Comparative Studies of White-Rot Fungal Strains (Trametes hirsuta MTCC-1171 and Phanerochaete chrysosporium NCIM-1106) for Effective Degradation and Bioconversion of Ferulic Acid.

Biodegradation of ferulic acid, by two white-rot fungal strains (Trametes hirsuta MTCC-1171 and Phanerochaete chrysosporium NCIM-1106) was investigated in this study. Both strains could use ferulic acid as a sole carbon source when provided with basal mineral salt medium. T. hirsuta achieved complete degradation of ferulic acid (350 mg L-1) in 20 h, whereas P. chrysosporium degraded it (250 mg L-1) in 28 h. The metabolites produced during degradation were distinguished by gas chromatography-mass spectrometry. Bioconversion of ferulic acid to vanillin by P. chrysosporium was also investigated. The optimum experimental conditions for bioconversion to vanillin can be summarized as follows: ferulic acid concentration 250 mg L-1, temperature 35 °C, initial pH 5.0, mycelial inoculum 0.32 ± 0.01 g L-1 dry weight, and shaking speed 150 rpm. At optimized conditions, the maximum molar yield obtained was 3.4 ± 0.1%, after 20 h of bioconversion. Considering that the degradation of ferulic acid was determined by laccase and lignin peroxidase to some extent, the possible role of ligninolytic enzymes in overall bioconversion process was also studied. These results illustrate that both strains have the potential of utilizing ferulic acid as a sole carbon source. Moreover, P. chrysosporium can also be explored for its ability to transform ferulic acid into value-added products.

Introduction

In the plant kingdom, ferulic acid (4-hydroxy-3-methoxycinnamic acid), a natural aromatic compound, is ubiquitously found in the cell wall of plants, including numerous agriculturally important crops.[1] However, the advancement of agro-industrial operations has led to a larger accumulation of ferulic acid in the form of residues and industrial effluent. Various industries are producing effluents containing ferulic acid, including those found in oil processing, wine distilleries, and paper pulp–processing industries.[2] Ferulic acid is being considered as an environmental pollutant because it shows a toxic effect on the microbial growth, even at a low concentration.[3] Numerous investigations have reported the utilization of ferulic acid as a sole carbon source for microbial growth.[4] However, most of the intermediates of the degradation process are toxic pollutants. Several methods have been studied for degradation of the pollutant, including chemical oxidation and photodegradation because of their capabilities to assist the degradation of ferulic acid in a contaminated environment. Advanced oxidation process, a chemical oxidation method, has been studied for effective degradation of ferulic acid; however, it is an expensive process and not ideal with respect to economical aspect.[5] Similarly, photocatalysis has demonstrated its potential of degrading ferulic acid effectively. However, this approach has the main disadvantages of poor stability and non-recovery of photocatalysts.[6] Microbial degradation of xenobiotics has been an attraction for the scientific community because it is a green process and can be carried out at mild reaction conditions. Therefore, microorganisms with the ability to effectively degrade ferulic acid along with its intermediates should be explored for their potential application in bioremediation. Several bacterial[1] and fungal strains[7] were isolated and identified to evaluate their degradation abilities against ferulic acid.[4] In general, various factors affect the microbial catalysis during biodegradation of ferulic acid. A number of different enzymes play a vital role in microbial transformation of ferulic acid and ultimately influence the overall bioconversion process.

In the recent past, researchers have used a number of biotechnology-based strategies to achieve synthesis and production of vanillin using substrates including ferulic acid, eugenol, lignin, phenolic stilbenes, isoeugenol, and aromatic amino acids by employing bacteria, fungi, plant cells, and genetically modified microorganisms.[8] Among these vanillin precursors, ferulic acid is the most relevant candidate because of its chemical resemblance to vanillin[9] and can be efficiently used for bioconversion. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most extensively adopted flavoring agents in the food and cosmetics industries. It is generally extracted from the species of vanilla orchid, including Vanilla planifolia, Vanilla pompona, and Vanilla tahitiensi. It is an essential raw material for the production of various pharmaceutical drugs, such as dopamine, aldomet, and papaverine, and for the synthesis of antifoaming agents.[10] Vanillin has antioxidant, antimicrobial, anti-mutagenic, and anticarcinogenic properties. These properties are mainly associated with the phenolic nature of vanillin.[11] Because there is limited availability of vanilla pods around the world, the “natural” vanillin produced is highly expensive. The thriving global market for natural vanillin demands higher supply. To fulfill the demand, chemically synthesized form of vanillin is an efficient and comparatively cheaper alternative.[8] Vanillin is being synthetically derived from several raw materials, such as lignin, eugenol, glyoxylic acid, aromatic amino acid, and guaiacol. A growing interest for natural flavors in food and cosmetic industries has led researchers to find an alternate biotechnological approach to produce natural vanillin. Vanillin can be obtained by biotransformation using natural substrates such as eugenol, isoeugenol, ferulic acid, and glucose, among others. Moreover, vanillin obtained via biotransformation meets the guidelines of international legislation, which defines “natural products” to include materials produced by microbial transformation using living cells or their enzymes.[12] Earlier findings suggest that yield obtained from synthesis and production of vanillin by enzymatic catalysis is comparatively lower than that of the whole cell biocatalysis. Therefore, bioconversion by whole cell system has been considered as a significant and efficient way for the bulk production of vanillin.[13,14] Also, vanillin production by bioconversion has several advantages over chemical processes because these are environment-friendly, substrate-specific processes and can be carried out at relatively mild reaction conditions.[4]

Recently, several microbial species have been investigated for their ability to produce vanillin via biotransformation of ferulic acid or other structurally similar substrates. On the basis of initial reactions associated with bioconversion of ferulic acid, five major pathways have been identified: a reductive pathway, non-oxidative decarboxylation, CoA-dependent β-oxidation, and CoA-dependent and CoA-independent retro-aldol reaction pathways.[8] A few microorganisms have evolved and could carry bioconversion of ferulic acid using multiple pathways.[15] During catabolism of ferulic acid, vanillin formation is an intermediate step. Also, many organisms are able to catabolize the vanillin formed into other products rapidly.[15]

Several bacterial species such as Streptomyces setonii and Pseudomonas putida were investigated for the bioconversion of ferulic acid. P. putida was found to be producing vanillic acid after 24 h, whereas accumulation of vanillin (68% molar yield over a period of 50–60 h) was observed in S. setonii when ferulic acid was provided with initial concentration of 6 g L–1.[12] In another report, Bacillus subtilis could obtain 60.43% molar yield of vanillin in 20–30 h of incubation when initial concentration was 1.5 g L–1 of ferulic acid.[16] The biodegradation of ferulic acid involving bacteria has been widely reported with efficient yield. However, biodegradation of ferulic acid by fungi has been underexplored when compared to the studies related to bacteria. Filamentous fungi produce potent enzymes that can team up more efficiently to break down lignocellulosic biomass than those produced by other microorganisms such as bacteria or yeast.[17] White-rot fungi are reported for their ability to secrete extracellular degrading enzymes, including Lac (laccase), LiP (lignin peroxidase), and MnP (manganese peroxidase). Apart from lignin decomposition, these ligninolytic enzymes can also oxidize phenolic and methoxyphenolic compounds, demethylate methoxyl groups, and decarboxylate them.[18] Therefore, white-rot fungi should be explored for their untapped potential in degradation of aromatic pollutants. Several non-white rot fungal strains such as Paecilomyces variotii(19) and Fusarium solani(20) can transform ferulic acid to vanillin along with several other metabolites over a period of 24 and 48 h of incubation, respectively. Although several non-white rot fungal strains were reported for bioconversion of ferulic acid, a limited number of white-rot fungi have been reported for degradation of ferulic acid.[19]Schizophyllum commune, a white-rot fungi, has degraded ferulic acid (initial concentration of 97.09 mg L–1 in 16 days of incubation) into 4-vinylguaiacol, which was further oxidized to vanillin and vanillic acid.[21] In another investigation, trametes sp. formed coniferyl alcohol and coniferyl aldehyde during ferulic acid degradation.[22] Although bioconversion of ferulic acid has been extensively investigated with numerous microorganisms, these reports essentially focused on the yield of vanillin by isolated and identified microbial strains. Phanerochaete chrysosporium and Trametes hirsuta are white-rot fungal strains widely reported for their ability to express ligninolytic enzymes (laccase and lignin peroxidase) while degrading a number of xenobiotics.[23,24] Despite having the potential of being a promising candidate in ferulic acid degradation, the role of ligninolytic enzymes of white-rot fungi in the overall degradation process has not been orderly investigated.

In the current investigation, we screened two white-rot fungi (T. hirsuta MTCC-1171 and P. chrysosporium NCIM-1106) for their ferulic acid–degrading capabilities along with their response to varying concentrations of ferulic acid. Moreover, the major role of Lac in ferulic acid degradation has also been assessed. To the best of our understanding, there has been no record on the systemic examination of experimental factors that influence the degradation and bioconversion of ferulic acid using T. hirsuta MTCC-1171 and P. chrysosporium NCIM-1106. It was also worthy of consideration to investigate the ability of white-rot fungi in the transformation of ferulic acid to natural vanillin.

Materials and Methods

Chemicals

Malt extract and other media components were procured from Himedia Ltd. (Mumbai, India). S. D. Fine-Chem. Ltd. (Mumbai) provided the following chemicals: guaiacol, veratryl alcohol, and hydrogen peroxide (50% w/v). Ferulic acid and vanillin (purity ≥ 97%) were procured from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, methanol and ethyl acetate of high-performance liquid chromatography (HPLC)-grade were procured from S. D. Fine-Chem. Ltd. (Mumbai). HPLC-grade glacial acetic acid was purchased from Spectrochem Ltd. (Mumbai).

Microorganisms

MTCC (Microbial-Type Culture Collection), Chandigarh, India, provided the lyophilized culture of T. hirsuta MTCC-1171. The strain of P. chrysosporium NCIM-1106 was procured from the National Collection of Industrially Important Microorganisms, NCL, Pune, India. Initially, both strains were subcultured using malt extract (2% w/v) agar for 72 h. The fungal strains were further grown by submerging in malt extract medium and incubated at 30 °C at 125 rpm using an orbital shaker for the next 48 h. The strains were maintained on agar medium containing malt extract and subcultured subsequently for 30 days. Strains maintained on agar slants were stored at 4 °C in refrigerator.

Inoculum Preparation

The fungal strains were activated by submerging in malt extract medium (50 mL, 20 g L–1) for 72 h at 30 °C at 125 rpm using an orbital shaker. Grown fungal mycelia were collected and rinsed twice with deionized (DI) water to discharge most of the accessible nutrients from malt extract, and then added to 100 mL basal mineral salt medium (1.0 g L–1 (NH4)2SO4, 1.0 g L–1 K2HPO4, 0.2 g L–1 KH2PO4, 1.0 g L–1 NaCl, 0.005 g L–1 boric acid, 0.002 g L–1 CaCl2·2H2O, 0.5 g L–1 MgSO4·7H2O, and 0.001 g L–1 CuSO4) in 250 mL conical flasks. These were further incubated at 125 rpm using an orbital shaker for 2 h at 30 °C to further consume any remaining nutrients adhered to the surface of mycelia and then used as inoculums.

Determination of Ligninolytic Enzymes Activity

Samples of culture were analyzed to determine the activity of ligninolytic enzymes. A flask containing media without microbial culture was used as a control in all studies. Aliquots of culture were taken out at a specific interval of time. Cultures were filtered through a muslin cloth. The filtrates obtained were then centrifuged at 8000 rpm at 4 °C for 12 min using a Beckman cold centrifuge to separate residues present in the supernatant. The activities of LiP and MnP were determined colorimetrically, as mentioned in earlier work[25] by a Spectroscan UV 2700 spectrophotometer (UV–vis spectrophotometer, Chemito India). Laccase activity was estimated using substrate (guaiacol 10 mM, pH 4.5) at 30 °C by measuring the absorbance at 470 nm.[26] Enzyme activity can be defined as the amount of enzyme needed to obtain catalyzed colored product min–1 mL–1, which is represented in U mL–1.

Effect of Initial Concentration of Ferulic Acid on Degradation

Inoculum containing fungal mycelia (approximately 5.2 g L–1, equal to 0.21 g L–1 dry fungal weight) of strains were added to 100 mL basal mineral salt media with ferulic acid (100–500 mg L–1) as the sole carbon source. Both cultures were incubated at 30 °C for 48 h in a shaking incubator at 150 rpm. Mycelial growth (dry weight) and the concentration of remaining ferulic acid were analyzed by collecting fungal biomass and culture broth. A flask containing media without culture was used as a control in all studies. An Agilent system (1290 infinity, Agilent, Palo Alto, CA, USA) provided with UV detector and Agilent ChemStation Software was employed to analyze the remaining concentration of ferulic acid. For HPLC, a C18 column (ZORBAX Eclipse XDB, 250 mm × 4.6 mm, 5 μm particle size) was used. Mobile phase containing acetic acid in DI water (0.2% v/v) and acetonitrile–methanol (1:1 v/v) at a ratio of 73:27 (v/v) was used. The injection volume, flow rate, column temperature, and wavelength were 10 μL, 1.0 mL min–1, 30 °C, and 280 nm, respectively.

Degradation of Ferulic Acid

Ferulic acid with a concentration of 350 and 250 mg L–1 were added in the media containing T. hirsuta and P. chrysosporium, respectively. These values were considered as the optimized concentration of ferulic acid in degradation studies because the rate of degradation and growth of fungal biomass were higher at these concentrations. Fungal inoculum of T. hirsuta and P. chrysosporium were added to 100 mL basal MSM media (pH 4.5) containing 350 and 250 mg L–1 of ferulic acid, respectively. Both cultures were incubated at 30 °C for 48 h in a shaking incubator at 150 rpm. A control flask of media containing ferulic acid without fungal mycelia was managed identically. To evaluate the effect of exogenously added carbon source (glucose) on the ability of T. hirsuta and P. chrysosporium in ferulic acid degradation, 1 g L–1 of additional carbon source (glucose) was added to a test medium containing 350 and 250 mg L–1 ferulic acid, respectively. The microbial cultures containing ferulic acid–only and glucose as a sole carbon source were analyzed at 4 h interval during incubation period to detect the remaining concentration of ferulic acid and activities of degrading enzymes.

Effect of Inhibitor and Inducer on Ferulic Acid Degradation

To further confirm the role of ligninolytic enzymes (Lac and LiP) in the overall degradation process, another experiment was conducted. Fungal inoculum of T. hirsuta and P. chrysosporium were added to 100 mL basal MSM media containing 350 and 250 mg L–1 of ferulic acid, respectively. Other experimental parameters were consistent with those specified in Section . Additionally, sodium azide 13.002 mg L–1 (200 μM) was added as Lac/LiP inhibitor.[27,28] After incubating from 0 to 32 h, aliquots of culture were collected at 4 h intervals and were analyzed for the remaining concentration of ferulic acid and activities of ligninolytic enzymes. Controls containing no fungal mycelia were examined in an identical way.

The effect of an inducer (veratryl alcohol) on the production of ligninolytic enzymes and overall degradation process was also examined. Veratryl alcohol (0.29 mL L–1; 2 mM) was added to 100 mL basal MSM media containing ferulic acid. Other experimental parameters, such as concentration of ferulic acid, fungal inoculum, and culture conditions, were consistent, as previously specified. During the incubation period, aliquots of culture were collected at 4 h intervals and were analyzed for the remaining concentration of ferulic acid and activities of ligninolytic enzymes.

Optimization of Bioconversion of Ferulic Acid

All tests were conducted in batch mode using horizontal shaking incubator. To examine the impact of initial pH, temperature, fungal inoculum, and rotational speed of shaking on the process of bioconversion of ferulic acid by P. chrysosporium, varying temperature (25–45 °C), pH (4.0–6.0), initial fungal inoculum (approximately 2.6–10.4 g L–1 wet weight), and shaking speed (0–200 rpm) were assessed in this investigation.

Analytical Procedures

Samples were taken out during the incubation period and products were further analyzed by applying HPLC. At a specific time interval, samples were removed and filtered through a muslin cloth to remove fungal biomass. The filtrate obtained was then centrifuged at 8000 rpm at 4 °C for 12 min. The supernatant was again filtered using Whatman nylon filter (0.2 μm) and was extracted with ethyl acetate (1:1 v/v). Ethyl acetate fractions were concentrated using a rotary evaporator. These were resuspended in methanol (1 mL) and were further analyzed by HPLC using the method mentioned in Section . To identify metabolic products of degradation process, samples of fermentation broth were extracted after respective period of incubation and gas chromatography–mass spectrometry (GC–MS) analysis was conducted by using a method mentioned in an earlier report.[29] Agilent 7890A system (Waldbronn, Germany) with column (HP-5) was used. GC–MS analysis was employed to treated samples along with the control. Fungal growth was estimated by dry cell weight analysis by employing filtration followed by drying process. To measure the biomass, a filter paper was oven dried at 60 °C for 24 h, and weight was noted. Remaining fungal mycelial biomass was weighed using oven-dried filter paper and was determined gravimetrically.

Statistical analysis is very critical to compile and evaluate the collected data. All experiments were handled in triplicates to verify the reproducibility, and all data are expressed as means ± standard deviation. The one-way ANOVA was conducted using Microsoft Excel. To obtain the statistically significant data, P-value less than 0.05 was considered.

Results and Discussion

Effect of Ferulic Acid Concentration on Degradation

To evaluate the capability of fungal strains for degradation, varying concentrations (100–500 mg L–1) of ferulic acid were used as a sole carbon source. Fungal biomass and concentration of remaining ferulic acid were analyzed after incubation period of 48 h (Figure ). In case of T. hirsuta, it could completely degrade ferulic acid when the initial concentration of ferulic acid was 350 mg L–1 or lower. However, the degraded ferulic acid reduced to 26.2–37.7% when it was provided with the initial concentration of 350 mg L–1 or more. In case of P. chrysosporium, it could degrade up to 250 mg L–1 of ferulic acid. The concentration of degraded ferulic acid notably reduced to 8.9–71.1% when the initial concentration was more than 250 mg L–1. Also, mycelial biomass concentration reached the maximum when the initial concentration of ferulic acid was 350 and 250 mg L–1 for T. hirsuta and P. chrysosporium, respectively. Therefore, these concentrations of ferulic acid were chosen for the subsequent experiments. These findings suggest that at higher concentrations of ferulic acid, it may have a toxic effect on fungal growth of both strains, whereas a concentration below 350 and 250 mg L–1 could have no toxic effect on T. hirsuta and P. chrysosporium, respectively. Similar findings demonstrated that xenobiotics at a higher concentration could inhibit microbial growth.[30]

Figure 1

Determination of optimum concentration of ferulic acid for degradation and fungal biomass of T. hirsuta MTCC-1171 and P. chrysosporium NCIM-1106. Data are represented as the means of three independent replicates with standard error bars.

Effect of Available Carbon Source on Degradation

The effect of the available form of carbon source on the expression of ligninolytic enzymes and overall degradation process was studied for the respective incubation period. Figure a depicts that in a culture of T. hirsuta, 350 mg L–1 of ferulic acid provided 1 g L–1 of glucose; the activity of Lac was slightly decreased along with a little delay in the overall degradation process. However, LiP activity was also slightly decreased in a culture of P. chrysosporium when the same amount of glucose was provided containing 250 mg L–1 of ferulic acid (Figure b). Moreover, the delay in degradation process was comparatively longer (∼4 h) in the case of P. chrysosporium when glucose was exogenously provided.

Figure 2

Effect of carbon source (glucose) on the production of Lac/LiP and overall ferulic acid degradation by T. hirsuta MTCC-1171 (a) and P. chrysosporium NCIM-1106 (b). Error bars represent the standard error of three replicates.

In this investigation, it was found that both fungal strains could grow on ferulic acid–only culture medium, which implies that these strains can use xenobiotic as its sole carbon source for their growth, even in a nutrient-deficient environment. Figure a shows the level of Lac produced by T. hirsuta. Ferulic acid is capable of inducing Lac production in trametes sp.[31] Moreover, Lac activity can be influenced by the presence of glucose as a carbon source in the media and can be detected when glucose in the culture medium is almost exhausted.[32] We did observe Lac activity from the fourth hour of incubation. However, a noticeable increase in Lac activity was observed after 12 h of incubation, indicating glucose depletion in the medium. P. chrysosporium could express a higher amount of LiP and MnP under carbon- and nitrogen-deficient conditions.[33] Although we observed slightly better LiP activity in ferulic acid–only culture when compared to the culture medium containing glucose (Figure b), we did not observe MnP activity in both culture media. Lack of MnP activity in the medium might be because of the possible inhibition of the enzyme by ferulic acid itself.[34] Lac activity was also not detected in both ferulic acid–only and glucose culture medium. Despite the fact that ferulic acid induced Lac expression in the culture of T. hirsuta, P. chrysosporium could not express Lac in the presence of ferulic acid,[35] even when glucose was provided exogenously. Researchers have also reported that P. chrysosporium could not induce Lac production even in the presence of glucose as a carbon source.[36]

These observations suggest that both strains were competent enough to degrade ferulic acid even when provided as a sole carbon source by expressing ligninolytic enzymes. Moreover, the level of expression of ligninolytic enzymes under nutrient-deficient conditions might have affected the overall rate of degradation. Therefore, T. hirsuta and P. chrysosporium can effectively develop its ecological functions for the degradation of ferulic acid even under a carbon-deficient environment.

Role of Lac/LiP in Ferulic Acid Degradation

Figure a,b depicts that ferulic acid was completely degraded by T. hirsuta and P. chrysosporium in 24 and 32 h, respectively. Ferulic acid could induce the expression of Lac and LiP in the case of T. hirsuta and P. chrysosporium, respectively. However, when the inhibitor was added to both of the culture media while keeping all other experimental conditions constant, the Lac and LiP activity was substantially decreased and only 0–16.3% (Figure a) and 0–21.1% (Figure b) degradation of ferulic acid was observed after the period of incubation by T. hirsuta and P. chrysosporium, respectively. Laccase-producing fungi have been used for bioremediation of wastewater in the recent past.[37] Researchers have also highlighted the role of Lac in the degradation of ferulic acid.[7] Laccases are multicopper enzymes that oxidize several aromatic pollutants by using molecular oxygen through a mechanism comprising radical formation.[37] As mentioned earlier, ligninolytic enzymes such as Lac and LiP can also oxidize phenolic and methoxyphenolic compounds, and demethylate methoxyl groups by attacking their methoxyl groups via decarboxylation.[18] A gradual increase in enzymatic activities was observed along with a steady degradation of ferulic acid during the incubation period. However, when Lac/LiP activity was inhibited, the substrate was degraded much more slowly when compared to the non-inhibition treatment. Although Lac of T. hirsuta could efficiently degrade ferulic acid at a much faster rate, P. chrysosporium lacking Lac activity could degrade it but at a much slower rate, indicating that Lac might have played a leading role in overall degradation.[7] Therefore, this study suggests that ligninolytic enzymes (Lac/LiP) determined the degradation of ferulic acid, to some extent.

Figure 3

The effect of inhibitor on the expression of ligninolytic enzymes and ferulic acid degradation by by T. hirsuta MTCC-1171 (a) and P. chrysosporium NCIM-1106 (b). Error bars represent the standard error of three replicates.

Role of Veratryl Alcohol on Bioconversion Process

Veratryl alcohol has been reported for the enhancement of Lac and LiP activity in white-rot fungi.[38,39] Faison and Kirk confirmed that ligninase activity in culture could be stimulated by veratryl alcohol.[40] We observed an enhancement in the activities of ligninolytic enzymes of both fungal strains when veratryl alcohol was provided exogenously. Although Lac activity was slightly increased in culture medium of T. hirsuta (Figure a), LiP activity was significantly increased in culture medium of P. chrysosporium (Figure b). Veratryl alcohol has been reported for enhancing LiP activity via enzyme stabilization in P. chrysosporium(41) and possibly played a similar role when added exogenously. Also, another report suggests that lignin and lignin-model compounds, along with veratryl alcohol and related aromatic compounds, can induce enhanced ligninase activity when added to the culture of P. chrysosporium.[40] We also noticed that veratryl alcohol could induce Lac expression to some extent in culture medium of P. chrysosporium. This is probably because of the inducing effect of veratryl alcohol on Lac expression in P. chrysosporium.[35,42]Figure a,b depicts that in the presence of veratryl alcohol, T. hirsuta and P. chrysosporium could completely degrade ferulic acid in 20 and 28 h, respectively. Enhancement in the rate of degradation because of the addition of veratryl alcohol in culture medium of both strains indicates that the level of expression of ligninolytic enzymes might have played a vital role in the overall degradation process.

Figure 4

Effect of inducer on the expression of ligninolytic enzymes and ferulic acid degradation by T. hirsuta MTCC-1171 (a) and P. chrysosporium NCIM-1106 (b). Error bars represent the standard error of three replicates.

Optimization of Bioconversion of Ferulic Acid

The presence of vanillin was detected only in the media containing veratryl alcohol. Therefore, basal MSM provided with veratryl alcohol (0.29 mL L–1, 2 mM) was used to further optimize the bioconversion of ferulic acid. Figure a represents the effect of temperature on the bioconversion of ferulic acid by P. chrysosporium at varying temperatures (25, 30, 35, 40, and 45 °C). As depicted in the figure, the molar yield was slightly affected over a temperature range of 30–35 °C, whereas it was found to be significantly lower at 25 °C, possibly because of the lower affinity of the substrate with the enzyme.[43] However, a drop in molar yield at a relatively elevated temperature (40 °C and above) could be the result of thermal deactivation of enzyme because of the conformational changes in protein folds. The maximum molar yield of 2.1 ± 0.06% was observed at 35 °C. A possible reason for this could be the that the optimized temperature for the expression of ligninolytic enzymes by P. chrysosporium has been found to be in the range of 33–38 °C.[44] Also, the temperature might have determined the energy-dependent mechanisms of fungal cells in bioconversion of ferulic acid.

Figure 5

Effect of different factors on the bioconversion of ferulic acid by P. chrysosporium NCIM-1106. Effect of temperature on the bioconversion of ferulic acid (a), effect of initial pH on the bioconversion of ferulic acid (b), effect of initial fungal inoculum on the bioconversion of ferulic acid (c), and effect of shaking speed on the bioconversion of ferulic acid (d).

It is well established that pH of the medium can significantly influence the microbial growth and bioconversion process. Apart from altering enzymatic activities, changes in pH can also actively affect the chemistry of the substrate in the surrounding. Moreover, it also directs the activities of functional groups of microbial cell wall.[45]Figure b represents the effect of initial pH on the bioconversion of ferulic acid by P. chrysosporium. As depicted in the figure, molar yield (%) significantly increased over a pH range of 4.5–5.0. However, the yield was found to be slightly lower over a pH range of 5.5–6.0. Also, at lower range (pH 4.0), yield was significantly low. The maximum molar yield of 3.1 ± 0.12% was observed at pH 5.0. These outcomes suggest that the optimal pH for microbial growth of P. chrysosporium affected the bioconversion process. Because P. chrysosporium has been reported for higher activity of LiP at a similar range of pH, it might have influenced the bioconversion process.[46]

The effect of the size of mycelial inoculum on the bioconversion process by P. chrysosporium was evaluated using a varying range of fungal mycelial balls of almost equal size (approximately 2.6–10.4 g L–1 wet weight), as shown in Figure c. Enzyme production can be affected by the size of inoculum used in the medium. When provided with a lower amount of fungal inoculum, it may not be enough to initiate growth, which ultimately affects the enzyme production, whereas excessive amount may originate competitive inhibition.[47] As the initial biomass quantity increased, an enhancement in the yield was witnessed. The maximum molar yield (3.4 ± 0.10%) was observed in the flask containing fungal mycelial (approximately 7.8 g L–1 wet weight equal to 0.32 ± 0.01 g L–1 dry weight). However, a fall in yield was noticed with a further rise in mycelial inoculum, possibly because of the faster depletion of nutrients,[48] ending up with a drop in metabolic activities.

The effect of rotational speed of shaker on the bioconversion by P. chrysosporium was investigated by incubating fungal mycelium in flasks on a rotary shaker provided with varying speeds (0, 50, 100, 150, and 200 rpm). Other experimental conditions were maintained as mentioned earlier. Figure d shows that the optimized rotational speed of shaking was observed to be 150 rpm with maximum molar yield (3.4 ± 0.10%), whereas as the rotational speed was increased further, a significant decrease in yield was observed. At a higher shaking speed, deeper physiological effects can occur in P. chrysosporium, possibly affecting secondary metabolism in general.[49] Shaking speed also determines the level of oxygen provided in the medium. The high level of oxygen concentration favors an enhancement in microbial growth and can further improve the process of bioconversion.

Dynamics of Metabolic Products and Degrading Enzymes Activities

When degradation products of both cultures were analyzed, detection of vanillin and vanillic acid in culture medium of P. chrysosporium was observed (Figure S1). However, no vanillin or vanillic acid was detected in a culture medium of T. hirsuta. In this study, we found that T. hirsuta could completely degrade the intermediate products and no secondary pollutants were observed. Moreover, traces of 4-hydroxybenzaldehyde, benzoic acid, and coniferyl aldehyde were observed in culture medium of T. hirsuta (Figure S2). trametes sp. has been reported for biotransformation of ferulic acid into coniferyl aldehyde and coniferyl alcohol.[22] In another observation during this study, the color of culture medium started turning into light yellow coloration after 8 h of incubation and into dark yellow/brown at the end of the incubation period. Similar outcomes were reported where yellow-colored products were observed in oxidation of ferulic acid by Lac from Myceliophthora thermophila.[50] In another report, a fungal strain of Sporotrichum thermophile was able to transform ferulic acid into vanillic acid via 4-vinylguaiacol, which was further converted to guaiacol.[51] Lac can form quinone by oxidizing guaiacol and produce yellow-colored products.[52]

P. chrysosporium ATCC 24725 has been reported for bioconversion of ferulic acid to vanillin where green coconut agro-industrial husk was used as a source of ferulic acid.[53] In another report, ferulic acid was transformed to vanillic acid using Aspergillus niger K8, and it was further transformed to vanillin using P. chrysosporium ATCC 24725.[54] These prior studies revealed that the P. chrysosporium species are capable of transforming ferulic acid to vanillin through various pathways. However, limited reports are available related to the role of ligninolytic enzymes during the degradation process. Ferulic acid has been reported for the production of aromatic aldehydes, where alkyl side chain has to be cleaved to obtain vanillin.[55] LiP of P. chrysosporium is capable of cleaving similar alkyl side chains in various lignin model compounds.[56] From the aspect of biochemical reactions, bioconversion of ferulic acid to vanillin could be a combined effect of decarboxylase, oxygenase, oxidase, and oxidoreductase.[1] Decarboxylation of vanillic acid by Lac or peroxidases has been validated in an earlier report.[57] Despite the fact that P. chrysosporium species does not produce ferulic acid decarboxylase, which plays a key role in decarboxylation, in another report, LiP of P. chrysosporium could carry out enzymatic decarboxylation of oxalic acid in the presence of veratryl alcohol.[58] Veratryl alcohol is known to activate a number of ligninase in white-rot fungi.[38] This study also revealed that the veratryl alcohol was an efficient inducer and was able to activate the multi-enzyme system, which helped in utilizing the ferulic acid in the overall bioconversion process.

Although a few researchers have reported polymerization of ferulic acid when treated with only Lac,[59] there are reports of production of substantial amount of vanillin in bioconversion of ferulic acid by Sporotrichum pulverulentum (the anamorph of P. chrysosporium) containing Lac/LiP enzyme system.[60] Note that the extent of Lac activity compared to that of other ligninolytic enzyme, such as LiP, might have played a key role in the overall bioconversion process. We did observe accumulation of vanillin for a very short period from 18 to 24 h. However, the vanillin was significantly decreased thereafter, possibly because of further oxidation of vanillin to other intermediates. These findings were consistent with another report where vanillic acid and vanillin were accumulated in small amounts by S. pulverulentum, in the presence of ferulic acid.[61] We also observed traces of vanillic acid and coniferyl aldehyde in degradation products. Because ferulic acid can be reversibly reduced to coniferyl aldehyde in basidiomycete,[62] this could be probably because of the action of Lac, which is an oxidoreductase. An alternative pathway, in which ferulic acid can be decarboxylated to 4-vinyl guaiacol and further oxidized to vanillin and vanillic acid, has been proposed for basidiomycete.[63] Despite the fact that we could not detect 4-vinyl guaiacol, a possibility of forming it in the process and its rapidly getting further oxidized couldn’t be ruled out. No traces of vanillin or vanillic acid were observed in culture containing Lac of T. hirsuta. It might be because of the comparatively high level of Lac activity, which led to rapid polymerization of ferulic acid which further degraded it completely. Also, Lac of T. hirsuta has been reported for higher redox potential when compared to other laccase-producing basidiomycetes, which might have contributed to comparatively faster degradation process.[64] On the contrary, P. chrysosporium produced a comparatively low level of Lac. Falconnier et al. demonstrated that Pycnoporus cinnabarinus I-937 with low Lac activity can efficiently transform ferulic acid to vanillin.[62] Lac/LiP might have played a significant role and could be responsible for the bioconversion, to some extent. However, other metabolic enzymes might have contributed to the overall bioconversion process. In another report, vanillate hydroxylase was found in P. chrysosporium and apparently decarboxylated the substrate.[65] Moreover, P. chrysosporium also has genes for various oxygenase enzymes, including monooxygenase, alcohol dehydrogenase, dioxygenase, intradiol dioxygenases, extradiol dioxygenases, and many other similar aromatic compound degrading enzymes.[66] Therefore, the accumulation of vanillin from ferulic acid degradation might be the effect of all of these enzymes altogether.

Conclusions

Two white-rot fungal strains (T. hirsuta MTCC-1171 and P. chrysosporium NCIM-1106) were studied for their capability to degrade ferulic acid. Both strains degraded ferulic acid effectively in basal MSM, where they used ferulic acid as a sole source of carbon and further identified its metabolic products. T. hirsuta was proven to be a better candidate for the degradation purpose of ferulic acid because it achieved complete degradation of ferulic acid (350 mg L–1) in 20 h of incubation. Although P. chrysosporium showed comparatively slower rate of degradation, 28 h for 250 mg L–1 of ferulic acid, vanillin was found to be accumulated over a period of 18–24 h. Further degradation of intermediates was observed and traces of vanillic acid and coniferyl aldehyde were also found at the end of the incubation period. No vanillin or vanillic acid was detected in a culture medium of T. hirsuta. The possible role of ligninolytic enzymes such as Lac and LiP was investigated in the overall bioconversion process. Moreover, optimum bioconversion conditions for the production of vanillin by P. chrysosporium were identified. Maximum vanillin accumulated was 6.61 ± 0.21 mg L–1 with molar yield (3.4 ± 0.10%) from ferulic acid (250 mg L–1) at optimum bioconversion conditions. This study revealed that the white-rot fungi could be efficiently employed for degradation of ferulic acid, indicating that T. hirsuta and P. chrysosporium have a high potential for efficient applications in bioremediation of pollutants. Moreover, the fundamental understanding acquired during this investigation should contribute a valuable platform to investigate the behavior of white-rot fungi associated with the bioconversion of ferulic acid and its potential applications in the production of vanillin.