Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes.

Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 â†’ 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine.

ß-glucosidase production by recombinant Pichia pastoris strain Y1433 under optimal feed profiles of fed-batch cultivation.

Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for ß-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for ß-glucosidase production. The results showed that at 72 h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55 g/L, respectively, while the total mutant ß-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60 h of cultivation, the highest total mutant ß-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant ß-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73 g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 gcell/gMeOH, methanol consumption rate ([Formula: see text]) 5.76 gMeOH/h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and ß- glucosidase activity were produced under exponential feed rate than constant and linear feed rates.

Two-step pretreatment of oil palm trunk for ethanol production by thermotolerent Saccharomyces cerevisiae SC90.

Oil palm (Elaeis guineensis) trunk chips were processed by steam explosion under different steam conditions, followed by alkaline extraction and fermentation to produce efficient lignocellulosic ethanol as sustainable alternative energy resource. The optimum condition of steam explosion was attained at 210°C for 4 min (α-cellulose: 58.83% and lignin: 27.12%). Taguchi 3 factor design [(sodium hydroxide concentration (NaOH), temperature and time)] was performed to optimize alkaline extraction. The optimum condition at 15% NaOH, 90°C for 60 min gave highest percentage α-cellulose: 87.14% and lowest percentage of lignin: 6.13%. Simultaneous saccharification and fermentation (SSF) involved 10% dry weight pretreated fibers, Celluclast 1.5L (15 FPU /gram substrate), Novozyme 188 (15 IU/gram substrate) and Saccharomyces cerevisiae SC90. The highest ethanol concentration (CP) produced during SSF was 44.25 g/L. Nonetheless, pre-hydrolysis simultaneous saccharification and fermentation gave 31.22 g/L (CP). All results suggested that optimized two step pretreatment produced efficient ethanol.

Genetic and metabolic engineering for microbial production of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed.

Ohmic heating pretreatment of algal slurry for production of biodiesel.

Suspensions of the model microalga Chlorella sp. TISTR 8990 were pretreated by ohmic heating to facilitate release of lipids from the cells in subsequent extraction and lipase-mediated transesterification to biodiesel. After ohmic pretreatment, the moist biomass was suspended in a system of water, hexane, methanol and immobilized lipase for extraction of lipids and simultaneous conversion to biodiesel. The ohmic pretreatment was optimized using an experimental design based on Taguchi method to provide treated biomass that maximized the biodiesel yield in subsequent extraction-transesterification operation. The experimental factors were the frequency of electric current (5-105 Hz), the processing temperature (50-70 °C), the algal biomass concentration in the slurry (algal fresh weight to water mass ratio of 1-3) and the incubation time (1-3 min). Extraction-transesterification of the pretreated biomass was carried out at 40 °C for 24 h using a reaction systems of a fixed composition (i.e. biomass, hexane, methanol, water and immobilized enzyme). Compared to control (i.e. untreated biomass), the ohmic pretreatment under optimal conditions (5 Hz current frequency, 70 °C, 1:2 mass ratio of biomass to water, incubation time of 2-min) increased the rate of subsequent transesterification by nearly 2-fold.

Model of acetic acid-affected growth and poly(3-hydroxybutyrate) production by Cupriavidus necator DSM 545.

Acetic acid, a potential growth inhibitor, commonly occurs in lignocellulosic hydrolysates. The growth of Cupriavidus necator DSM 545 and production of poly(3-hydroxybutyrate) (PHB) by this bacterium in a glucose-based medium supplemented with various initial concentrations of acetic acid are reported. The bacterium could use both glucose and acetic acid to grow and produce PHB, but acetic acid inhibited growth once its initial concentration exceeded 0.5 g/L. As acetic acid is an unavoidable contaminant in hydrolysates used as sugar sources in commercial fermentations, a mathematical model was developed to describe its impact on growth and the production of PHB. The model was shown to satisfactorily apply to growth and PHB production data obtained in media made with acetic-acid-containing hydrolysates of Napier grass and oil palm trunk as carbon substrates.

Microbial production of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on L-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid L-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.

Heterotrophic production of Chlorella sp. TISTR 8990-biomass growth and composition under various production conditions.

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon-to-nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L-1 glucose, 2.5 g L-1 KNO3 ) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500-mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L-1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g-1 and 305 mg L-1 day-1 , respectively. A repeated fed-batch culture with four cycles of feeding with the fivefold concentrated medium in a 3-L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L-1 was obtained with a TFA productivity of 223 mg L-1 day-1 . The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589-1600, 2017.

Optimization of production of C-phycocyanin and extracellular polymeric substances by Arthrospira sp.

The key factors influencing the production of C-phycocyanin (C-PC) and extracellular polymeric substances (EPS) by photoautotrophic culture of Arthrospira sp. were optimized using Taguchi method. Six factors were varied at either three or two levels as follows: light intensity at three levels; three initial culture pHs; two species of Arthrospira; three concentrations of Zarrouk's medium; three rates of aeration of the culture with air mixed with 2% v/v carbon dioxide; and two incubation temperatures. All cultures ran for 14 days. The optimal conditions for the production of C-PC and EPS were different. For both products, the best cyanobacterium proved to be Arthrospira maxima IFRPD1183. The production of C-PC was maximized with the following conditions: a light intensity of 68 µmol photons m-2 s-1 (a diurnal cycle of 16-h photoperiod and 8-h dark period), an initial pH of 10, the full strength (100%) Zarrouk's culture medium, an aeration rate of 0.6 vvm (air mixed with 2% v/v CO2) and a culture temperature of 30 °C. The concentration of Zarrouk's medium was the most important factor influencing the final concentration of C-PC. The optimal conditions for maximal production of EPS were as follows: a light intensity of 203 µmol photons m-2 s-1 with the earlier specified light-dark cycle; an initial pH of 9.5; a 50% strength of Zarrouk's medium; an aeration rate of 0.2 vvm (air mixed with 2% v/v CO2); and a temperature of 35 °C. Production of C-PC and EPS in raceway ponds is discussed.

Enhanced Production of Poly-γ-glutamic Acid by Bacillus licheniformis TISTR 1010 with Environmental Controls.

Bacillus licheniformis TISTR 1010 was used for glutamic acid-independent production of poly-γ-glutamic acid (γ-PGA). A fed-batch production strategy was developed involving feedings of glucose, citric acid, and ammonium chloride at specified stages of the fermentation. With the dissolved oxygen concentration controlled at ≥50% of air saturation and the pH controlled at ~7.4, the fed-batch operation at 37 °C afforded a peak γ-PGA concentration of 39.9 ± 0.3 g L-1 with a productivity of 0.926 ± 0.006 g L-1 h-1. The observed productivity was nearly threefold greater than previously reported for glutamic acid-independent production using the strain TISTR 1010. The molecular weight of γ-PGA was in the approximate range of 60 to 135 kDa.

Furfural and glucose can enhance conversion of xylose to xylitol by Candida magnoliae TISTR 5663.

Xylitol production from xylose by the yeast Candida magnoliae TISTR 5663 was enhanced by supplementing the fermentation medium with furfural (300mg/L) and glucose (3g/L with an initial mass ratio of glucose to xylose of 1:10) together under oxygen limiting conditions. In the presence of furfural and glucose, the final concentration of xylitol was unaffected relative to control cultures but the xylitol yield on xylose increased by about 5%. Supplementation of the culture medium with glucose alone at an initial concentration of 3g/L, stimulated the volumetric and specific rates of xylose consumption and the rate of xylitol production from xylose. In a culture medium containing 30g/L xylose, 300mg/L furfural and 3g/L glucose, the volumetric production rate of xylitol was 1.04g/L h and the specific production rate was 0.169g/g h. In the absence of furfural and glucose, the volumetric production rate of xylitol was ∼35% lower and the specific production rate was nearly 30% lower. In view of these results, xylose-containing lignocellulosic hydrolysates contaminated with furfural can be effectively used for producing xylitol by fermentation so long as the glucose-to-xylose mass ratio in the hydrolysate does not exceed 1:10 and the furfural concentration is ≤300mg/L.

Production of theabrownins using a crude fungal enzyme concentrate.

Theabrownins were produced from infusions of sun-dried green tea leaves using a crude enzyme concentrate of Aspergillus tubingensis TISTR 3647. This fungus had been isolated from a solid state fermentation of Pu-erh type tea. The crude enzyme concentrate contained activities of peroxidase, catechol oxidase and laccase. The enzyme concentrate effectively oxidized the phenolic compounds in green tea infusion to theabrownins. A theabrownins concentration of 56.0g/L was obtained in 44h. The reaction mixture contained the green tea infusion and crude enzyme concentrate in the volume ratio of 1: 0.205. The tea infusion had been produced using 200g of tea leaves per liter of distilled water. The reaction was carried out in a stirred bioreactor at 37°C with an aeration rate of 1 vvm, an agitation speed of 250rpm and a controlled pH of 7.0. Peroxidase, catechol oxidase, and laccase acted synergistically to convert the phenolic compounds in green tea infusion to theabrownins. Previously, theabrownins had been produced from green tea infusions only by using live fungal cultures. Production using the microorganism-free enzyme concentrate was comparable to production using the fungus A. tubingensis TISTR 3647. The proposed novel production process using the fungal crude enzymes and green tea infusion, offers a more controlled, reproducible and highly productive option for commercial production of theabrownins.

Carbon-to-nitrogen ratio affects the biomass composition and the fatty acid profile of heterotrophically grown Chlorella sp. TISTR 8990 for biodiesel production.

Chlorella sp. TISTR 8990 was cultivated heterotrophically in media with various initial carbon-to-nitrogen ratios (C/N ratio) and at different agitation speeds. The production of the biomass, its total fatty acid content and the composition of the fatty acids were affected by the C/N ratio, but not by agitation speed in the range examined. The biomass production was maximized at a C/N mass ratio of 29:1. At this C/N ratio, the biomass productivity was 0.68gL(-1)d(-1), or nearly 1.6-fold the best attainable productivity in photoautotrophic growth. The biomass yield coefficient on glucose was 0.62gg(-1) during exponential growth. The total fatty acids (TFAs) in the freeze-dried biomass were maximum (459mgg(-1)) at a C/N ratio of 95:1. Lower values of the C/N ratio reduced the fatty acid content of the biomass. The maximum productivity of TFAs (186mgL(-1)d(-1)) occurred at C/N ratios of 63:1 and higher. At these conditions, the fatty acids were mostly of the polyunsaturated type. Allowing the alga to remain in the stationary phase for a prolonged period after N-depletion, reduced the level of monounsaturated fatty acids and the level of polyunsaturated fatty acids increased. Biotin supplementation of the culture medium reduced the biomass productivity relative to biotin-free control, but had no effect on the total fatty acid content of the biomass.

Fungal isolates from a Pu-erh type tea fermentation and their ability to convert tea polyphenols to theabrownins.

The natural microbiota involved in the fermentation influence the quality and taste of fully postfermented teas such as China's Pu-erh tea. Ten microbial isolates representing 6 species were recovered from a solid-state fermentation of a Pu-erh type tea. The isolates were Aspergillus tubingensis, Aspergillus marvanovae, Rhizomucor pusillus, Rhizomucor tauricus, Aspergillus fumigatus, and Candida mogii. With the exception of A. marvanovae and C. mogii, all these microorganisms have been previously reported in solid-state fermentations of native Pu-erh tea. The ability of the isolates for converting the tea polyphenols to bioactive theabrownins in infusions of sun-dried green tea leaves in a submerged fermentation process was subsequently investigated. All isolates except C. mogii TISTR 5938 effectively produced theabrownins in a 4-d fermentation in shake flasks at 40 °C, 250 rpm. A. tubingensis TISTR 3646, A. tubingensis TISTR 3647, A. marvanovae TISTR 3648, and A. fumigatus TISTR 3654 produced theabrownins at particularly high levels of 6.5, 12.4, 11.1, and 8.4 g/L, respectively.

Benzoate-induced stress enhances xylitol yield in aerobic fed-batch culture of Candida mogii TISTR 5892.

Production of the natural sweetener xylitol from xylose via the yeast Candida mogii TISTR 5892 was compared with and without the growth inhibitor sodium benzoate in the culture medium. Sodium benzoate proved to be an uncompetitive inhibitor in relatively poorly oxygenated shake flask aerobic cultures. In a better controlled aerobic environment of a bioreactor, the role of sodium benzoate could equally well be described as competitive, uncompetitive or noncompetitive inhibitor of growth. In intermittent fed-batch fermentations under highly aerobic conditions, the presence of sodium benzoate at 0.15gL(-1) clearly enhanced the xylitol titer relative to the control culture without the sodium benzoate. The final xylitol concentration and the average xylitol yield on xylose were nearly 50gL(-1) and 0.57gg(-1), respectively, in the presence of sodium benzoate. Both these values were substantially higher than reported for the same fermentation under microaerobic conditions. Therefore, a fed-batch aerobic fermentation in the presence of sodium benzoate is promising for xylitol production using C. mogii.

Bioconversion of tea polyphenols to bioactive theabrownins by Aspergillus fumigatus.

Theabrownins (TB) are water-soluble phenolic compounds associated with the various health benefits of Pu-erh tea, a post-fermented Chinese dark tea. This work reports on the production of theabrownins from infusions of sun-dried green tea leaves using a pure culture of Aspergillus fumigatus isolated from a solid-state Pu-erh tea fermentation. A theabrownins yield of 158 g kg(-1) sun-dried green tea leaves was obtained in 6 days at 45 °C in an aerobic fermentation. In a 2 l fermenter, the yield of theabrownins was 151 g kg(-1) sun-dried green tea leaves in 48 h of aerobic culture (45 °C, 1 vvm aeration rate, 250 rpm agitation speed). Extracellular polyphenol oxidase and peroxidase of A. fumigatus contributed to this bioconversion. Repeated batch fermentation process was used for producing theabrownins but was less productive than the batch process.

Biodecolorization of a food azo dye by the deep sea Dermacoccus abyssi MT1.1(T) strain from the Mariana Trench.

This study reports the characterization of the ability of Dermacoccus spp. isolated from the deepest point of the world's oceans for azo dye decolorization. A detailed investigation of Dermacoccus abyssi MT1.1(T) with respect to the azoreductase activity and enzymatic mechanism as well as the potential role of the bacterial strain for biocleaning of industrial dye baths is reported. Resting cells with oxygen-insensitive azoreductase resulted in the rapid decolorization of the polysulfonated dye Brilliant Black BN (BBN) which is a common food colorant. The highest specific decolorization rate (vs) was found at 50 °C with a moderately thermal tolerance for over 1 h. Kinetic analysis showed the high rates and strong affinity of the enzymatic system for the dye with a Vmax = 137 mg/g cell/h and a Km = 19 mg/L. The degradation of BBN produces an initial orange intermediate, 8-amino-5-((4-sulfonatophenyl)diazenyl)naphthalene-2-sulfonic acid, identified by mass spectrometry which is later converted to 4-aminobenzene sulfonic acid. Nearly 80% of the maximum vs is possible achieved in resting cell treatment with the salinity increased up to 5.0% NaCl in reaction media. Therefore, this bacterial system has potential for dye decolorization bioprocesses occurring at high temperature and salt concentrations e.g. for cleaning dye-containing saline wastewaters.

Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: toward dye decolorization using a packed-bed metal affinity reactor.

This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23 kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170 µM at 600 mL/h) was achieved in 3 h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5 h-degradation period that was maintained throughout 16 cycles.

Photoautotrophic production of lipids by some Chlorella strains.

The microalgae Chlorella protothecoides UTEX 25, Chlorella sp. TISTR 8991, and Chlorella sp. TISTR 8990 were compared for use in the production of biomass and lipids under photoautotrophic conditions. Chlorella sp. TISTR 8990 was shown to be potentially suitable for lipid production at 30°C in a culture medium that contained only inorganic salts. For Chlorella sp. TISTR 8990 in optimal conditions in a stirred tank photobioreactor, the lipid productivity was 2.3 mg L(-1) h(-1) and after 14 days the biomass contained more than 30% lipids by dry weight. To attain this, the nitrogen was provided as KNO(3) at an initial concentration of 2.05 g L(-1) and chelated ferric iron was added at a concentration of 1.2 × 10(-5) mol L(-1) on the ninth day. Under the same conditions in culture tubes (36 mm outer diameter), the biomass productivity was 2.8-fold greater than in the photobioreactor (0.125 m in diameter), but the lipid productivity was only 1.2-fold higher. Thus, the average low-light level in the photobioreactor actually increased the biomass specific lipid production compared to the culture tubes. A light-limited growth model closely agreed with the experimental profiles of biomass production, nitrogen consumption, and lipid production in the photobioreactor.