Methionine inducing carbohydrate esterase secretion of Trichoderma harzianum enhances the accessibility of substrate glycosidic bonds.

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.

Biosynthesis of poly-3-hydroxybutyrate (PHB) from glycerol by Paracoccus denitrificans in a batch bioreactor: effect of process variables.

In this study, the kinetics of poly-3-hydroxybutyrate (PHB) biosynthesis from glycerol by Paracoccus denitrificans DSMZ 413 were explored in a batch bioreactor. Effects of inorganic and organic nitrogen source, carbon to nitrogen ratio, and other process variables such as pH, aeration, and initial glycerol concentration on PHB production were investigated in a 2.5-L bioreactor. Yeast extract was found to be the best nitrogen source compared to several organic nitrogen sources tested. At pH 6, specific growth rate, product formation rate, and accumulation of PHB within the cell were maximum. Specific growth rate increased with increase in oxygen transfer rate, but moderate oxygen transfer rate promoted PHB production. High glycerol concentration inhibited specific product formation rate but not growth. High initial carbon/nitrogen (C/N) ratio favored PHB accumulation and its productivity. At a C/N ratio of 21.4 (mol mol(-1)), 10.7 g L(-1) of PHB corresponding to 72% of cell dry weight was attained.

Cell surface hydrophobicity: a key component in the degradation of polyethylene succinate by Pseudomonas sp. AKS2.

AIM: Polyethylene succinate (PES) contains hydrolysable ester bonds that make it a potential substitute for polyethylene (PE) and polypropylene (PP). Towards bioremediation of PES, we have already reported that a new strain of Pseudomonas, Pseudomonas sp. AKS2, can efficiently degrade PES and hypothesized that cell surface hydrophobicity plays an important role in this degradation process. In this study, our efforts were targeted towards establishing a correlation between cell surface hydrophobicity and PES degradation. METHODS AND RESULTS: We have manipulated cell surface hydrophobicity of AKS2 by varying concentrations of glucose and ammonium sulphate in the growth medium and subsequently examined the extent of PES degradation. We observed an increase in PES degradation by AKS2 with an increase in cell surface hydrophobicity. The increased surface hydrophobicity caused an enhanced biofilm formation on PES surface that resulted in better polymer degradation. CONCLUSION: The current study establishes a direct correlation between cell surface hydrophobicity of an organism and its potential to degrade a nonpolar polymer like PES. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell surface hydrophobicity manipulation can be used as an important strategy to increase bioremediation of nonpolar polymer like PES.

Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml(-1)) were obtained with media containing soy peptone (3-6 g l(-1)) and glutamate (3.6 g l(-1)). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control. No significant differences were observed in the highest cellulase titers and the protein pattern (according to the SDS-PAGE) of supernatants from pH controlled stirred tank bioreactor cultivations, when different nitrogen sources were used in the medium. Here the cellulase activities (~1.0 ± 0.2 FPU ml(-1)) were also much greater (8-150 times) than in shake flask cultivation. Consequently, the addition of ammonium sulphate as sole nitrogen source to Avicel basal medium is recommended when performing cultivations in stirred tank bioreactors with strict pH controlled conditions.

Enhanced Phytase Production by Bacillus subtilis subsp. subtilis in Solid State Fermentation and its Utility in Improving Food Nutrition.

BACKGROUND: Phytic acid acts as anti-nutritional factor in food and feed ingredients for monogastric animals as they lack phytases. OBJECTIVE: Phytase production by Bacillus subtilis subsp. subtilis JJBS250 was studied in solid-state fermentation and its applicability in dephytinization of food. METHODS: Bacterial culture was grown in solid state fermentation using wheat bran and various culture conditions were optimized using 'One variable at a time' (OVAT) approach. Effects of different substrates (wheat bran, wheat straw, sugarcane bagasse), incubation time (24, 48, 72 and 96 h), incubation temperatures (25, 30, 35 and 40°C), pH (4.0, 5.0, 6.0, 7.0 and 8.0) and moisture content (1:1.5, 1:2.0, 1:2.5 and 1:3) were studied on phytase production. Bacterial phytase was used in dephytinization of food samples. RESULTS: Optimization of phytase production was studied in solid state fermentation (SSF) using 'One variable at a time' (OVAT) approach. Bacillus subtilis subsp. subtilis JJBS250 grew well in various agroresidues in SSF and secreted high enzyme titres using wheat bran at 30°C and pH 5.0 after incubation time of 48 h with substrate to moisture ratio of 1:3. Glucose and ammonium sulphate supplementation to wheat bran further enhanced phytase production in SSF. Optimization of phytase production resulted in 2.4-fold improvement in phytase production in solid state fermentation. The enzyme resulted in dephytinization of wheat and rice flours with concomitant release of inorganic phosphate, reducing sugar and soluble protein. CONCLUSION: Optimization resulted in 2.34-fold enhancement in phytase production by bacterial culture that showed dephytinization of food ingredients with concomitant release of nutritional components. Therefore, phytase of B. subtilis subsp. subtilis JJBS250 could find application in improving nutritional quality of food and feed of monogastric animals.

Polyene susceptibility is dependent on nitrogen source in the opportunistic pathogen Candida albicans.

OBJECTIVES: Polyene antifungal drugs, including amphotericin B or nystatin, target ergosterol in the fungal plasma membrane and are used to treat systemic, vaginal and oral fungal infections. In the oral cavity, the available nitrogen sources are primarily in the form of proteins, which are poor nitrogen sources. This study evaluates the effect of protein as a nitrogen source on drug susceptibilities. METHODS: Candida albicans was grown in protein [bovine serum albumin (BSA) or casein (CSN)] as a sole nitrogen source, in ammonium sulphate (AS) as a nitrogen source, or in both protein and AS. RESULTS: Cells grown in BSA or CSN were 4- to 16-fold less susceptible to amphotericin B and nystatin than those grown in AS. Similar results were observed for cycloheximide, but not for fluconazole or caspofungin, and were observed for many C. albicans clinical isolates. The results were observed in two different media, and in broth and on agar. Cells grown under these nitrogen-poor conditions have a reduction in ergosterol sterol levels and a reduction in overall sterol synthesis. Quantitative real-time reverse transcription-polymerase chain reaction analysis shows that some genes involved in sterol biosynthesis are induced under nitrogen-limiting conditions, consistent with the lower sterol levels. CONCLUSIONS: The results demonstrate that nitrogen source has a significant effect on polyene susceptibilities. As these nitrogen-limiting conditions mimic oral nitrogen availability, they suggest that in vitro polyene susceptibilities may overestimate the in vivo susceptibilities to polyene drugs in the mouth.

Effect of substrate particle size and additional nitrogen source on production of lignocellulolytic enzymes by Pleurotus ostreatus strains.

Two strains of Pleurotus ostreatus (IE-8 and CP-50) were grown on defined medium added with wheat straw extract (WSE). Mycelia from these cultures were used as an inoculum for solid fermentation using sugar cane bagasse (C:N=142). This substrate was used separately either as a mixture of heterogeneous particle sizes (average size 2.9 mm) or as batches with two different particle sizes (0.92 mm and 1.68 mm). Protein enrichment and production of lignocellulolytic enzymes on each particle size was compared. The effect of ammonium sulphate (AS) addition was also analyzed (modified C:N=20), this compound favored higher levels of protein content. Strain CP-50 showed the highest increase of protein content (48% on particle size of 1.68 mm) when compared to media with no additional N source. However, strain IE-8 produced the highest levels of all enzymes: xylanases (5.79 IU/g dry wt on heterogeneous particles) and cellulases (0.18 IU/g dry wt on smallest particles), both without the addition of AS. The highest laccase activity (0.040 IU/g dry wt) was obtained on particles of 1.68 mm in the presence of AS. Since effect of particle size and addition AS was different for each strain, these criteria should be considered for diverse biotechnological applications.

The role of salinity on the changes of the biomass characteristics and on the performance of an OMBR treating tannery wastewater.

Tannery wastewaters are difficult to treat biologically due to the high salinity and organic matter concentration. Conventional treatments, like sequential batch reactors (SBR) and membrane bioreactors (MBR), have showed settling problems, in the case of SBR, and ultrafiltration (UF) membrane fouling in the case of MBR, slowing their industrial application. In this work, the treatment of tannery wastewater with an osmotic membrane bioreactor (OMBR) is assessed. Forward osmosis (FO) membranes are characterized by a much lower fouling degree than UF membranes. The permeate passes through the membrane pores (practically only water by the high membrane rejection) from the feed solution to the draw solution, which is also an industrial wastewater (ammonia absorption effluent) in this work. Experiments were carried out at laboratory scale with a FO CTA-NW membrane from Hydration Technology Innovations (HTI). Tannery wastewater was treated by means of an OMBR using as DS an actual industrial wastewater mainly consisting of ammonium sulphate. The monitoring of the biological process was carried out with biological indicators like microbial hydrolytic enzymatic activities, dissolved and total adenosine triphosphate (ATP) in the mixed liquor and microbial population. Results indicated a limiting conductivity in the reactor of 35 mS cm-1 (on the 43th operation day), from which process was deteriorated. This process performance diminution was associated by a high decrease of the dehydrogenase activity and a sudden increase of the protease and lipase activities. The increase of the bacterial stress index also described appropriately the process performance. Regarding the relative abundance of bacterial phylotypes, 37 phyla were identified in the biomass. Proteobacteria were the most abundant (varying the relative abundance between 50.29% and 34.78%) during the first 34 days of operation. From this day on, Bacteroidetes were detected in a greater extent varying the relative abundance of this phylum between 27.20% and 40.45%.

Purification and characterization of extracellular beta-glucosidase from Sinorhizobium kostiense AFK-13 and its algal lytic effect on Anabaena flos-aquae.

A beta-glucosidase from the algal lytic bacterium Sinorhizobium kostiense AFK-13, grown in complex media containing cellobiose, was purified to homogeneity by successive ammonium sulfate precipitation, and anion-exchange and gel-filtration chromatographies. The enzyme was shown to be a monomeric protein with an apparent molecular mass of 52 kDa and isoelectric point of approximately 5.4. It was optimally active at pH 6.0 and 40'C and possessed a specific activity of 260.4 U/mg of protein against 4-nitrophenyl-beta-D-glucopyranoside (pNPG). A temperature-stability analysis demonstrated that the enzyme was unstable at 50 degrees C and above. The enzyme did not require divalent cations for activity, and its activity was significantly suppressed by Hg+2 and Ag+, whereas sodium dodecyl sulfate (SDS) and Triton X-100 moderately inhibited the enzyme to under 70% of its initial activity. In an algal lytic activity analysis, the growth of cyanobacteria, such as Anabaena flos-aquae, A. cylindrica, A. macrospora, Oscillatoria sancta, and Microcystis aeruginosa, was strongly inhibited by a treatment of 20 ppm/disc or 30 ppm/disc concentration of the enzyme.

Photocontrol of urease-collagen membrane activity.

(1) Urease (EC 3.5.1.5.) was modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyrene)] propionic anhydride. Three amino acid residues of urease were modified by the anhydride at a molar ratio of 2000. (2) The activity of modified urease was decreased with ultraviolet irradiation and then restored to the initial activity with visible light irradiation. (3) Modified urease was used to prepare a urease-collagen membrane. The apparent Michaelis constant (Km) of the modified urease-collagen membrane ultraviolet light was identical to that of the membrane under visible light. (4) The optimum pH of the modified urease-collagen membrane was displaced toward lower pH values with ultraviolet irradiation. At higher ionic strength, the pH activity curve of the membrane was displaced toward higher pH values. (5) The thermostability of urease was increased with its modification.

Role of the disordered terminal regions of flagellin in filament formation and stability.

Terminal regions of flagellin from Salmonella typhimurium, residues 1 to 65 and 451 to 494, have no ordered tertiary structure in solution, which makes them very susceptible to proteolytic degradation. Flagellin was subjected to mild controlled proteolytic treatment with highly specific proteases to remove terminal segments from the disordered regions. It is demonstrated here that various fragments can be readily prepared that differ from each other in 1 x 10(3) to 2 x 10(3) Mr segments in their NH2- or COOH-terminal regions. Terminally deleted fragments of flagellin were used to clarify the role of the disordered regions in the self-assembly of flagellin. The polymerization ability of the fragments was tested by inducing filament formation with ammonium sulfate. We found that fragments of flagellin containing large terminal deletions could form straight filaments, although the stability of these filaments required high salt concentrations. Even a fragment lacking the whole mobile COOH-terminal part of flagellin and 36 residues from the NH2-terminal region could form long filaments. The fragments could be also polymerized onto native flagellar seeds, suggesting that the subunit packing of the filaments of fragments is similar to that of the native ones. The fragments could also copolymerize with native flagellin, resulting in various helical forms. Filaments of fragments were found to be straight at both pH 4.0 and pH 12.5, indicating that they might have lost their polymorphic ability. Our results show that the major part of the disordered terminal regions of flagellin is not essential for polymerization, but it does play an important role in stabilization of the filaments and in influencing their polymorphic conformation.

Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii.

Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, ß-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

Reinforced polycarboxylate cements.

Mechanical properties of polycarboxylate cements are greatly improved by incorporation of high modulus fibers such as potassium titanate into acrylic-itaconic acid and acrylic-itaconic-acronitic acid copolymers. Other desirable properties of the cements are not changed by the addition of fibers.

Production of citrinin-free Monascus pigments by submerged culture at low pH.

Microbial fermentation of citrinin-free Monascus pigments is of great interest to meet the demand of food safety. In the present work, the effect of various nitrogen sources, such as monosodium glutamate (MSG), cornmeal, (NH4)2SO4, and NaNO3, on Monascus fermentation was examined under different initial pH conditions. The composition of Monascus pigments and the final pH of fermentation broth after Monascus fermentation were determined. It was found that nitrogen source was directly related to the final pH and the final pH regulated the composition of Monascus pigments and the biosynthesis of citrinin. Thus, an ideal nitrogen source can be selected to control the final pH and then the citrinin biosynthesis. Citrinin-free orange pigments were produced at extremely low initial pH in the medium with (NH4)2SO4 or MSG as nitrogen source. No citrinin biosynthesis at extremely low pH was further confirmed by extractive fermentation of intracellular pigments in the nonionic surfactant Triton X-100 micelle aqueous solution. This is the first report about the production of citrinin-free Monascus pigments at extremely low pH.

Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns.

Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10-30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column.

Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii / Influência das fontes de nitrogênio na atividade enzimática e crescimento de Lentinula edodes na biomassa Eucalyptus benthamii

Abstract Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L–1) at 20 days of cultivation.

Resumo Lignocelulose é o componente mais abundante do meio ambiente e recurso orgânico renovável no solo. Alguns fungos filamentosos têm desenvolvido a habilidade de degradar e utilizar celulose, hemicelulose e lignina como fonte de energia. O objetivo deste trabalho foi analisar o efeito de três fontes de nitrogênio (sulfato de amônio, nitrato de potássio e farelo de soja) na atividade enzimática de Lentinula edodes EF 50 utilizando como substrato serragem de E. benthamii. Foi aplicado um planejamento experimental de mistura com três repetições no ponto central constituído de sete tratamentos (T) de iguais concentrações em nitrogênio de sulfato de amônia, nitrato de potássio e farinha de soja cozida. Foram determinadas a atividade enzimática da avicelase, carboximetilcelulase, β-glicosidase, xilanases e manganês peroxidase. Foram avaliados o teor de umidade, pH, atividade de água (aw) e análise qualitativa do crescimento micelial em 8 tempos de cultivo. Os resultados mostraram efeito negativo na produção das enzimas nos tratamentos com máxima concentração de sulfato de amônia e nitrato de potássio. Os tratamentos com farinha de soja cozida expressaram maiores atividades enzimáticas, nos tempos de 3, 6 e 9 dias de cultivo exceto na atividade do manganês peroxidase. A maior produção foi observada no tratamento com sulfato de amônia e farinha de soja cozida (83.86 UI.L–1) em 20 dias de cultivo.

Nitrogen source optimization for cellulase production by Penicillium funiculosum, using a sequential experimental design methodology and the desirability function.

The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.

A simple and direct pre-embedding technique for ultrastructure of scarce biological specimens.

A simple and rapid technique for pre-embedding scarce biological specimens for Transmission electron microscopy (TEM) is reported. It is based on pre-embedding biological samples in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized with paraformaldehyde, glutaraldehyde, ammonium persulfate and Temed. Pre-embedding in BSA and BA offers several advantages over traditional pre-embedding techniques for TEM including the ability to visualize the sample and a more resistant matrix. This results in more reproducible and consistent analysis. It can be applied to tissues, cells, and subcellular structures handled as pellets or suspensions. In addition, use of the pre-embedding matrix for light microscopy is reported. The ability to pre-embed scarce biological specimens efficiently and reproducibly provides a valuable way to study and characterize cytological tissues such as biopsies or cystic and amniotic fluid cells.