Enhanced Exoglucanase Production by Brown Rot Fungus Fomitopsis sp. RCK2010 and its Application for Cellulose Saccharification

Exoglucanase production by brown rot fungus Fomitopsis sp. RCK2010 was optimized under solid-state fermentation using Plackett–Burman design (PBD) and response surface methodology (RSM). Four fermentation variables (moisture, inoculum level, casein, and Triton X-100) were identified to effect cellulase production significantly by PBD, which were further optimized using RSM of central composite design. An overall 130 % increase in enzyme production was achieved by the optimization of variables using statistical approaches. Moreover, crude cellulase from Fomitopsis sp. RCK2010 was applied to saccharify pretreated Prosopis juliflora (cellulosic fraction), which resulted in the release of 327.35 mg/g of reducing sugars that could further be utilized for bioethanol production.     

Characteristics of Bacterial Strains with Desirable Flavor Compounds from Korean Traditional Fermented Soybean Paste (Doenjang)

To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.     

Purification of α-Amylase from Bacillus sp. GHA1 and its partial characterization

Bacillus sp. GHA1 was isolated from water samples and screened for the production of α-amylase. Maximum production of amylase by this strain occurs at 42 °C, pH 6.5 and 72 h after cultivation in production medium. The enzyme was purified through successive applications of ammonium sulfate precipitation, ion exchange and hydrophobic interaction chromatography, resulting in a single band with an apparent molecular weight of 66 kDa, as judged by SDS-PAGE. Calcium analysis of the purified enzyme revealed that it contained three metal ions per molecule. The new extracellular α-amylase is active in a wide range of pH with its maximum activity at pH values 5.5–8.0. The optimum temperature for enzyme activity is 57 °C and the presence of calcium has relatively low influence on its activity and thermostability. The Bacillus sp. GHA1 α-amylase with these properties may be suitable for use in detergent and food industries.     

Statistical optimization of conditions for protease production fromBacillus sp. and its scale-up in a bioreactor

A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained through the method of testing the parameters one at a time were starch, soybean meal, CaCl₂, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production, yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization of a large number of variables and provides profound insight into the interactive effect of various parameters involved in protease production.     

Intrinsic Characteristics of Cr6+-Resistant Bacteria Isolated from an Electroplating Industry Polluted Soils for Plant Growth-Promoting Activities

The Cr⁶⁺-resistant plant growth-promoting bacteria was isolated from soil samples that were collected from an electroplating industry at Coimbatore, India, that had tolerated chromium concentrations up to 500?mg Cr⁶⁺/L in Luria-Bertani medium. Based on morphology, physiology, and biochemical characteristics, the strain was identified as Bacillus sp. following the Bergey's manual of determinative bacteriology. Evaluation of plant growth-promoting parameters has revealed the intrinsic ability of the strain for the production of indole-3-acetic acid (IAA), siderophore, and solubilization of insoluble phosphate. Bacillus sp. have utilized tryptophan as a precursor for their growth and produced IAA (122???g/mL). Bacillus sp. also exhibited the production of siderophore that was tested qualitatively using Chrome Azurol S (CAS) assay solution and utilized the insoluble tricalcium phosphate as the sole source of phosphate exhibiting higher rate of phosphate solubilization after 72?h of incubation (1.45???g/mL). Extent of Cr⁶⁺ uptake and accumulation of Cr⁶⁺ in the cell wall of Bacillus sp. was investigated using atomic absorption spectrophotometer and scanning electron microscope-energy dispersive spectroscopy, respectively. The congenital capability of this Cr⁶⁺-resistant plant growth-promoting Bacillus sp. could be employed as bacterial inoculum for the improvement of phytoremediation in heavy metal contaminated soils.     

Enhanced Bioethanol Production from Potato Peel Waste Via Consolidated Bioprocessing with Statistically Optimized Medium

In this study, an extensive screening was undertaken to isolate some amylolytic microorganisms capable of producing bioethanol from starchy biomass through Consolidated Bioprocessing (CBP). A total of 28 amylolytic microorganisms were isolated, from which 5 isolates were selected based on high α-amylase and glucoamylase activities and identified as Candida wangnamkhiaoensis, Hyphopichia pseudoburtonii (2 isolates), Wickerhamia sp., and Streptomyces drozdowiczii based on 26S rDNA and 16S rDNA sequencing. Wickerhamia sp. showed the highest ethanol production (30.4 g/L) with fermentation yield of 0.3 g ethanol/g starch. Then, a low cost starchy waste, potato peel waste (PPW) was used as a carbon source to produce ethanol by Wickerhamia sp. Finally, in order to obtain maximum ethanol production from PPW, a fermentation medium was statistically designed. The effect of various medium ingredients was evaluated initially by Plackett-Burman design (PBD), where malt extracts, tryptone, and KH₂PO₄ showed significantly positive effect (p value < 0.05). Using Response Surface Modeling (RSM), 40 g/L (dry basis) PPW and 25 g/L malt extract were found optimum and yielded 21.7 g/L ethanol. This study strongly suggests Wickerhamia sp. as a promising candidate for bioethanol production from starchy biomass, in particular, PPW through CBP.     

Characterization of a Thermotolerant and Alkalotolerant Xylanase from a Bacillus sp.

In a recent screening for thermophilic bacteria from Azores hot springs, a Bacillus sp strain 3M, exhibiting cellulase-free extracellular xylanolitic activity, was isolated. Further enzyme characterization from liquid cultures grown on birchwood xylan revealed that the endo-l,4-βxylanase retains 100% of activity for at least 3 d at 55°C. At 80°C, it retains 47% of its maximal activity, and the enzyme is still active at 90°C. The optimum pH of the enzyme has a broad pH range, between 6.0 and 7.5, and it is remarkably active for the alkaline region, exhibiting 89% of relative activity at pH 9.O. The enzyme was partially inactivated by different divalent metal ions. Because of its tolerance for high temperature and pH conditions, and the absence of contaminating cellulase activity, the xylanase produced byBacillus sp 3M appears to be attractive for use in the pulp and paper industry. Indeed, the efficiency of the enzyme application to the kraftEucalyptus pulp was studied for bleaching pretreatment, resulting in a moderate increase of pulp bleachability.     

Production of Cellobiose Dehydrogenase from a Newly Isolated White Rot Fungus Termitomyces sp. OE147

Class I cellobiose dehydrogenases (CDHs) are extracellular hemoflavo enzymes produced at low levels by the Basidiomycetes (white rot fungi). In presence of suitable electron acceptors, e.g., cytochrome c, 2,6-dichlorophenol-indophenol, or metal ions, it oxidizes cellobiose to cellobionolactone. A stringent requirement for disaccharides makes CDH also useful for conversion of lactose to lactobionic acid, an important ingredient in pharma and detergent industry. In this work, class I CDH was produced using a newly identified white rot fungus Termitomyces sp. OE147. Four media were evaluated for CDH production, and maximum enzyme activity of 0.92 international unit (IU)/ml was obtained on Ludwig medium under submerged conditions. Statistical optimization of N source, which had significant effect on CDH production, using Box–Behnken design followed by optimization of inoculum size and age resulted in an increase in activity to 2.9 IU/ml and a productivity of ~25 IU/l/h. The nearly purified CDH exhibited high activity of 26.4 IU/mg protein on lactose indicating this enzyme to be useful for lactobionic acid synthesis. Some of the internal peptide sequences bore 100 % homology to the CDH produced in Myceliophthora thermophila. The fungal isolate was amenable to scale up, and an overall productivity of ~18 IU/l/h was obtained at 14-l level.     

Optimization of Extraction or Purification Process of Multiple Components from Natural Products: Entropy Weight Method Combined with Plackett–Burman Design and Central Composite Design

In this paper, the optimization of the extraction/purification process of multiple components was performed by the entropy weight method (EWM) combined with Plackett–Burman design (PBD) and central composite design (CCD). We took the macroporous resin purification of Astragalus saponins as an example to discuss the practicability of this method. Firstly, the weight of each component was given by EWM and the sum of the product between the componential content and its weight was defined as the comprehensive score, which was taken as the evaluation index. Then, the single factor method was adopted for determining the value range of each factor. PBD was applied for screening the significant factors. Important variables were further optimized by CCD to determine the optimal process parameters. After the combination of EWM, PBD and CCD, the resulting optimal purification conditions were as follows: pH value of 6.0, the extraction solvent concentration of 0.15 g/mL, and the ethanol volume fraction of 75%. Under the optimal conditions, the practical comprehensive score of recoveries of saponins was close to the predicted value (n = 3). Therefore, the present study provided a convenient and efficient method for extraction and purification optimization technology of multiple components from natural products.     

Estimation of Fundamental Kinetic Parameters of Polyhydroxybutyrate Fermentation Process of Azohydromonas australica Using Statistical Approach of Media Optimization

Polyhydroxybutyrate or PHB is a biodegradable and biocompatible thermoplastic with many interesting applications in medicine, food packaging, and tissue engineering materials. The present study deals with the enhanced production of PHB by Azohydromonas australica using sucrose and the estimation of fundamental kinetic parameters of PHB fermentation process. The preliminary culture growth inhibition studies were followed by statistical optimization of medium recipe using response surface methodology to increase the PHB production. Later on batch cultivation in a 7-L bioreactor was attempted using optimum concentration of medium components (process variables) obtained from statistical design to identify the batch growth and product kinetics parameters of PHB fermentation. A. australica exhibited a maximum biomass and PHB concentration of 8.71 and 6.24?g/L, respectively in bioreactor with an overall PHB production rate of 0.75?g/h. Bioreactor cultivation studies demonstrated that the specific biomass and PHB yield on sucrose was 0.37 and 0.29?g/g, respectively. The kinetic parameters obtained in the present investigation would be used in the development of a batch kinetic mathematical model for PHB production which will serve as launching pad for further process optimization studies, e.g., design of several bioreactor cultivation strategies to further enhance the biopolymer production.     

Production and application of amylases of Rhizopus oryzae and Rhizopus microsporus var. oligosporus from industrial waste in acquisition of glucose

Amylases from Rhizopus oryzae and Rhizopus microsporus var. oligosporus were obtained using agro-industrial wastes as substrates in submerged batch cultures. The enzymatic complex was partially characterised for use in the production of glucose syrup. Type II wheat flour proved better than cassava bagasse as sole carbon source for amylase production. The optimum fermentation condition for both microorganisms was 96 hours at 30°C and the amylase thus produced was used for starch hydrolysis. The product of the enzymatic hydrolysis indicated that the enzyme obtained was glucoamylase, only glucose as final product was attained for both microorganisms. R. oligosporus was of greater interest than R. oryzae for amylase production, taking into account enzyme activity, cultivation time, thermal stability and pH range. Glucose syrup was produced using concentrated enzyme and 100 g L^?1 starch in a 4 hours reaction at 50°C. The bioprocess studied can contribute to fungus glucoamylase production and application.     

A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4?kDa with an NH₂-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40?°C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5?mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-??-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K _m and V _max values toward beechwood xylan were 3.24?mg?ml^?1 and 58.21???mol?min^?1?mg^?1 protein, respectively.     

Sonoassisted Microbial Reduction of Chromium

This study presents sonoassisted microbial reduction of hexavalent chromium (Cr(VI)) using Bacillus sp. isolated from tannery effluent contaminated site. The experiments were carried out with free cells in the presence and absence of ultrasound. The optimum pH and temperature for the reduction of Cr(VI) by Bacillus sp. were found to be 7.0 and 37 °C, respectively. The Cr(VI) reduction was significantly influenced by the electron donors and among the various electron donors studied, glucose offered maximum reduction. The ultrasound-irradiated reduction of Cr(VI) with Bacillus sp. showed efficient Cr(VI) reduction. The percent reduction was found to increase with an increase in biomass concentration and decrease with an increase in initial concentration. The changes in the functional groups of Bacillus sp., before and after chromium reduction were observed with FTIR spectra. Microbial growth was described with Monod and Andrews model and best fit was observed with Andrews model.     

Preparation of Magnetic Nanoparticles and Their Use for Immobilization of C-Terminally Lysine-Tagged <Emphasis Type="Italic">Bacillus</Emphasis> sp. TS-23 α-Amylase

The aim of this investigation was to synthesize the adipic acid-modified magnetic nanoparticles for the efficient immobilization of C-terminally lysine-tagged α-amylase (BACΔNC-Lys₇) from thermophilic Bacillus sp. strain TS-23. The carboxylated magnetic nanoparticles were prepared by the simple co-precipitation of Fe³⁺/Fe²⁺ in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy micrographs showed that the carboxylated magnetic nanoparticles remained discrete and had no significant change in size after the binding of BACΔNC-Lys₇. Free enzyme was active in the temperature range of 45–70 °C and had an optimum of 60 °C, whereas the thermal stability of BACΔNC-Lys₇ was improved as a result of immobilization. The immobilized BACΔNC-Lys₇ could be recycled 20 times without a significant loss of the amylase activity and had a better stability during storage with respect to free enzyme. Taken together, the magnetic nanoparticles coated with this functional moiety can be a versatile platform for the effective manipulation of various kinds of engineered proteins.     

Saccharification of marine microalgae using marine bacteria for ethanol production

The saccharification of marine microalgae using amylase from marine bacteria in saline conditions was investigated. An amylase-producing bacterium, Pseudoalterimonas undina NKMB 0074 was isolated and identified. The green microalga NKG 120701 was determined to have the highest concentration of intracellular carbohydrate and was found from our algal culture stocks. P. undina NKMB 0074 was inoculated into suspensions containing NKG 120701 cells and increasingly reduced suspended sugars with incubation time. Terrestrial amylase and glucoamylase were inactive in saline suspension. Therefore, marine amylase is necessary in saline conditions for successful saccharification of marine microalgae.     

Production,purification and characterisation of a chitosanase from Bacillus cereus

In the present work, a chitosanase was induced from a squid pen powder-containing Bacillus cereus TKU031 medium, and the addition of 0.05 % (w/v) boric acid or sodium tetraborate resulted in 195 and 177 % enhancement, respectively, in TKU031 chitosanase production. The purified TKU031 chitosanase exhibited optimum activity at pH 5 and 50 °C and was stable at pH 5–9 and <50 °C. The TKU031 chitosanase that was used for chitooligomers preparation was studied. The enzyme products revealed various chitooligomers with different degrees of polymerisation from 3 to 8, as determined by a MALDI-TOF–mass spectrometer, confirming the endo-type nature of the TKU031 chitosanase.     

Extraction and characterization of total phenolic and flavonoid contents from bark of Swietenia macrophylla and their antimicrobial and antioxidant properties

In the current study, the bioactive compounds such as total phenols, flavonoids, hydrolyzable tannins, and gallic acid were extracted from the bark of Swietenia macrophylla using four different solvents such as ethanol, methanol, acetone, and water. Among them, acetone exhibited the highest contents of bioactive compounds. To optimize the extraction process, a statistical approach was adopted using the central composite design (CCD) of response surface methodology (RSM). The five parameters at five different levels were chosen in the design of the experiments. A total of 32 experimental runs given by the design were fitted into the second-order regression model equation. The analysis of the model shows the best fit of the experimental data with an R² of 0.9971 and a model F-value of 191.73. The optimal conditions of acetone concentration (56 %), the volume of acetone (22 mL), agitation speed (173 rpm), extraction temperature (31 °C), and extraction time (28 h) were noted from desirability function and showed a 2.0-fold increase in the contents of bioactive compounds when compared to unoptimized conditions. Further, the antimicrobial activity of 5 % (w/v) extract was tested against two-gram positive strains Bacillus sp, and Staphylococcus aureus, and two-gram negative strains Escherichia coli, and S. marcescens. The extract exhibited the 21 mm and 18 mm clear zone of diameter with 5 mm standard disc against the gram-positive strains tested whereas no clear zone was found against gram-negative strains. Finally, the antioxidant property was electrochemically analyzed using cyclic voltammetry and Differential pulse voltammetry, which confirmed the presence of multiple antioxidants in the extract.     

Amicoumacins from the marine-derived bacterium Bacillus sp. with the inhibition of NO production

Chemical examination of the fermentation broth of the marine-derived bacterium Bacillus sp. resulted in the isolation of seven new amicoumacin-type isocoumarin derivatives, namely bacillcoumacins A–G (1–7), together with four known analogues. Their structures were elucidated on the basis of extensive spectroscopic analysis, while the absolute configurations of the new compounds were determined by CD, Mosher’s method, and chemical conversion. Compounds 7 and 9 showed inhibitory effects against the NO production induced by lipopolysaccharide (LPS) in mouse macrophage RAW 264.7 cells.     

Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation

This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO₄, pretreated S. spontaneum, K₂HPO₄, (NH₄)₂SO₄ and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.     

Production of Microbial Cellulose by a Bacterium Isolated from Fruit

This study presents the production of bacterial cellulose (BC) by a bacterium isolated from a rotten fruit and its process optimization. Here, isolation and screening of potent cellulose producers were carried out from different natural sources, viz., soil, rotten fruits, and vegetables and vinegar. A total of 200 bacterial isolates were obtained, which were screened for cellulose production using Hestrin?CSchramm medium. A novel and potent cellulose-producing bacterium was newly isolated from a rotten fruit and identified as Gluconacetobacter sp. F6 through 16S ribosomal DNA sequencing and morphological, cultural, and biochemical characteristics. After optimization of culture conditions, including pH, temperature, agitation, carbon/nitrogen sources, and inducers, the BC production was greatly increased from 0.52 to 4.5?g/l (8.65-fold increase). The optimal culture medium contained 1% (w/v) glucose, 1.5% (w/v) yeast extract, 0.5% (w/v) peptone, 0.27% (w/v) disodium hydrogen phosphate, 0.115% (w/v) citric acid, and 0.4% (w/v) ethanol. BC produced was analyzed for the presence of cellulose fibrils by epiflourescent microscopy using Calcofluor white stain and scanning electron microscopy and confirmed by NMR. There are very scanty reports about the optimization of BC production by bacteria isolated from rotten fruits.