolic compounds derived from plants can generate active free radicals and then act on inert chemicals as mediators for laccase, thus expanding the substrate spectrum of laccase. In this study, the degradation of rice straw catalyzed by enzyme cocktails secreted by T. reesei ZJ-09 during SSF could release various phenolic compounds, which then formed laccase–mediator system and expanded the oxidation ability of laccase.

In this way, the recombinant T. reesei have greater potential to effectively degrade lignocellulosic wastes and organic pollutants simultaneously without addition of mediators. The future application of LKPB in wastewater and soil remediation is worth exploring.

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