Botryosphaeria Rhodina MAMB-05 Research Articles

Biocidal and synergistic effect of three types of biologically synthesised silver/silver chloride nanoparticles.

Three types of silver/silver chloride nanoparticles were obtained by green synthesis from three types of microbial biomass. Their biocidal capacity was tested against six microorganisms. Two filamentous fungi were used that had previously demonstrated the ability to synthesise nanoparticles, Penicillium sp. 8L2 and Botryosphaeria rhodina MAMB-05. Also, the synthesis capacity of a yeast, Rhodotorula mucilaginosa 1S1, was evaluated. The original protocols underwent slight modifications. At the same time, the fractional inhibitory concentration was obtained. The interaction between specific antibiotics and the nanoparticles that showed the greatest biocidal capacity came from Penicillium sp.8L2, and it was studied further. All nanoparticles were characterised by UV-vis spectrophotometry and transmission electron microscopy (TEM). Also, their size distribution was analysed, which was in the range of 4 to 34nm. The biocidal capacity of the nanoparticles for a group of bacteria and fungi was studied, presenting very low values in the range of 2.5-10µg/mL for bacteria and 4-256µg/mL for fungi. The interactions between the nanoparticles synthesised by Penicillium sp. 8L2 and a group of specific antibiotics for the tested microorganisms were also studied, proving that there was a synergistic interaction with vancomycin and ciprofloxacin and Staphylococcus epidermidis CECT 4183 and Escherichia coli CECT 101 bacteria, respectively.

Electrochemical Characterization of the Laccase-Catalyzed Oxidation of 2,6-Dimethoxyphenol: an Insight into the Direct Electron Transfer by Enzyme and Enzyme-Mediator System.

The oxidation process of 2,6-dimethoxyphenol (2,6-DMP) by laccase from Botryosphaeria rhodina MAMB-05 and the corresponding enzyme-mediator systems was studied using cyclic voltammetry (CV). The enzyme was classified as a high oxidation potential laccase (> 0.70) V vs. NHE) based on its Redox potential at different pHs. The cyclic voltammograms for 2,6-DMP (- 58.7mV pH-1) showed that its oxidation potential decreased more significantly compared to the enzyme (- 50.2mV pH-1) by varying the pH. The 2,2'-azino-bis[3-ethyl-benzothiazoline-6-sulfonic acid] diammonium salt (ABTS) and 2,2,6,6-tetramethylpiperidine 1-oxyl radical (TEMPO) mediators were effectively oxidized by laccase from B. rhodina MAMB-05. The influence of laccase on the comproportionation of ABTS and the ionic step of the oxidation of TEMPO was also studied using CV. A higher potential difference was observed between laccase and the substrate, and correlated with higher enzyme activity. For the laccase-mediator systems, there was no clear correlation of potential difference between laccase and mediators with enzyme activity towards 2,6-DMP. This observation suggests that there are other limiting parameters for enzyme activity despite Redox potential difference, especially during ionic steps of the mechanism.

Covalent attachment of laccase to carboxymethyl-botryosphaeran in aqueous solution for the construction of a voltammetric biosensor to quantify quercetin

Laccase from Botryosphaeria rhodina MAMB-05 was covalently immobilized on carboxymethyl-botryosphaeran by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) in aqueous solution. This approach was employed to fabricate a novel laccase-based biosensor to electrochemically quantify quercetin (QCT), using a simple carbon black paste electrode as a transducer. The proposed biosensor was characterized by electrochemical impedance spectroscopy and Nyquist plots were used to evaluate the immobilization of the enzyme. For determining QCT, variables were optimized, that included experimental conditions for laccase immobilization, pH of the supporting electrolyte, and instrumental parameters of the electroanalytical technique. From square-wave-voltammograms, a linear dependence between the cathodic current peak and QCT concentration was observed within the range 4.98–50.0 × 10−8 mol L−1, with a theoretical detection limit of 2.6 × 10−8 mol L−1. The proposed method was successfully applied to determine QCT in beverages, pharmaceuticals, and biological samples. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in chemically complex solutions. Operational and analytical stability of the laccase-biosensor were evaluated, and good intra-day (SD = 1.23%) and inter-day (SD = 2.32%) repeatability, and long storage stability (SD = 3.47%) are presented.

Tumor development in rats and cancer cachexia are reduced by treatment with botryosphaeran by increasing apoptosis and improving the metabolic profile

AimsCancer is a multifactorial disease characterized by an uncontrolled growth of cells that can lead to cachexia-anorexia syndrome. Botryosphaeran, a fungal (1 → 3)(1 → 6)-β-D-glucan produced by Botryosphaeria rhodina MAMB-05, has presented antimutagenic, antiproliferative, pro-apoptotic, hypoglycemic and hypocholesterolemic effects. This study evaluated the effects of botryosphaeran (30 mg/kg b.w./day) on tumor development and cachexia syndrome in Walker-256 tumor-bearing rats, and also the metabolic and hematological profiles of these animals. Materials and methodsMale Wistar rats were divided into 3 groups: control (C), control tumor (CT) and control tumor botryosphaeran (CTB). On the first day, 1 × 107 Walker-256 tumor cells were inoculated subcutaneously into the right flank of the CT and CTB rats, and concomitantly treatment with botryosphaeran (30 mg/kg b.w./day) started. After the 15th day of treatment, biological parameters, tumor development, cachexia, glucose and lipid profiles, hemogram and protein expression were analyzed. Key findingsBotryosphaeran significantly reduced tumor development (p = 0.0024) and cancer cachexia, modulated the levels of glucose, triglycerides and HDL-cholesterol, and corrected macrocytic anemia. Botryosphaeran also increased significantly the bax expression in the tumor tissue (p = 0.038) demonstrating that this (1 → 3)(1 → 6)-β-D-glucan is increasing the apoptosis of tumor cells. p53, p27, bcl-2, caspase-3 and Forkhead transcription factor 3a (FOXO3a) protein expression were similar among the groups. SignificanceThis study demonstrated that botryosphaeran was effective in decreasing tumor development and cachexia by direct and indirect mechanisms increasing apoptosis and improving the metabolic and hematological profiles.

Chemometric-assisted construction of a biosensing device to measure chlorogenic acid content in brewed coffee beverages to discriminate quality

In this work we propose the use of statistical mixture design in the construction of a biosensor device based on graphite oxide, platinum nanoparticles and biomaterials obtained from Botryosphaeria rhodina MAMB-05. The biosensor was characterized by electrochemical impedance spectroscopy. Under optimized experimental parameters by factorial design, the biosensor was applied to the voltammetric determination of chlorogenic acid (CGA) measured as 5-O-caffeoylquinic acid (5-CQA). The biosensor response was linear (R2 = 0.998) for 5-CQA in the concentration range 0.56–7.3 µmol L−1, with limit of detection and quantification of 0.18 and 0.59 µmol L−1, respectively. The new biosensing device was applied to quality control analysis based upon the determination of CGA content in specialty and traditional coffee beverages. The results indicated that specialty coffee had a significantly higher content of CGA. Principal component analysis of the voltammetric fingerprint of brewed coffees revealed that the laccase-based biosensor can be used for their discrimination.

Soil and entomopathogenic fungi with potential for biodegradation of insecticides: degradation of flubendiamide in vivo by fungi and in vitro by laccase

PurposeFlubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides.MethodFilamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity.ResultIsolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro.ConclusionThis is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

Assessment of By-Product from Botryosphaeria rhodina MAMB-05 as an Effective Biosorbent of Pb(II).

In this work, two types of biomass preparations (VMSM and M3) from the filamentous fungus Botryosphaeria rhodina MAMB-05, which were previously used in a process of production of β-glucan, were assessed as biosorbents of lead. The operating conditions, optimized through response surface methodology and experimental design, were shown to be pH 5.29 and a biosorbent dose of 0.23 g/L for the VMSM biomass type; and pH 5.06 and a dose of biosorbent of 0.60 g/L for the M3 biomass type, at a constant temperature of 27 °C. Fourier transform-infrared spectroscopy analyzed the presence of functional groups on the biomass surface. In addition to give an extra value to the by-product biomass, the VMSM-type from B. rhodina MAMB-05 showed an excellent lead biosorption capacity (qm) with a value of 403.4 mg/g for the Langmuir model, comparing favorably with literature results, while the M3 subtype biomass showed a value of 96.05 mg/g.

Laccase stabilized on β-D-glucan films on the surface of carbon black/gold nanoparticles: A new platform for electrochemical biosensing

In this study, (1→3)(1→6)-β-D-glucan (botryosphaeran) from Botryosphaeria rhodina MAMB-05 was used, for the first time, to immobilize laccase on a carbon black paste electrode modified with gold nanoparticles. The physicochemical characterization of the proposed laccase-biosensor was performed using transmission electron microscopy and electrochemical impedance spectroscopy. The performance of this novel bio-device was evaluated by choosing hydroquinone as a typical model of a phenolic compound. For hydroquinone determination, experimental variables such as enzyme concentration, pH and operational parameters of the electroanalytical technique were optimized. From square-wave voltammograms, a linear dependence between the cathodic current peak and the hydroquinone concentration was observed within the range 2.00–56.5μmolL−1, with a theoretical detection limit of 0.474μmolL−1. The proposed method was successfully applied to determine hydroquinone in dermatological cream, and samples from biological and environmental niches. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in complex matrices. Operational and analytical stability of the laccase biosensor were evaluated, and demonstrated good intra-day (SD=0.3%) and inter-day (SD=3.4%) repeatability and long storage stability (SD=4.9%).

Biosorption of lanthanum and samarium by viable and autoclaved mycelium of Botryosphaeria rhodina MAMB-05.

The ascomycetous fungus, Botryosphaeria rhodina MAMB-05, secretes a (1→3)(1→6)-β-D-glucan, and the scaled-up production of this β-glucan results in large amounts of mycelial biomass being produced that represent a potentially cost-effective biosorbent for rare-earth elements. High sorption capacities for both La(III) and Sm(III) were demonstrated for viable and autoclaved lyophilized fungal mycelium. Fourier-transformed infra-red absorption spectra and the point of zero charge (PZC) were similar for the viable and inactive fungal mycelia. The rare-earth lanthanide elements (La and Sm) binding increased at initial pH values greater than 5.0, which was also observed for the PZC determination. The maximum La(III) uptake capacity was observed at lower amounts of La(III) ions in solution, decreasing from 100.0 to 25.3% when the initial lanthanide concentration increased from 15 to 100 mg/L. Lanthanide biosorption by B. rhodina MAMB-05 mycelia followed the Langmuir model, and the affinity of biosorbent functional groups was similar for La(III) and Sm(III).

Laccase from Botryosphaeria rhodina MAMB-05 as a biological component in electrochemical biosensing devices

A crude laccase preparation from Botryosphaeria rhodina MAMB-05 was employed to construct an electrochemical biosensor using nanostructured carbon black to epinephrine determination.

Botryosphaeria spp. as Producers of Laccase When Cultivated on Vegetable Oils as Sole Carbon Source: Optimizing Laccase Production by Botryosphaeria rhodina MAMB-05 on Soybean Oil

Eight vegetable oils and glycerol were examined as sole carbon source to screen nine isolates of Botryosphaeria spp. Among these, B. rhodina MAMB-05 produced highest laccase titres on all carbon sources evaluated followed by B. rhodina MMLR isolated (from orange, Citrus sp.). The influence of inorganic nitrogen (NH 4 Cl, NaNO 3 , (NH 4 ) 2 SO 4 , and NH 4 NO 3 ), organic nitrogen (yeast extract (YE), urea, peptone, and corn steep liquor) sources, inorganic phosphate (KH 2 PO 4 , Na 2 HPO 4 , NaH 2 PO 4 , K 2 HPO 4 ), as well as increasing concentrations of copper (from zero to 100 µg mL -1 ), as nutritional requirements for enhancing laccase production by B. rhodina MAMB-05 were also evaluated. Soybean oil was the only carbon source in a central composite factorialdesign (2 3 ) to optimize laccase production with the selected variables NaNO 3 , KH 2 PO 4 and copper (CuSO 4 .5H 2 O), using the response surface methodology. The optimal conditions obtained were NaNO 3 (100 g L -1 ), KH 2 PO 4 (125 g L -1 ) and 50 µg mL -1 of CuSO 4 .5H 2 O. The specific laccase activity obtained (2.95 U mg -1 ) in the validation experiments using the optimal condition for laccase production by B. rhodina MAMB-05 was in agreement with the predicted value (3.07 U mg -1 ), and demonstrated the fitness of the proposed model. DOI: http://dx.doi.org/10.17807/orbital.v10i7.1206

3,3′,5,5′-Tetramethoxybiphenyl-4,4′-diol: A new antioxidant enhancing oxidative stability of soybean biodiesel

3,3′,5,5′-Tetramethoxybiphenyl-4,4′-diol (TMBP) is described in the literature as having antioxidant activity, and can be obtained through enzyme-catalyzed processes. Its use as an additive to biodiesel offers a more sustainable substitute for the antioxidants commonly used for the conservation of this type of biofuel. In this work, TMBP was synthesized from 2,6-dimethoxyphenol catalyzed by the laccase from Botryosphaeria rhodina MAMB-05, and applied to soybean biodiesel to evaluate its antioxidant action by the determination of the induction period, and kinetic and thermodynamic oxidation parameters. Biodiesel samples containing TMBP presented induction periods higher than the 6 h in the Rancimat test conducted at 110 °C. The reaction rate constants at all temperatures examined were lower than those obtained for samples containing tert-butylhydroxytoluene (BHT), a common synthetic antioxidant added to biodiesel. The kinetic and thermodynamic parameters of oxidation indicated the effectiveness of TMBP in the inhibition of oxidative processes in soybean biodiesel compared to BHT. The antioxidant action of TMBP was not impaired as a function of thermal degradation, as thermogravimetry analyzes indicated degradation events occurring only above 270 °C.

Selective control for the laccase-catalyzed synthesis of dimers from 2,6-dimethoxyphenol: Optimization of 3,3’,5,5’-tetramethoxy-biphenyl-4,4’-diol synthesis using factorial design, and evaluation of its antioxidant action in biodiesel

The synthesis of transformed products from 2,6-dimethoxyphenol (2,6-DMP) can be selectively obtained through controlled oxidative coupling in laccase-catalyzed reactions, and the products formed are dependent upon the enzyme source, as well as, upon the reactions conditions applied. The laccase from Botryosphaeria rhodina MAMB-05 was investigated for its ability to catalyze the oxidative transformation of 2,6-DMP. Studies conducted in aqueous medium at pH 3.0 and 6.5, for reaction times of 48, 96 and 144 h, led to the formation of three dimers (I, II, III) via carbon-carbon and carbon-oxygen coupling reactions depending upon the pH and reaction time. Dimer I was identified as 3,3′,5,5′-tetramethoxydiphenylquinone by 13C NMR, while dimer II was identified by 1H NMR as 3,3′,5,5′-tetramethoxybiphenyl-4,4′-diol (TMBP). Dimer III was synthesized at pH 3.0 and was influenced by the reaction time (96h), and is predicted to be formed via carbon-oxygen coupling. The chemical structure of III (4-(2,6-dimethoxy-phenoxy)-2,6-dimethoxyphenol) was identified by ESI-Q-TOF HRMS. TMBP (II) was the only product obtained at pH 6.5 independent of the reaction times. TMBP synthesis at pH 6.5 was optimized by design-matrix using a three-factor Box-Behnken incomplete factorial-design that defined the parameters: 2,6-DMP concentration (2.6–3.4 mmol) laccase activity (1.35–3.38 U) and reaction time (120 h) to give a yield of 11.93 ± 0.49%, which agreed with the mathematical model value of 12%. TMBP was evaluated as an antioxidant to stabilize biodiesel, and showed an efficacy similar to the commercial antioxidant standard, butyl hydroxytoluene, indicating that TMBP could serve as an alternative antioxidant to stabilize biodiesel.

In vivo antimutagenic and antiatherogenic effects of the (1 → 3)(1 → 6)-β-d- glucan botryosphaeran

The antimutagenic effect of botryosphaeran, an exocellular (1 → 3)(1 → 6)-β-d-glucan, from the ascomyceteous and plant-borne endophytic fungus, Botryosphaeria rhodina MAMB-05, was evaluated in young (6–8 weeks) and elderly (18 months) Swiss albino mice of both genders. The hypolipidemic, hypoglycemic and antiatherogenic potential was also evaluated in 18-month old male LDL receptor knockout (LDLr−/−) mice. Administration of botryosphaeran by gavage (doses: 7.5, 15, 30 mg/kg b.w./day) in a 30-day pretreatment protocol (young mice), or 15-day protocol (older mice), did not cause genotoxicity as assessed by the micronucleus test in peripheral blood (PB) and bone marrow cells (BMCs). Furthermore, there was no cytotoxic effect of this β-d-glucan in the treatments. A lower frequency of micronuclei was observed in BMCs from young and old mice that received botryosphaeran, indicating its antimutagenic effect. Botryosphaeran (30 mg/kg b.w./day) promoted 102.22% (young) and 103.45% (elderly) reductions in cyclophosphamide-induced damage in male mice. Botryosphaeran also exerted chemoprotective effects in LDLr−/− and wild-type (C57BL/6) mice. Botryosphaeran treatment for 15 days at a dose of 30 mg/kg b.w./day improved the lipidic profile (reductions of 53.8–84.3%), and decreased aortic lipid deposition (32.8%) in the LDLr−/− atherosclerotic mice. The results indicate botryosphaeran has relevant biologic effects, making it a promising candidate for the development of new therapeutic agents.

In vitro protective effects of botryosphaeran, a (1 → 3;1 → 6)-β-d-glucan, against mutagens in normal and tumor rodent cells

Botryosphaeran (BOT) is an exocellular β-d-glucan (carbohydrate biopolymer) of the (1→3;1→6)-linked type produced by Botryosphaeria rhodina MAMB-05. The cytotoxic, mutagenic, genotoxic, and protective effects of this substance were evaluated in Chinese hamster lung fibroblasts (V79) and rat hepatocarcinoma cells (HTC) by the micronucleus test (MN) and the comet assay. BOT was not genotoxic in either cell line; it decreased the clastogenic effects of doxorubicin, H2O2, and benzo[a]pyrene. These results indicate that BOT may have potential as a therapeutic agent.

Antiproliferative and pro-apoptotic effects of three fungal exocellular β-glucans in MCF-7 breast cancer cells is mediated by oxidative stress, AMP-activated protein kinase (AMPK) and the Forkhead transcription factor, FOXO3a

Fungal β-d-glucans of the (1→3)-type are known to exhibit direct antitumor effects, and can also indirectly decrease tumor proliferation through immunomodulatory responses. The underlying molecular mechanisms involved in decreasing tumor formation, however, are not well understood. In this study, we examined the antiproliferative role and mechanism of action of three different fungal exocellular β-glucans in MCF-7 breast cancer cells. The β-glucans were obtained from Botryosphaeria rhodina MAMB-05 [two botryosphaerans; (1→3)(1→6)-β-d-glucan; one produced on glucose, the other on fructose] and Lasiodiplodia theobromae MMPI [lasiodiplodan; (1→6)-β-d-glucan, produced on glucose]. Using the cell proliferation-MTT assay, we showed that the β-glucans exhibited a time- and concentration-dependent antiproliferative activity (IC50, 100μg/ml). Markers of cell cycle, apoptosis, necrosis and oxidative stress were analyzed using flow cytometry, RT-PCR and Western blotting. Exposure to β-glucans increased apoptosis, necrosis, oxidative stress, mRNA expression of p53, p27 and Bax; the activity of AMP-activated protein-kinase, Forkhead transcription factor FOXO3a, Bax and caspase-3; and decreased the activity of p70S6K in MCF-7 cells. In the presence of hydrogen peroxide, the fungal β-glucans increased oxidative stress, which was associated with reduced cell viability. We showed that these β-glucans exhibited an antiproliferative effect that was associated with apoptosis, necrosis and oxidative stress. This study demonstrated for the first time that the apoptosis induced by β-glucans was mediated by AMP-activated protein-kinase and Forkhead transcription factor, FOXO3a. Our findings provide novel mechanistic insights into their antiproliferative roles, and compelling evidence that these β-glucans possess a broad range of biomodulatory properties that may prove useful in cancer treatment.

Evaluation of Fermentative Parameters for the Production of Botryosphaeran (a(1-->3;1-->6)-β-D-glucan) and Mycelial Biomass by Botryosphaeria rhodina MAMB-05

Botryosphaeran is an exopolysaccharide (EPS) of the (13;16)-β-D-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. In attempts to enhance its extracellular production, the quantitative effects of different nitrogen sources, fermentation time, inoculum size, C/N ratio, and initial pH were evaluated on the yields of botryosphaeran. Ammonium nitrate was selected as the best nitrogen source in examining these effects using a 2³-factorial central-composite experimental design, and analysis by the response surface method. According to a factorial design, the most important variables influencing both botryosphaeran and mycelium biomass production was inoculum concentration and the time of growth. Under these conditions, optimum botryosphaeran production occurred at 88 h of growth, 0.88 g of mycelium L -1 of nutrient mediumat C/N ratio of 30, and resulted in a 5.2 g L -1 yield of EPS in the fermentation broth. For production of the mycelium biomass, the optimum condition occurred at the same time of growth and inoculum concentration; however the best C/N ratio was 75, and resulted in a yield of 33.8 g L -1 of biomass. The results also indicated that the maximal mycelial growth is not associated to the yields of botryosphaeran production by Botryosphaeria rhodina MAMB-05. DOI: http://dx.doi.org/10.17807/orbital.v7i1.637

Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-β-d-glucan in human lymphocytes

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor.

Value Adding of Poplar Hemicellulosic Prehydrolyzates: Laccase Production by Botryosphaeria rhodina MAMB-05 and Its Application in the Detoxification of Prehydrolyzates

Fast-growing hybrid poplar species are attractive as feedstocks for the production of bio-based chemicals including biofuels. Pretreatment makes cellulose accessible to enzymatic hydrolysis, producing hemicellulose and lignin as major co-products. Poplar woodchips pretreated hydrothermally in an experimental industrial pilot-plant generated two prehydrolyzate streams; purge-condensate collected from cooking the woodchips and press-filtrate collected after the fiber explosion step. Both prehydrolyzates contain hemicellulose sugars (pentoses, hexoses, oligosaccharides), acetic acid, and degradation products from hemicellulose and lignin (furanic and phenolic compounds, respectively). Purge prehydrolyzate was highly diluted, of low sugar content and high biochemical/chemical oxygen demand, and constituted a process waste that was addressed for value-adding through production of enzymes by fermentation. Wood-decay fungi were screened for laccases on purge prehydrolyzate, with Botryosphaeria rhodina producing highest titers. B. rhodina detoxified the prehydrolyzate but at expense of sugar consumption. Adding xylose and glycerol to purge did not inhibit fungal growth or impede laccase production. Phenolics in the purge had inducing effect on enhancing laccase titers. Laccase production was optimized by Box-Behnken-(33)-factorial design varying the concentration of xylose in the purge, together with adding copper as laccase inducer and glycerol to increase the level of fermentable substrate, resulting in optimal enzyme titers (36.37 ± 3.52U/mL; validated). The crude laccase preparation was employed to detoxify the more concentrated press prehydrolyzate, reducing the overall content of phenolics by ∼30 %. Chromatographic (high-performance liquid chromatography-ultraviolet, gas chromatography/mass spectrometry) analysis identified various phenolic compounds present in the press prehydrolyzate following laccase treatment, and in the presence of the mediator, ABTS, the phenolics content decreased further.

Thermal and Rheological Properties of a Family of Botryosphaerans Produced by Botryosphaeria rhodina MAMB-05

Differential scanning calorimetry (DSC), thermogravimetry (TG) and Fourier-transform infra-red spectroscopy (FT-IR) analyses were performed to investigate changes in the physico-chemical properties of botryosphaerans, a family of exopolysaccharides (EPS) produced by the fungus Botryosphaeria rhodina MAMB-05 grown on glucose (EPSGLC), sucrose (EPSSUC) and fructose (EPSFRU). A slight endothermic transition and small mass loss attributable to the removal of water of hydration were observed in the DSC and TG analyses, respectively, for the three EPS samples. The FT-IR spectra confirmed no structural changes occurred during thermal treatment. Viscometry was utilized to obtain information on the rheological behaviour of the EPS in aqueous solutions. The Power Law and Cross Equations determined the natural pseudoplastic characteristics of the EPS. Comparatively, results obtained for EPS produced when B. rhodina MAMB-05 was grown on each of the three carbohydrate sources demonstrated similar apparent viscosity values for EPSGLC and EPSSUC, while EPSFRU displayed the lowest apparent viscosity of the three botryosphaerans, suggesting a higher degree of ramification and lower Mw. EPSGLC and EPSSUC possessed similar degrees of ramification. The slight differences found in their viscosities can be explained by the differences in the type of branching among the three botryosphaerans, thus varying the strength of intermolecular interactions and consequently, consistency and viscosity. The physico-chemical studies of botryosphaerans represent the originality of this work, and the knowledge of these properties is an important criterion for potential applications.