Characterization of Diesel Degrading Indigenous Bacterial Strains, Acinetobacter pittii and Pseudomonas aeruginosa, Isolated from Oil Contaminated Soils.

In this study, 13 diesel degrading bacteria were isolated from the oil contaminated soils and the promising strains identified as Acinetobacter pittii ED1 and Pseudomonas aeruginosa BN were evaluated for their diesel degrading capabilities. These strains degraded the diesel optimally at 30 °C, pH 7.0 and 1% diesel concentration. Both the strains produced biofilm at 1% diesel concentration indicating their ability to tolerate diesel induced abiotic stress. Gravimetric analysis of the spent medium after 7 days of incubation showed that A. pittii ED1 and P. aeruginosa BN degraded 68.61% and 76% diesel, respectively, while biodegradation reached more than 90% after 21 days. Fourier Transform Infrared (FTIR) analysis of the degraded diesel showed 1636.67 cm-1 (C=C stretch, N-H bond) peak corresponding to alkenes and primary amines, while GC-TOF-MS analysis showed decline in hydrocarbon intensities after 7 days of incubation. The present study revealed that newly isolated A. pittii ED1 and P. aeruginosa BN were able to degrade diesel hydrocarbons (C11-C18, and C19-C24) efficiently and have potential for bioremediation of the oil-contaminated sites. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01317-3.

Parametrically optimized feather degradation by Bacillus velezensis NCIM 5802 and delineation of keratin hydrolysis by multi-scale analysis for poultry waste management.

Enormous amounts of keratinaceous waste make a significant and unexploited protein reserve that can be utilized through bioconversion into high-value products using microbial keratinases. This study was intended to assess the keratinase production from a newly isolated B. velezensis NCIM 5802 that can proficiently hydrolyze chicken feathers. Incubation parameters used to produce keratinase enzyme were optimized through the Response Surface Methodology (RSM) with chicken feathers as substrate. Optimization elevated the keratinase production and feather degradation by 4.92-folds (109.7 U/mL) and 2.5 folds (95.8%), respectively. Time-course profile revealed a direct correlation among bacterial growth, feather degradation, keratinase production and amino acid generation. Biochemical properties of the keratinase were evaluated, where it showed optimal activity at 60 °C and pH 10.0. The keratinase was inhibited by EDTA and PMSF, indicating it to be a serine-metalloprotease. Zymography revealed the presence of four distinct keratinases (Mr ~ 100, 62.5, 36.5 and 25 kDa) indicating its multiple forms. NMR and mass spectroscopic studies confirmed the presence of 18 free amino acids in the feather hydrolysates. Changes in feather keratin brought about by the keratinase action were studied by X-ray diffraction (XRD) and spectroscopic (FTIR, Raman) analyses, which showed a decrease in the total crystallinity index (TCI) (1.00-0.63) and confirmed the degradation of its crystalline domain. Scanning electron microscopy (SEM) revealed the sequential structural changes occurring in the feather keratin during degradation. Present study explored the use of keratinolytic potential of the newly isolated B. velezensis NCIM 5802 in chicken feather degradation and also, unraveled the underlying keratin hydrolysis mechanism through various analyses.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

Biotechnological potential of microbial inulinases: Recent perspective.

Among microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Screening, statistical optimized production, and application of ß-mannanase from some newly isolated fungi.

Eighty-eight fungi isolated from soil and decaying organic matter were screened for mannanolytic activity. Twenty-eight fungi produced extracellular mannanase on locust bean gum as evidenced by zone of hydrolysis produced on mannan agar gel. Six prominent producers, including four Fusarium species namely Fusarium fusarioides NFCCI 3282, Fusarium solani NFCCI 3283, Fusarium equiseti NFCCI 3284, Fusarium moniliforme NFCCI 3287 with Cladosporium cladosporioides NFCCI 3285 and Acrophialophora levis NFCCI 3286 produced the ß-mannanase in the range of 84-140 nkat/mL. All these grew well on particulate substrates in solid-state fermentation (SSF), producing relatively higher titers on mannan-rich palm kernel cake (PKC) and copra meal. Two high yielding strains, F. equiseti (1747 nkat/gds) and A. levis (897 nkat/gds) were selected for statistical optimization of mannanase on PKC. Interaction of two critical solid state fermentation parameters, pH and moisture on mannanase production by these two molds was studied by response surface method. Optimized production on PKC resulted in three- to fourfold enhancement in enzyme yield was observed in case of F. equiseti (5945 nkat/gds) and A. levis (4726 nkat/gds). HPLC analysis of mannan hydrolysate indicated that F. equiseti and A. levis mannanase performed efficient hydrolysis of konjac gum (up to 99%) with exclusive mannooligosaccahride (DP of 4) production. A seminative SDS-PAGE revealed that A. levis and F. solani produced three isoforms, F. moniliforme produced two isoforms while F. fusarioides, F. equiseti, and C. cladosporioides produced a single enzyme.

Experimental design of response surface methodology used for utilisation of palm kernel cake as solid substrate for optimised production of fungal mannanase.

The results obtained from this work strongly indicate that the solid state fermentation (SSF) system using the palm kernel cake (PKC) as a substrate is an economical method for the production of ß-mannanase at extremely low operational cost based on the fact that PKC is one of the cheap and abundant agro-waste by-products of the palm oil industry. Under initial conditions, i.e. 2 mm particle size of PKC, the moisture ratio of 1:1 of PKC:moistening agent and pH 7, Malbranchea cinnamomea NFCCI 3724 produced 109 U/gram distribution of the substrate (gds). The production of ß-mannanase was optimised by the statistical approach response surface methodology (RSM) using independent variables, namely initial moisture (12.5), pH (9.0) and solka floc (100 mg). Noticeably, six fold enhancement of ß-mannanase production (599 U/gds) was obtained under statistically optimised conditions. HPLC results revealed that ß-mannanase is an endo-active enzyme that generated manno-oligosaccharides with a degree of polymerisation (DP) of 3 and 4. Semi-native PAGE analysis revealed that M. cinnamomea produced three isoforms of mannanase. Selective production of oligosaccharide makes M. cinnamomea ß-mannanase an attractive enzyme for use in food and nutraceutical industries.

Purification and characterization of ß-mannanase from Aspergillus terreus and its applicability in depolymerization of mannans and saccharification of lignocellulosic biomass.

Aspergillus terreus FBCC 1369 was grown in solid-state culture under statistically optimized conditions. ß-Mannanase was purified to apparent homogeneity by ultrafiltration, anion exchange and gel filtration chromatography. A purification factor of 10.3-fold was achieved, with the purified enzyme exhibiting specific activity of 53 U/mg protein. The purified ß-mannanase was optimally active at pH 7.0 and 70 °C and displayed stability over a broad pH range of 4.0-8.0 and a 30 min half-life at 80 °C. The molecular weight of ß-mannanase was calculated as ~49 kDa by SDS-PAGE. The enzyme exhibited K m and V max values of 5.9 mg/ml and 39.42 µmol/ml/min, respectively. ß-Mannanase activity was stimulated by ß-mercaptoethanol and strongly inhibited by Hg2+. The ß-Mannanase did not hydrolyze mannobiose and mannotriose, but only mannotetraose liberating mannose and mannotriose. This indicated that at least four mannose residues were required for catalytic activity. Oligosaccharide with a degree of polymerization (DP) three was the predominant product in the case of locust bean gum (16.5 %) and guar gum (15.8 %) hydrolysis. However, the enzyme liberated DP4 oligosaccharide (24 %) exclusively from konjac gum. This property can be exploited in oligosaccharides production with DP 3-4. ß-Mannanase hydrolyzed pretreated lignocelluloses and liberated reducing sugars (% theoretical yield) from copra meal (30 %). This property is an important factor for the bioconversion of the biomass.

Production of inulinase, fructosyltransferase and sucrase from fungi on low-value inulin-rich substrates and their use in generation of fructose and fructo-oligosaccharides.

Owing to applications in the food and nutraceutical industries, inulinases, fructosyltransferases and sucrases have gained considerable attention in recent times. Twenty-five fungal strains were screened for production of these enzymes on three different media formulated using inulin-rich plant extracts prepared from asparagus root, dahlia tuber and dandelion root extract. Culture filtrates of the fungi were examined for hydrolytic activities. Fungi belonging to genus Aspergillus, A. niger GNCC 2655 (11.3 U/ml), A. awamori MTCC 2879 (8.2 U/ml), A. niger ATCC 26011 (7.9 U/ml) secreted high titers of inulinase followed by Penicillium sp. NFCCI 2768 (2.6 U/ml) and Penicillium citrinum MTCC 1256 (1.1 U/ml). High sucrase activity was noticed in A. niger GNCC 2613 (113 U/ml) and A. awamori MTCC 2879 (107.8 U/ml). Analysis of end products of inulinase action by HPLC revealed that most of the enzymes were exo-inulinases liberating fructose exclusively from inulin. Five fungi, P. citrinum MTCC 1256, Penicillium rugulosum MTCC 3487, Penicillium sp. NFCCI 2768, A. fumigatus GNCC 1351 and A. niger ATCC 26011 however, produced a mixture of endo- and exo-inulinases liberating oligosaccharides (GF3 and GF2) along with fructose. High inulinase/sucrase yielding strains were evaluated for extracellular and intracellular hydrolytic and transfructosylating activities and intracellular enzyme profiles were found to be considerably different in terms of titers and end products.