Tyrosinase and laccase-producing Bacillus aryabhattai TFG5 and its role in the polymerization of phenols.

BACKGROUND: Tyrosinases and laccases are oxidoreductase enzymes that are used widely in the food, feed, textile, and biofuel industries. The rapidly growing industrial demand for bacterial oxido-reductases has encouraged research on this enzyme worldwide. These enzymes also play a key role in the formation of humic substances (HS) that are involved in controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants besides increasing carbon sequestration and mitigating greenhouse gas emission in the environment. The present study aimed to screen and characterize extracellular tyrosinase and laccase-producing soil bacteria that could be utilized in the polymerization of phenols. RESULTS: Twenty isolates from different soil samples collected from forest ecosystems were characterized through ARDRA using restriction digestion with AluI, HpaII, and HaeIII restriction enzymes. The results of Hierarchical Cluster Analysis (HCA) revealed a 60 % similarity coefficient among 13 out of 20 isolates, of which, the isolate TFG5 exhibited only 10 % similarity when compared to all the other isolates. The isolate TFG5 exhibited both tyrosinase (1.34 U.mL- 1) and laccase (2.01 U.mL- 1) activity and was identified as Bacillus aryabhattai. The increased polymerization activity was observed when B. aryabhattai TFG5 was treated with phenols. The monomers such as catechol, p-Hydroxy benzoic acid, ferulic acid, and salicylic acid were polymerized efficiently, as evidenced by their FT-IR spectra depicting increased functional groups compared to the standard mushroom tyrosinase. CONCLUSIONS: The polymerization ability of B. aryabhattai TFG5 could be applied to phenol-rich wastewater treatment for efficient precipitation of phenols. Furthermore, tyrosinases can be used for enhancing the synthesis of HS in soil.

Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Bacillus aryabhattai TFG5-mediated synthesis of humic substances from coir pith wastes.

BACKGROUND: Humic substances (HS) form the largest proportion among all the constituents of soil organic matter and are a key component of the terrestrial ecosystem. HS plays a multifunctional role in the environment by controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants. The rate of formation of HS in soils determines its productivity and carbon sequestration capacity. Enhancement of HS synthesis in the soil through the microbial route not only increases CO2 sequestration but also mitigates the greenhouse gas emissions in the environment. RESULT: In this study, we attempted to understand the mechanism of formation and enhancement of HS from coir pith wastes using the tyrosinase produced by Bacillus aryabhattai TFG5. The bacterium TFG5 isolated from the termite garden produced the tyrosinase (1.34 U mL-1) and laccase (2.1 U mL-1) at 48 h and 60 h of fermentation, respectively. The extracellular tyrosinase from B. aryabhattai TFG5 was designated as TyrB. Homology modeling of TyrB revealed a structure with a predicted molecular mass of 35.23 kDa and two copper ions in the active center with its conserved residues required for the tyrosinase activity. TyrB efficiently transformed and polymerized standard phenols, such as p-cresol, p-hydroxyl benzoic acid, Levo DOPA, and 2,6 DMP, besides transforming free phenols in coir pith wash water (CWW). Additionally, UV-Vis and FT-IR spectra of the degradation products of the coir pith treated with TyrB revealed the formation of HS within 3 days of incubation. Furthermore, the E472/664 ratio of the degradation products revealed a higher degree of condensation of the aromatic carbons and the presence of more aliphatic structures in the HS. CONCLUSION: The results confirmed the influence of TyrB for the effective synthesis of HS from coir pith wastes. The results of the present study also confirm the recently accepted theory of humification proposed by the International Humic Substances Society.

Thermotolerant glycosyl hydrolases-producing Bacillus aerius CMCPS1 and its saccharification efficiency on HCR-laccase (LccH)-pretreated corncob biomass.

BACKGROUND: The current production of bioethanol based on lignocellulosic biomass (LCB) highly depends on thermostable enzymes and extremophiles owing to less risk of contamination. Thermophilic bacterial cellulases are preferred over fungi due to their higher growth rate, presence of complex multi-enzymes, stability, and enhanced bioconversion efficiency. Corncob, underutilized biomass, ensures energy conservation due to high lignocellulosic and more fermentable sugar content. In the present study, the thermophilic bacterium Bacillus aerius CMCPS1, isolated from the thermal springs of Manikaran, Himachal Pradesh, India, was characterized in terms of its activity, stability, and hydrolytic capacity. A two-step process comprising: (i) a combined strategy of hydrodynamic cavitation reaction (HCR)-coupled enzymatic (LccH at 6.5 U) pretreatment for delignification and (ii) subsequent hydrolysis of pre-treated (HCR-LccH) corncob biomass (CCB) using a thermostable cocktail of CMCPS1 was adopted to validate the efficiency of the process. Some of the parameters studied include lignin reduction, cellulose increase, and saccharification efficiency. RESULT: Among the five isolates obtained by in situ enrichment on various substrates, B. aerius CMCPS1, isolated from hot springs, exhibited the maximum hydrolytic activity of 4.11. The GH activity of the CMCPS1 strain under submerged fermentation revealed maximum filter paper activity (FPA) and endoglucanase activity of 4.36 IU mL-1 and 2.98 IU mL-1, respectively, at 44 h. Similarly, the isolate produced exoglucanase and ß-glucosidase with an activity of 1.76 IU mL-1 and 1.23 IU mL-1 at 48 h, respectively. More specifically, the enzyme endo-1,4-ß-d glucanase E.C.3.2.1.4 (CMCase) produced by B. aerius CMCPS1 displayed wider stability to pH (3-9) and temperature (30-90 °C) than most fungal cellulases. Similarly, the activity of CMCase increased in the presence of organic solvents (118% at 30% acetone v/v). The partially purified CMCase from the culture supernatant of CMCPS1 registered 64% yield with twofold purification. The zymogram and SDS-PAGE analyses further confirmed the CMCase activity with an apparent molecular mass of 70 kDa. The presence of genes specific to cellulases, such as cellulose-binding domain CelB, confirmed the presence of GH family 46 and ß-glucosidase activity (GH3). The multifunctional cellulases of CMCPS1 were evaluated for their saccharification efficiency on laccase (LccH, a fungal laccase from Hexagonia hirta MSF2)-pretreated corncob in a HCR. The lignin and hemicelluloses removal efficiency of HCR-LccH was 54.1 and 6.57%, respectively, with an increase in cellulose fraction (42.25%). The saccharification efficiency of 55% was achieved with CMCPS1 multifunctional cellulases at 50 °C and pH 5.0. CONCLUSION: The multifunctional cellulase complex of B. aerius CMCPS1 is a potential biocatalyst for application in lignocellulosic biomass-based biorefineries. The saccharification ability of HCR-LccH-pretreated corncob at elevated temperatures would be an advantage for biofuel production from lignocellulosic biomass.

Simultaneous lipid production for biodiesel feedstock and decontamination of sago processing wastewater using Candida tropicalis ASY2.

BACKGROUND: Without sufficient alternatives to crude oil, as demand continues to rise, the global economy will undergo a drastic decline as oil prices explode. Dependence on crude oil and growing environmental impairment must eventually be overcome by creating a sustainable and profitable alternative based on renewable and accessible feedstock. One of the promising solutions for the current and near-future is the substitution of fossil fuels with sustainable liquid feedstock for biofuel production. Among the different renewable liquid feedstock's studied, wastewater is the least explored one for biodiesel production. Sago wastewater is the byproduct of the cassava processing industry and has starch content ranging from 4 to 7%. The present investigation was aimed to produce microbial lipids from oleaginous yeast, Candida tropicalis ASY2 for use as biodiesel feedstock and simultaneously decontaminate the sago processing wastewater for reuse. Initial screening of oleaginous yeast to find an efficient amylolytic with maximum lipid productivity resulted in a potent oleaginous yeast strain, C. tropicalis ASY2, that utilizes SWW as a substrate. Shake flask experiments are conducted over a fermentation time of 240 h to determine a suitable fatty acid composition using GC-FID for biodiesel production with simultaneous removal of SWW pollutants using ASY2. RESULTS: The maximum biomass of 0.021 g L-1 h-1 and lipid productivity of 0.010 g L-1 h-1 was recorded in SWW with lipid content of 49%. The yeast strain degraded cyanide in SWW (79%) and also removed chemical oxygen demand (COD), biological oxygen demand (BOD), nitrate (NO3), ammoniacal (NH4), and phosphate (PO4) ions (84%, 92%, 100%, 98%, and 85%, respectively). GC-FID analysis of fatty acid methyl esters (FAME) revealed high oleic acid content (41.33%), which is one of the primary fatty acids for biodiesel production. CONCLUSIONS: It is evident that the present study provides an innovative and ecologically sustainable technology that generates valuable fuel, biodiesel using SWW as a substrate and decontaminates for reuse.