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

Abstract 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. </jats:sec

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

Abstract 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. </jats:sec

Tyrosinase mediated humic substances synthesis by <i>Bacillus aryabhattai</i> TFG5

AbstractThe present investigation aims at understanding the mechanism of Humic Substances (HS) formation and enhancement through tyrosinase produced by Bacillus aryabhattai TFG5. A bacterium isolated from termite mound produced tyrosinase (1.34 U.ml−1) and laccase (2.1 U.ml−1) at 48 and 60 h of fermentation respectively. The protein from B. aryabhattai TFG5 was designated as TyrB and it had a predicted molecular weight of 35.23 kDa. Swiss modelling of protein revealed a bi copper protein with its conserved residues required for activity. Interestingly, TyrB efficiently transformed and polymerized standard phenols besides transforming free phenols of Coir pith Wash Water (CWW). In addition, spectroscopic evidences suggest that TyrB enhanced the HS production from coir pith biomass. Furthermore, degradative products and changes in biomass structure by TyrB analysed through FT-IR suggests that TyrB might follow the polyphenol theory of HS synthesis.</jats:p

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

Abstract 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. </jats:sec

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

AbstractBackgroundAmylases 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 usingAspergillus oryzaeand 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 usingA. oryzaewas 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.ResultsThe substrate optimization for α-amylase production by the Box–Behnken design of RSM showed GOC as the most suitable substrate forA. 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.ConclusionsThe 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 &lt; 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.</jats:sec

Functional and molecular characterization of millet associated probiotic bacteria

Abstract The lactic acid bacteria are one of the sustainable ways of food production. As the native lactic acid bacteria (LAB) easily manipulate the substrate, helps in production of health essential probiotics with enhancing the bioavailability of the substrate. Here also, in present study, the native LAB isolates isolated from the millets and characterize them for the functional analysis for the human health association. In the present study, fermented millet-associated lactic acid bacteria were screened and characterized for their probiotic potential, safety evaluation and antimicrobial activity. A total of 33 isolates were purified as lactic acid bacteria based on colony shape and biochemical assays. However, only 13 isolates were found to be catalase-negative. Among the 13 isolates, 5 isolates exhibited optimum growth at 6.5% and 9.5% of salt concentrations, pH of 4.5 to 8.5 and 17 °C to 40 °C of the temperature. The probiotic properties of the five isolates exhibited that the survival rates in acid and bile salt concentration ranged from 56.2 to 73.7% and 55.3 to 70.3%, respectively. Similarly, the surface hydrophobicity of the isolates was 41–75%. Antibiotic assay revealed that all five isolates were resistant to Amoxicillin, Cloxacillin, and Penicillin-V. Interestingly, all the isolates except ME26 displayed susceptibility towards Penicillin (2 units) and Tetracycline (10 µg). Further, the four isolates (ME25, ME26, ME9, and ME2) had more antifungal activity against Aspergillus flavus. However, only three, except ME1 and ME2, showed maximum antibacterial activity and produced more antimicrobial compounds compared to reference strain L. plantarum Pb3. The potential probiotic isolates were identified as Weisella cibaria ME9, Weisella cibaria ME26, and Weisella confusa ME25