Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.     

香菇基因型及对蔗渣降解水平的研究

在江南一些地方采集九株野生香菇,分析担孢子基因型并测定A、B不亲和因子等位基因的数目及差异,保藏以A_xB_x(或A-yB_y)标记的单核菌丝体。实验结果表明,每一菌株的四个基因型呈随机分配。香菇A·B因子具有地区性,重复频率很低,等位基因差异明显。九个菌株的单核菌丝体的基因型从A_1B_1到A_(18)B_(18)不同。进一步对14个菌株(包括上述野生菌株)在蔗渣培养基上生长70天后对主要基质降解程度的测定表明,除个别菌株比较适合用蔗渣进行栽培外,其它菌株对蔗渣中纤维素、半纤维素和木质素的分解利用能力都较差。     

Production, characterization and evaluation of methylcellulose from sugarcane bagasse for applications as viscosity enhancing admixture for cement based material

In previous works, methylcellulose (MC) was prepared from sugarcane bagasse cellulose in heterogeneous medium using dimethyl sulfate (DMS) as etherification agent. MC was produced in a range of degrees of substitution (DS) from 0.70 to 1.40 and the materials showed low water solubility. In this work methylcellulose was prepared with 5 h (MC5h) of reaction with reagent substitution at each hour. MC5h showed a DS of 1.89 ± 0.04. An aqueous viscous suspension was produced with MC5h for application as viscosity enhancing admixture of cimentitious adhesive mortars. It was observed a 40.37% increase on the consistency index (CI) and a 27.70% increase on the Potential Tensile Adhesion Strength. Such characteristics show the potential of this material for the utilization in situations that require good workability, improve viscosity and adhesive properties such as for tile setting in civil engineering.     

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification.     

Bioindicator production with <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>’ thermal-resistant spores cultivated by solid-state fermentation

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.     

Influence of the carbon and nitrogen sources on keratinase production by <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis> in submerged and solid state cultures

Myrothecium verrucaria is a nondermatophytic filamentous fungus able to grow and to produce keratinase in submerged (93.0 ± 19 U/ml) and solid state (98.8 ± 7.9 U/ml) cultures in which poultry feather powder (PFP) is the only substrate. The purpose of the present work was to verify how different carbon and nitrogen sources can influence the production of keratinase by this fungus. Addition of carbohydrates, such as glucose and sucrose, caused only slight improvements in keratinase production, but the addition of starch caused a significant improvement (135.0 ± 25 U/ml). The highest levels of keratinase activity, however, were obtained by supplementing the PFP cultures with cassava bagasse, 168.0 ± 28 U/ml and 189.0 ± 26 U/ml in submerged and solid state cultures, respectively. Contrarily, the supplementation of PFP medium with organic or inorganic nitrogen sources, such as casein, soy bean protein, gelatin, ammonium nitrate and alanine, decreased the production of keratinase in both types of cultures (around 20 U/ml), showing that the production of keratinase by M. verrucaria is repressed by nitrogen sources. The results obtained in this work suggest that the association of the two residues PFP plus cassava bagasse could be an excellent option as a cheap culture medium for the production of keratinase in submerged and solid state cultures.     

Fractional and physico-chemical characterization of hemicelluloses from ultrasonic irradiated sugarcane bagasse

The present study was undertaken to investigate the extractability of the hemicelluloses from bagasse obtained by ultrasound-assisted extraction methods. The results showed that the ultrasonic treatment and sequential extractions with alkali and alkaline peroxide under the conditions given led to a release of over 90% of the original hemicelluloses and lignin. This fact as well as the sugar composition and structural features of the isolated seven hemicellulosic fractions indicated that ultrasonication attacked the integrity of cell walls, cleaved the ether linkages between lignin and hemicelluloses, and increased accessibility and extractability of the hemicelluloses. Increasing alkali concentration from 0.5 to 2M and alkaline peroxide percentage from 0.5% to 3.0% resulted in degradation of hemicellulosic backbone as shown by a decrease in their molecular weights from 43,580 to 14,470 and 30,180 to 18,130gmol(-1), respectively. However, there were no significant differences in the structural features of the seven sequential alkali- or alkaline peroxide-soluble hemicellulosic fractions, which are composed mainly of L-arabino-(4-O-methyl-D-glucurono)-D-xylans. Ferulic and p-coumaric acids were found to be chemically linked with hemicelluloses.     

Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry

An on-line system based on microdialysis sampling (MD), micro-high performance anion exchange chromatography (micro-HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on-line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5-mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro-HPAEC-IPED/MS and on-line desalting of hydrolysates enabled injection (5 microL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm-selectivity of the MD membrane (30 kDa cut-off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on-line with micro-HPAEC-IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co-elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean-up combined with on-line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.     

Combination of dilute acid and ionic liquid pretreatments of sugarcane bagasse for glucose by enzymatic hydrolysis

Loss of hemicellulose and inability to effectively decrystallize cellulose, result in low yield and high cost of sugars derived from biomass. In this work, dilute sulfuric acid pretreatment could easily remove most of hemicellulose as sugars. The sugars were successfully used for 2,3-butanediol production with relative high yield (36.1%). Then, the remained solid residue after acid-pretreatment was further pretreated by ionic liquid (IL) to decrease its crystallinity for subsequent enzymatic saccharification. The combination of dilute acid- and IL-pretreatments resulted in significant higher glucose yield (95.5%) in enzymatic saccharification, which was more effective than using dilute acid- or IL-pretreatment alone. This strategy seems a promising route to achieve high yield of sugars from both hemicellulose and cellulose for biorefinery.