Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents

In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated. Substrates included wood chips, cereal grain, wheat husk and wheat bran. Suitability for transformation of chlorophenols depended on laccase production by the fungus. The greatest amount of laccase (<25 Units g⁻¹ substrate) was produced on wheat husk and wheat bran over 30 days colonisation. Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75–80% of pentachlorophenol (50 ppm) within 24 h. Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation. Addition of 8–12% yeast extract to the pellets increased laccase production five-fold. Colonised pellets were added to chlorophenol solutions in 200–4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. Received: 10 April 2000 / Received revision: 4 July 2000 / Accepted: 10 July 2000     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Development,identification, and characterization of blue-grained wheat- Triticum boeoticum substitution lines

Diploid wild einkorn wheat, Triticum boeoticum Boiss (AbAb, 2n = 2x = 14), is a wheat-related species with a blue aleurone layer. In this study, six blue-grained wheat lines...     

Carrageenan of <Emphasis Type="Italic">Eucheuma isiforme</Emphasis> (Solieriaceae,Rhodophyta) from Nicaragua

The yield and physicochemical properties of native and alkali treated carrageenan from Eucheuma isiforme harvested from the Nicaraguan coast were investigated. The native carrageenan yield was 57.2% of dry weight and decreased to 43.5% when the alga was alkali treated. Native carrageenan viscosities showed significant differences between native (144.6 ± 3.3 cPs) and treated carrageenan (113.9 ± 2.6 cPs) (p < 0.01). Alkali treatment reduced carrageenan sulphate content by 19.3% and increased 3,6 AG content by 13%. Alkali-treated carrageenan formed very weak gels in 1.5% solutions (<50 g cm⁻²). Chemical analysis and FTIR spectra revealed that Eucheuma isiforme from Nicaragua is a good source of relatively pure iota-carrageenan with sufficient quality to serve as a substitute for traditional iota-carrageenan sources.     

Occurrence of some enzymes in starchy endosperm and hormonal regulation of isoperoxidase in aleurone of wheat

Peroxidase, indoleacetic acid-oxidase, alkaline, and acid phosphatases were detected in dry starchy endosperm (minus aleurone) of wheat grain. The isoperoxidase pattern differed in different parts of the dry grain. Several new isoperoxidases were found in embryos after soaking. The intensity of isoperoxidases in aleurones was enhanced in the presence of embryo or 2 μM GA₃ after 24 hours of soaking, but decreased after 72 hours. Indoleacetic acid and kinetin had no effect on isoperoxidase of aleurone. Actinomycin D and cycloheximide had no effect on isoperoxidases of aleurones from embryonectomized or naturally occurring embryoless grains. However, these two inhibitors increased the intensity of isoperoxidases in aleurones of intact embryonated grains after soaking.     

Solid Loss of Carrots During Simulated Gastric Digestion

The knowledge of solid loss kinetics of foods during digestion is crucial for understanding the factors that constrain the release of nutrients from the food matrix and their fate of digestion. The objective of this study was to investigate the solid loss of carrots during simulated gastric digestion as affected by pH, temperature, viscosity of gastric fluids, mechanical force present in stomach, and cooking. Cylindrical carrot samples were tested by static soaking method and using a model stomach system. The weight retention, moisture, and loss of dry mass were determined. The results indicated that acid hydrolysis is critical for an efficient mass transfer and carrot digestion. Internal resistance rather than external resistance is dominant in the transfer of soluble solids from carrot to gastric fluid. Increase in viscosity of gastric fluid by adding 0.5% gum (w/w) significantly increased the external resistance and decreased mass transfer rate of carrots in static soaking. When mechanical force was not present, 61% of the solids in the raw carrot samples were released into gastric fluid after 4 h of static soaking in simulated gastric juice. Mechanical force significantly increased solid loss by causing surface erosion. Boiling increased the disintegration of carrot during digestion that may favor the loss of solids meanwhile reducing the amount of solids available for loss in gastric juice. Weibull function was successfully used to describe the solid loss of carrot during simulated digestion. The effective diffusion coefficients of solids were calculated using the Fick’s second law of diffusion for an infinite cylinder, which are between 0.75 × 10⁻¹¹ and 8.72 × 10⁻¹¹ m²/s, depending on the pH of the gastric fluid.     

Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues

The objective of this study was to investigate the function of heat shock protein 60 (HSP60) on pancreatic tissues by applying HSP60 small interfering RNA (siRNA) to reduce HSP60 expression. Rat pancreas was isolated and pancreatic tissue snips were prepared, cultured, and stimulated with low and high concentrations of cerulein (10⁻¹¹ and 10⁻⁵ mol/L) or lipopolysaccharide (LPS, 10 and 20 μg/mL). Before the stimulation and 1 and 4 h after the stimulation, the viability and the level of trypsinogen activation peptide (TAP) in the tissue fragments were determined and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the culture supernatants were measured. Real-time PCR and Western blotting were used to evaluate the HSP60 mRNA and protein expression. After the administration of siRNA to inhibit HSP60 expression in the isolated tissues, these injury parameters were measured and compared. The pancreatic tissues in the control (mock-interfering) group showed a decreased viability to varying degrees after being stimulated with cerulein or LPS, and the levels of TAP, TNF-α, and IL-6 increased significantly (p < 0.05) in the tissues and/or in the culture supernatant. The expressions of HSP60 mRNA and protein were raised moderately after stimulating 1 h with low concentrations of cerulein or LPS, but decreased with high concentrations of the toxicants. In particular, the expression of HSP60 protein was reduced significantly (p < 0.05) when the tissues were stimulated by the two toxicants for 4 h. In contrast, the tissue fragments in which HSP60 siRNA was applied showed much lower tissue viability (p < 0.01) and higher levels of TNF-a, IL-6, and TAP (p < 0.01) in the tissues or culture supernatant after stimulating with the toxicants at the same dose and for the same time duration as compared with those of the control groups (p < 0.05). The results indicated that both cerulein and LPS can induce injuries on isolated pancreatic tissues, but the induction effects are dependent on the duration of the stimulation and on the concentrations of the toxicants. HSP60 siRNA reduces HSP60 expression and worsens the cerulein- or LPS-induced injuries on isolated pancreatic tissues, suggesting that HSP60 has a protective effect on pancreatic tissues against these toxicants.     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.     

A 56-kDa protein is a novel granule-bound starch synthase existing in the pericarps, aleurone layers, and embryos of immature seed in diploid wheat (Triticum monococcum L.)

A novel 56-kDa granule-bound starch synthase (GBSS; NDPglucose-starch glucosyltransferase, EC 2.4.1.21) responsible for amylose synthesis was found in the pericarps, aleurone layers and embryos of immature diploid wheat (Triticum monococcum L.). The GBSS and other proteins bound to starch granules of various tissues of immature normal and waxy diploid wheat seeds were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their activities were examined. In the waxy mutant, the waxy protein (59.5 kDa, GBSSI) was absent, but amylose and GBSS activity were evident in all tissues except the endosperm. Of the proteins bound to starch granules, only the 56-kDa protein was associated with the presence of amylose and GBSS activities in the pericarps, aleurone layers and embryos. Mutations at the waxy locus did not affect the 56-kDa protein in these tissues. Changes in the amount of 56-kDa protein during the course of seed development, and the distribution of the 56-kDa protein in each tissue of immature seeds were quite different from those of the waxy protein. On the other hand, the N-terminal amino acid sequence of the 56-kDa protein had a 40–50% similarity to GBSSI of some other plant species and was antigenically related to the waxy protein. These results strongly suggest that the 56-kDa protein in diploid wheat is a GBSSI class enzyme and, hence, an isoform of the waxy protein. The waxy protein and 56-kDa protein, however, are expressed in different seed tissues and at different stages of seed development. Received: 15 May 1998 / Accepted: 18 June 1998     

Patterns of stable carbon isotope turnover in gag, <Emphasis Type="Italic">Mycteroperca microlepis</Emphasis>, an economically important marine piscivore determined with a non-lethal surgical biopsy procedure

To determine the feasibility of using stable isotopes to track diet shifts in wild gag, Mycteroperca microlepis, populations over seasonal timescales, we conducted a repeated measures diet-shift experiment on four adult gag held in the laboratory. Fish were initially fed a diet of Atlantic mackerel, Scomber scombrus, (mean δ¹³C = −21.3‰ ± 0.2, n = 20) for a period of 56 days and then shifted to a diet of pinfish, Lagodon rhomboids, (mean δ¹³C = −16.6‰ ± 0.6, n = 20) for the 256 day experiment. We developed a non-lethal surgical procedure to obtain biopsies of the muscle, liver, and gonad tissue monthly from the same four fish. We then determined the δ¹³C value of each tissue by isotope ratio mass spectrometry. For the gonad tissue we used the relationship between C/N and lipid content to correct for the influence of lipids on δ¹³C value. We observed a significant shift in the δ¹³C values of all of the tissues sampled in the study. Carbon turnover rates varied among the three tissues, but the shift in diet from mackerel to pinfish was clearly traceable through analysis of δ¹³C values. The turnover rates for muscle tissue were 0.005‰ day⁻¹, and for gonad tissue was 0.009‰ day⁻¹. Although it is generally thought that tissue turnover rates in ectotherms are driven primarily by growth, we found that metabolic rate can be a major factor driving tissue turnover in adult gag.     

Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping. Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998     

Inhibition of α-Amylase Acting in Hexaploid Triticale Lines by Exogenous Abscisic Acid

Hexaploid triticale introgressive lines developed after recombination of A-genome with A^m-genome of diploid wheat (Triticum monococcum) were analysed in respect of grains responsiveness to exogenous ABA treatment. This was assessed by in vivo bioassay as grain germination indices, and by α-amylase assay as quantity of synthesised α-amylase measured with the technique of radial diffusion in agarose gel. The results showed an important diminishing of seedling length caused by ABA (variable in different lines) as well as genotype dependant variability of α-amylase synthesis inhibition. The differences of ABA responsiveness were seen both in whole grains and in embryoless half-grains as a direct reaction of the aleurone layer. Variation of grain sensitivity to ABA treatment compared with two sprouting resistance indices showed a significant correlation with Falling Number values in grains, but not with a dormant grains germination in spikes. This is an evidence that in triticale precocious starch decompose in unripened and ungerminated grains is dependent on genotype ABA-responsiveness of the aleurone layer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.     

Metallothionein as Potential Biomarker of Cadmium Exposure in Persian Sturgeon (<Emphasis Type="Italic">Acipenser persicus</Emphasis>)

Metallothionein (MT) concentration in gills, liver, and kidney tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sublethal levels of waterborne cadmium (Cd) (50, 400, and 1,000 μg l⁻¹) after 1, 2, 4, and 14 days. The increases of MT from background levels were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured, and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively, 14 days after exposure to 1,000 μg l⁻¹ Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Pearson correlation coefficients showed that the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in A. persicus for waterborne Cd contamination.     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Change in wall composition of transfer and aleurone cells during wheat grain development

In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1–3)(1–4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1–3)(1–4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1–3)(1–4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.     

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Studies on the production of nigerloxin using agro-industrial residues by solid-state fermentation

Nigerloxin, a new and potent lipoxygenase inhibitor, was discovered in our laboratory through solid-state fermentation of wheat bran by Aspergillus niger V. Teigh (MTCC-5166). The aim of this study is to investigate the possibility of using different agro-industrial residues as nutritional supplements along with wheat bran to enhance the production of nigerloxin. Nigerloxin produced by SSF was quantified spectrophotometrically at 292 nm. The results indicate that the inhibitor production was influenced by the type of solid substrate supplemented, moisture content, pH and size of the inoculum. Individually optimized supplements were tested in different combinations to determine their effects on nigerloxin production. A twofold increase in the production of nigerloxin (4.9 ± 0.3 mg gds⁻¹) was achieved by supplementing wheat bran with 10% w/w sweet lemon peel and 5% v/w methanol at optimized process parameters, that is, an initial moisture content of 65% v/w and incubation period of 6 days with an initial inoculum size of 2 ml (8 × 10⁵ spores gds⁻¹). Nigerloxin production was stable between pH of 4 and 5.     

Efficacy of Allicin in Decreasing Lead (Pb) Accumulation in Selected Tissues of Lead-Exposed Common Carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

The objective of this study was to evaluate the effects of allicin, the main biologically active component of garlic clove extracts, on lead levels in different common carp tissues including liver, kidney, brain, bone, and blood following experimental lead poisoning. Fish were divided randomly into five groups depending on the combination of lead acetate and allicin treatments. Lead acetate exposure (7.0 mgL⁻¹, 10 days) caused a significant increase in mean Pb concentrations in all examined tissues in comparison to control unexposed fish (p < 0. 001). The results showed that allicin supplementation is effective in decreasing lead accumulation in all examined tissues of common carp. The promising ameliorative effects of allicin on tissue lead levels of common carp make it a good candidate for therapeutic intervention of lead poisoning. However, more studies are required to elucidate the pharmacokinetic effects of allicin and also molecular basis of the ameliorative properties of allicin in lead poisoning.