Various culture parameters were optimised for laccase synthesis by
Laccase and various microorganisms that produce the enzyme have been studied intensively due to their potential applications in industrial and remediative processes. However, one of the factors inhibiting the application of laccase is the cost associated with using large quantities of the enzyme. A possible strategy is to improve laccase yields using waste substrates as a culture media for solid or submerged fermentations. Numerous studies have investigated the most favourable conditions for laccase production by various fungi with solid and submerged fermentations [
A variety of agroindustrial waste residues may be utilized to produce laccase and thereby lower the substrate costs involved in production. Barley bran [
Inducers are compounds that significantly increase laccase production while occurring at concentrations that are extremely low relative to available carbon sources. Many inducers are phenolic or aromatic compounds related to lignin or are lignin derivatives. Non-phenolic compounds such as ethanol [
The objective of this study was to enhance laccase synthesis by
The optimal pH was assessed using a full-strength distillery wastewater (COD 29.5 g/L, total phenolic compounds 280 mg/L, and pH 3.75) adjusted to 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 using hydrochloric acid or Na2CO3 powder (both Saarchem, uniLAB, Merck). Aliquots of 65 mL of the wastewater were placed in 250 mL Erlenmeyer flasks, covered with aluminium foil (to prevent contamination), and autoclaved for fifteen minutes. Duplicate flasks were inoculated with
Different carbon sources in the form of fructose, glucose, mannitol, maltose, sucrose, cellobiose, and lactose (all Saarchem, univAR, Merck) were added to a low-strength brandy distillery wastewater (COD 10.5 g/L, total phenolic compounds 35 mg/L, and pH 3.9) to assess their individual effects on laccase synthesis. The amount added was equivalent to the molar equivalent of carbon atoms in 10 g/L of glucose. Different nitrogen sources in the form of NH4NO3, NH4Cl, KNO3 (Saarchem, univAR, Merck), and H2NCNH2 (analaR, BDH) were added at a molar equivalent of nitrogen atoms in 2 g/L of KNO3, while malt extract, yeast extract, and peptone were added at 2 g/L. Cellulose and lignin-containing supplements in the form of cellulose powder, blue gum powder, rooibos tea leaves (
All inducers were assessed in 250 mL Erlenmeyer flasks containing 65 mL of a synthetic medium containing: 2% glucose (Saarchem, uniLAB, Merck), 0.3% peptone, 0.3% malt extract (both Biolab, Merck), KH2PO4 (1 g/L), Na2HPO4·2H2O (100 mg/L), MgSO4·7H2O (500 mg/L), CaCl2 (10 mg/L), FeSO4·7H2O (10 mg/L), MnSO4·4H2O (1 mg/L), ZnSO4·7H2O (1 mg/L), and CuSO4·5H2O (2 mg/L) (all Saarchem, uniLAB, Merck). Reported inducers in the form of 3,4-dimethoxybenzyl alcohol, 2,5-xylidine (2,5-dimethylalinine), syringic acid, hydroxybenzotriazole (HBT), violuric acid (all Fluka, Sigma Aldrich Ltd, Cape Town), guaiacol,
Autoclaved flasks containing only the synthetic medium described in Section
One reported inducer, 2,5-xylidine, was additionally tested in the synthetic medium by varying the both time and number of additions. It was added aseptically such that the concentration increased by 1 mM with each addition. One, two, or three doses were administered at 48-hour intervals. Dosing commenced at different times after inoculation (see Table
Four wastewaters were obtained from a winery and two distilleries near Worcester in the Western Cape Province of South Africa and stored at 4°C. After assessment for growth inhibition, two distillery wastewaters were tested at full strength, while two of the wastewaters (a wine lees and a distilled wine lees after tartaric acid extraction) were tested at 30% concentration. Wastewater controls consisted of the raw, unadjusted wastewater. Additions of 2% glucose, 1 mM copper sulfate, or three 1 mM additions of 2,5-xylidine were assessed in the four wastewaters. In addition, a synergistic reaction was studied by combining pH adjustment, glucose, copper, and 2,5-xylidine addition. Triplicate flasks of all wastewaters were inoculated with biomass of
Laccase synthesis varied significantly over the pH range tested in the brandy distillery wastewater. A peak in production was evident at pH 5.0—as laccase synthesis decreased by more than 40% at a pH only 0.5 units more acidic and basic (Figure
The highest laccase concentration produced at various starting pH values (
The greatest laccase synthesis was obtained when fructose, glucose, sucrose, and cellobiose were used as carbon sources (Table
Laccase synthesis with different carbon, nitrogen, lignin/cellulose sources, and phosphorus (
HLA* ± std dev (units/L) | Day of HLA | Increase (fold) | ||
---|---|---|---|---|
Carbon sources | Fructose | |||
Glucose | ||||
Mannitol | ||||
Maltose | ||||
Sucrose | ||||
Cellobiose | ||||
Lactose | ||||
Nitrogen sources | NH4NO3 | |||
NH4Cl | ||||
KNO3 | ||||
H2NCNH2 | ||||
Malt extract | ||||
Yeast extract | ||||
Peptone | ||||
Lignin/cellulose | Cellulose | |||
Bluegum | ||||
Rooibos | ||||
Bagasse | ||||
Phosphorous source | H3PO4 | |||
Control |
*HLA: highest laccase activity.
The nitrogen source that improved laccase synthesis to the greatest extent was peptone (1.8-fold increase). Lower yields were obtained with an inorganic nitrogen source. The effects of inorganic nitrogen upon laccase synthesis in this study were corroborated by Revankar and Lele [
Although an inorganic nitrogen source such as asparigine aids downstream processes such as enzyme extraction and purification [
A synthetic media was used to assess a number of reported inducers (Table
Laccase synthesis obtained with the addition of various reported inducers prior to inoculation or four days thereafter (
Reported inducer | Added prior to inoculation | Added after four days | ||||
HLA* (units/L) | Day of HLA | Increase (fold) | HLA (units/L) | Day of HLA | Increase (fold) | |
2,5-Xylidine | 8419 | 2 | 3.7 | 2944 | 11 | 2.4 |
Ethanol | 6701 | 20 | 2.9 | 292 | 6 | 0.2 |
Copper | 5492 | 20 | 2.4 | 1044 | 13 | 0.9 |
4-Methylcatechol | 1153 | 20 | 0.5 | 2253 | 13 | 1.9 |
1636 | 14 | 0.7 | 2283 | 13 | 1.9 | |
Gallic acid | 3303 | 14 | 1.4 | 820 | 8 | 0.7 |
Tannic acid | 3114 | 20 | 1.4 | 1111 | 13 | 0.9 |
Quercetin | 2966 | 14 | 1.3 | 588 | 13 | 0.5 |
Syringic acid | 1862 | 14 | 0.8 | 1404 | 11 | 1.2 |
Guaiacol | 2580 | 16 | 1.1 | 1292 | 11 | 1.1 |
Dimethoxybenzyl alcohol | 1939 | 16 | 0.8 | 1247 | 13 | 1.0 |
Phenol | 2149 | 14 | 0.9 | 1270 | 11 | 1.0 |
Violuric acid | 2035 | 14 | 0.9 | 1039 | 11 | 0.9 |
Phenol red | 2601 | 12 | 1.1 | 663 | 13 | 0.5 |
Cellulose | 2474 | 16 | 1.1 | 328 | 13 | 0.3 |
2370 | 14 | 1.0 | 1227 | 13 | 1.0 | |
Rooibos | 2119 | 16 | 0.9 | 372 | 13 | 0.3 |
2064 | 12 | 0.9 | 558 | 11 | 0.5 | |
Dichloroindophenol | 127 | 12 | 0.1 | 795 | 8 | 0.7 |
Hydroxybenzotriazole | 977 | 14 | 0.4 | 656 | 13 | 0.5 |
Cycloheximide | 20 | 4 | 0.0 | 455 | 5 | 0.4 |
Control | 2305 | 12 | 1.0 | 1214 | 13 | 1.0 |
*HLA: highest laccase activity.
Laccase synthesis when varying 2,5-xylidine dosage time and number (
Days added | HLA (units/L) | Std Dev | Day of HLA | Std Dev | Increase (fold) |
---|---|---|---|---|---|
Control | 1295 | 152 | 13 | 1 | 1.0 |
0 | 9999 | 411 | 2 | 0 | 7.7 |
11012 | 688 | 3 | 1 | 8.5 | |
13294 | 627 | 7 | 1 | 10.3 | |
2 | 7529 | 321 | 4 | 0 | 5.8 |
7589 | 346 | 4 | 1 | 5.9 | |
7817 | 661 | 4 | 1 | 6.0 | |
4 | 1972 | 323 | 6 | 0 | 1.5 |
3579 | 417 | 9 | 1 | 2.8 | |
5033 | 327 | 13 | 2 | 3.9 | |
6 | 4058 | 366 | 11 | 1 | 3.1 |
3399 | 192 | 9 | 1 | 2.6 | |
3930 | 298 | 9 | 1 | 3.0 | |
8 | 1454 | 154 | 13 | 1 | 1.1 |
10 | 1134 | 78 | 13 | 0 | 0.9 |
*HLA: highest laccase activity.
There have been many studies regarding the effects of inducers using a plethora of fungal genera, species, and even strains. Differences in laccase stimulation were already observed in very early studies more than half a century ago. Fåhraeus et al. [
Following on from the success of 2,5-xylidine at greatly improving laccase titres, the effects of time of addition and multiple dosing were assessed to determine whether laccase stimulation could be improved further. The time of the initial dose and the number of additional doses were varied—as per Table
Numerous studies have shown 2,5-xylidine to be a potent inducer amongst a variety of fungal genera [
Another significant finding was that the presence of 2,5-xylidine countered glucose repression of laccase synthesis. In the control samples, the highest laccase concentrations were observed in the stationary phase at the end of the fermentation period; which has been published in prior work [
Raw wastewaters were adjusted to pH 5, supplemented with either copper, glucose, ethanol, dosed three times with 1 mM 2,5-xylidine or supplemented with a combination of glucose, copper and dosed three times with 2,5-xylidine. The initial pH, chemical oxygen demand (COD), and total phenolic compounds were measured and displayed in Table
Wastewater characteristics [
pH | COD (g/L) | Total phenols (mg/L) | |
---|---|---|---|
Brandy distillery wastewater | 3.67 | 19.9 | 320 |
Distilled wine | 3.58 | 34.8 | 290 |
Distilled wine lees | 5.09 | 45.5 | 540 |
Wine lees | 3.72 | 211.8 | 1720 |
Laccase synthesis of
The simultaneous addition of glucose, copper, and 2,5-xylidine resulted in the greatest increase in laccase synthesis in all four wastewaters. This would be expected as the wastewaters now contained carbon, copper (an essential metal component of the enzyme), and a reported inducer. Synergistic effects using 2,5-xylidine and other compounds have been observed before. Fåhraeus et al. [
The addition of 2,5-xylidine led to the most significant increases in laccase activity in the two distillery wastewaters that were tested at full strength (Figure
In the current study, laccase repression normally occurring in the presence of excess glucose was countered by the addition of 2,5-xylidine. The phenolic compound altered laccase synthesis such that the highest concentration was recorded earlier in the batch culture, as opposed to idiophasic production. When glucose was added as the only modification, the maximum laccase concentration occurred much later (approximately 11.3 days) than it did with the combination of glucose, copper, and 2,5-xylidine (approximately 6.1 days).
Copper supplementation only increased laccase synthesis in the diluted wine lees, where the combination of the metal with organic acids, sugars, ethanol, and the various phenolic compounds could have elicited greater enzyme synthesis. Copper addition by itself was of little benefit to any of the distillery wastewaters. Copper is vital to laccase synthesis as the enzyme requires four copper atoms to be catalytically active, but lack of an adequate carbon or nitrogen source or inducer may have hampered laccase synthesis. Other studies have confirmed little difference regarding laccase synthesis by
Glucose addition did not improve laccase yields. Some studies have found an abundance of glucose inhibits laccase synthesis. Moreira et al. [
There was great variability in fungal growth and enzyme synthesis in wine-related wastewaters. The wine lees and the distilled wine lees had both inhibited the growth of
Conditions tested in this study indicated that a number of factors could significantly increase laccase synthesis using
Funding from the National Research Foundation (NRF) of South Africa via a Department of Labour (DoL)/NRF Scarce Skills Bursary and the Joint Research Committee of Rhodes University is acknowledged. Opinions expressed and conclusions arrived at are those of the author and not to be attributed to the DoL or NRF. Tamlyn Stewart and Joanna Elizabeth Burgess are gratefully acknowledged for their contribution towards the paper structure and grammar.