Volume 5 Special Issue 2 2011
Proceedings of the 1st International Conference
“Bio-Processing and Application of Microbial Biotechnology in Agriculture”
1-3 November 2010 in National Research Centre, Cairo, Egypt
How to reference: Elkahoui S, Djébali N, Tabbene O, Hadjbrahim A, Mnasri B, Mhamdi R, Limam F (2011) Screening of Bacterial Isolates Collected from Marine Bio-Films for Antifungal Activity against Rhizoctonia solani. Dynamic Biochemistry, Process Biotechnology and Molecular Biology 5 (Special Issue 2), 1-4
CONTENTS AND ABSTRACTS
Salem Elkahoui, Naceur Djébali, Olfa Tabbene, Adel Hadjbrahim, Bacem Mnasri, Ridha Mhamdi, Ferid Limam (Tunisia) Screening of Bacterial Isolates Collected from Marine Bio-Films for Antifungal Activity against Rhizoctonia solani (pp 1-4)
Original Research Paper: Rhizoctonia solani is one of the major damaging diseases of potato crops in Tunisia and worldwide. The use of fungicides and varieties with different levels of tolerance for the control of this disease is limited by the appearance of resistant fungal strains and with the non-availability of resistant varieties to R. solani attacks. The biological control of pathogens offers a promising approach in managing plant disease attacks. In this context, 30 bacteria isolates were isolated from marine bio-films from the Tunisian cost and their antifungal activity against R. solani was evaluated by the dual culture assay. Among the tested bacteria two showed an important antagonistic effect against this pathogen, which belong to the species Bacillus subtillis and Bacillus cereus according to their 16S rDNA gene sequence. The antagonistic activity of the two Bacillus species was also observed using their culture supernatant, demonstrating that the active substances are secreted in the medium. The maximum of activity was reached for both strains at 48 h of culture in LB liquid medium. The two antagonistic bacteria were cultured on LB, LB supplemented with glucose and sea water (LBGSW) and M2 supplemented with sea water (M2SW) for 48 and 60 h in order to optimize the production of active substances against three fungal pathogens R. solani, Pythium ultimum and Aphanomyces euteiches. The results showed that for the two Bacillus species maximal antifungal activity against R. solani was observed at 48 h culture on LB medium. The antifungal activity against P. ultimum was observed only with B. cereus after 48 h on M2SW. Maximum antifungal activity against A. cochlioides was observed with B. subtilis in culture on LBGSW at 60 h.
Mohd Naim, Hafidzi Mohd. Noor, Azhar Kassim, Jalila Abu (Malaysia) Comparison of the Breeding Performance of the Barn Owl Tyto alba javanica under Chemical and Bio-based Rodenticide Baiting in Immature Oil Palms in Malaysia (pp 5-11)
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Original Research Paper: The breeding performance of barn owl, Tyto alba javanica, in areas treated with rodenticides in immature oil palms in Malaysia was investigated. Four plots were established, each at least 100 ha in size and treated with warfarin, brodifacoum, a biorodenticide (Sarcocystis singaporensis) and a non-baited control plot. Three rat baiting campaigns, which coincided with the barn owl breeding season, were carried out in October 2008, February and March 2009, and in October 2009. The nest boxes were distributed at a mean density of one unit per 25 ± 3.83 ha. The clutch size, hatching and fledging rates of barn owls in each plot was monitored monthly from September 2008 to January 2010. There was no significant difference in mean clutch size for all four treatments. The lowest percentage of hatching success was recorded in the brodifacoum-treated plot in all three breeding seasons. Fledging success was highest in the control plot, followed by the S. singaporensis-, warfarin- and brodifacoum-treated plots. The mean clutch size and mean hatching success was not significantly correlated with mean rat damage (clutch size, r = 0.754, p > 0.05; mean hatching success, r = 0.832; p > 0.05). The mean fledging success was significantly correlated with mean rat damage (r = 0.969; p < 0.05). Brodifacoum achieved the lowest level of rat damage but not significantly lower than warfarin and S. singaporensis. This indicates that S. singaporensis is a better rodenticide than warfarin and brodifacoum in controlling rats and yet achieved the highest reproductive rates in the baited areas as reflected by the rate of fledging success.
Sanjukta Ghoshal, Pinaki Bhattacharya, Ranjana Chowdhury (India) Packed Bed Reactor Model for De-mercurization of Simulated Mercury-Laden Wastewater (pp 12-17)
Original Research Paper: Microbial reduction of soluble bivalent mercury to its less toxic elemental metallic form was performed using mercury-resistant Bacillus cereus (JUBT1) isolated from the sludge of chloralkali industries. A lab-scale 1 m long and 0.05 m diameter packed bed biofilm reactor was designed to remove mercuric ions (Hg2+) using isolated bacterial consortium. The bioreactor was continuously fed with sterile simulated wastewater containing HgCl2 to reduce bivalent mercury to its elemental form by growing bacterial biofilm on porous packing material of the reactor. The performance efficiency of the reactor was studied varying different chemical and hydrodynamic parameters such as inlet concentration of mercury and inlet flow rate of simulated mercury-laden water, among others. The reactor was followed by an activated carbon-based adsorber to remove residual mercury in the reactor effluent. A maximum of 97-98% removal efficiency was obtained with respect to the concentration of Hg2+ in the inlet water. A deterministic mathematical model was developed to explain the performance of the packed bed reactor.
Arijit Das, Sourav Bhattacharya, K. S. Roopa, S. S. Yashoda (India) Microbial Utilization of Agronomic Wastes for Cellulase Production by Aspergillus niger and Trichoderma viride Using Solid-State Fermentation(pp 18-22)
Original Research Paper: Microbial utilization of agronomic wastes has attracted worldwide attention for conversion of these renewable resources into bio-based products and bioenergy. Microbial cellulases find enormous applications in various industries. The objectives of the present study were to optimize media and growth conditions for effective utilization of agronomic wastes such as wheat bran, rice straw, sugarcane bagasse, banana peel, Bengal gram husk and corn husk through cellulase production by Aspergillus niger and Trichoderma viride using solid-state fermentation. Optimization studies revealed maximum production of cellulase from rice straw by A. niger at 25°C, pH 4.5, when incubated for 4 days, with initial moisture content of 49.44% and inoculum size of 4%, supplemented with 1% (w/v) tryptone as the nitrogen source. T. viride produced maximum cellulase from banana peels at 30°C, pH 5.5, when incubated for 3 days, with a moisture content of 24.01%, inoculum size of 3%, supplemented with 1% (w/v) yeast extract as the nitrogen source. The enzymatic assay of cellulase was performed by the dinitrosalicylic acid method with absorbance at 540 nm. The A. niger and T. viride enzyme extracts, when subjected to ammonium sulphate precipitation, ion-exchange chromatography and dialysis, revealed a 2- and 8-fold increase in enzyme activity, respectively. The activities of the partially purified cellulases from A. niger and T. viride were 402.13 and 104.8 U/g dry substrate, respectively. SDS-PAGE revealed three protein bands with apparent molecular weights of 25, 30 and 75 kDa.
Mona A. Esawy (Egypt), Harold Corke (China) The Effect of Wheat Flour in Enhancing and Stabilizing Intracellular and Extracellular Bacillus licheniformis 5A5 β-amylase Activity (pp 23-27)
Original Research Paper: The effect of wheat flour on the enhancement of β-amylase activity was studied. Crude intracellular and extracellular Bacillus licheni-formis 5A5 β-amylases were treated with different wheat flour concentrations (5-20%) for 1-5 days at 4°C. Maximum specific activities were obtained after 4 days at 20% wheat flour addition (183 and 135 U mg-1, respectively). This addition protected the enzymes completely during lyophilization and was considered as a partial purification step. The results were confirmed using a Rapid Visco Analyzer, where 1000 U ml-1 of partially purified enzyme led to near complete degradation of raw maize starch (4 g/40 mL) after 10 min. Noticeable splitting of some maize and wheat starch granules, also complete lysis of others was observed by scanning electron microscopy during the incubation of wheat starch granules with the enzyme for only 30 min. SDS gel electrophoresis showed that the wheat flour had removed and added new protein bands to the lyophilized enzyme molecule and the disappearance of bands through the purification steps led to a big loss in enzyme activities. Thermal stability of the pure β-amylases was studied in the presence and absence of 0.01% (w/v) wheat flour filtrate.
Nariman A. H. Aly (Egypt), Jaime A. Teixeira da Silva (Japan), Effat A. M. Soliman (Egypt) Intergeneric Protoplast Fusion by Combining Genes to Improve Lipase and α-Amylase Enzyme Activities (pp 28-34)
Original Research Paper: In order to produce new modified strains with improved lipase and α-amylase productivity for industrial and commercial use, the lipase gene (lip3) from Pseudomonas aeruginosa, Pa (Kmr Tcr Sms/Rifs Cmr Nmr), the α-amylase genes (amyE and dltB) from Bacillus subtilis, Bs (Kms Tcs Smr) and genes from Bacillus thuringiensis, Bt (Rifr Cms Nms) were combined using protoplast fusion. The pattern of antibiotic resistance was used as a selectable marker to screen for fusant strains from these three parental strains. Six Pa::Bs fusants were selected on medium containing kanamycin, tetracycline and streptomycin and five Pa::Bt fusants were selected on medium containing rifampicin, chloramphenicol and neomycin. Each of the 11 new fusant strains combined the properties of their corresponding parental strains. PCR amplification of the parental P. aeruginosa strain revealed a 162-bp fragment that represented the lip3 gene for lipase enzyme production. The other parental B. thuringiensis strain displayed a 1167-bp fragment that represented the dltB gene for a-amylase. The five fusants of these two parents contained the two fragments. PCR amplification of thetwo parental strains (P. aeruginosa, B. subtilis) showed the presence of the 162-bp fragment of the lip3 gene in P. aeruginosa and the 1066-bp fragment of the amyE gene in B. subtilis. The six Pa::Bs fusant strains also contained the two genes, as revealed by the 162- and 1066-bp fragments. Lipase and a-amylase activities were estimated in the three parental strains and their 11 fusants; some fusants displayed higher activities of both enzymes than the three parental strains. SDS-PAGE analysis of the proteins confirmed that all 11 fusant strains acquired and expressed many specific protein bands from the three parental strains.
Vishal Kapoor, Renu Singh, Rintu Banerjee, Vijay Kumar (India) Application of Response Surface Methodology (RSM) for Optimization of Physico-chemical Parameters for the Production of Endoglucanase by Trichoderma ressei Rut C-30 using Agro-residues (pp 35-40)
Original Research Paper: Response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the physico-chemical parameters for the production of endoglucanase by Trichoderma ressei Rut C-30 under solid state fermentation using a novel mixture of waste paper and wheat bran. Most effective variables for the endoglucanase production in screening experiments were incubation day, substrate ratio, solid: liquid ratio and pH of the medium. A quadratic model was developed through RSM in terms of related independent variables to maximize the endoglucanase production as the response. Incubation day and solid: liquid ratio were found to be the most significant factors. The predicted optimal parameters were tested in the laboratory and the final endoglucanase activity obtained was very close to the predicted value (22.93 IU/g, predicted; 25.43 IU/g, tested). After optimization, endoglucanase activity increased by ~1.77-fold. Our result shows that optimization of enzyme production is the most useful way to obtain concentrated enzyme extracts from solid state cultivation and that T. ressei Rut C-30 using cheap agro-residuescan be an attractive source for endoglucanase production.
Shalom Nwodo Chinedu, Obinna C. Nwinyi, Uzoma A. Okafor, Veronica I. Okochi (Nigeria) Kinetic Study and Characterization of 1, 4-b-Endoglucanase of Aspergillus niger ANL301 (pp 41-46)
Original Research Paper: Submerged fermentation of Aspergillus niger ANL 301 in basal medium containing cellulose as sole carbon source, yielded crude extracellular proteins with 0.54 ± 0.02 units mg protein-1 of 1,4-β-endoglucanase activity. Partial purification by ammonium sulphate precipitation (80% saturation) and gel filtration on Sephadex 25-300 gave two active fractions of 1,4-β-endoglucanase, which exhibited close activity towards carboxymethyl-cellulose (CMC). The pH profile of the pooled enzyme fractions showed three activity peaks at pH 3.5, 5.5 and 7.0. The enzyme was most active at pH 5.5 and showed optimal activity at 50°C. Vmax of 4.4 ± 0.4 µmol min-1 mg protein-1 and Km of 12.5 ± 0.4 gL-1 was obtained with CMC for the enzyme. Different divalent metal ions and EDTA affected the enzyme activity at 2.0 mM concentrations in different ways. Mn2+ and Fe2+ exhibited 253.4 and 24.0% stimulatory effects, respectively on the enzyme activity. Mg2+, Ca2+, Cu2+, and Zn2+ inhibited the enzyme by between 22.3 and 29.4%, whereas 75.0 and 71.3% inhibition were obtained with Hg2+ and EDTA, respectively. Manganese ion showed an exceptional activation of the 1,4-β-endoglucanase. The organism produced two types of 1,4-β-endoglucanase with different molecular weights.
Sandipan Chatterjee, Pritam Chattopadhyay, Sharmistha Maity, Angshuman Sarkar, Subrata Laskar, Sukanta Kumar Sen (India) A Water-Soluble, Non-aromatic, Nitrogenous Compound from a Hyper-red Pigment-Producing Mutant of Monascus purpureus (pp 47-52)
Original Research Paper: Attempts have been made to develop hyper-pigment-producing mutants of Monascus purpureus MTCC 1090 by UV irradiation. Harvested spores of M. purpureus were irradiated and LD50 was determined. Out of several mutants, M10c was selected as the most potent. Total protein profile of M10c was compared with the prototroph of M. purpureus to confirm its mutant nature. Pigment extraction was standardized using different test solvents. The extracted pigment was purified by thin layer chromatography and column chromatography. The final yield of red pigment at the laboratory scale was 6.8 g Kg-1. For characterization of the principal compound, spectral analysis using UV-Vis, IR and GC-MS were performed. The purified compound was also checked for antimicrobial efficacy and toxicity. The purified compound was active against Gram-positive test bacteria, Bacillus sp. and Staphylococcus sp. and found to be non-toxic against test Swiss albino mice.
Sateesh Lanka, Vimala R. Adivikatla, Naseeruddin Shaik , Srilekha Y. Kothagauni , Smita H. Panda, Gerard P. Yenumula, Venkateswar R. Linga (India) Studies on Different Detoxification Methods for the Acid Hydrolysate of Lignocellulosic Substrate Saccharum spontaneum (pp 53-57)
Original Research Paper: Pretreatment is an important step in the conversion of lignocellulosic substrates to ethanol. Acid hydrolysis of lignocellulosic biomass results in the generation of fermentable sugars and also compounds that are inhibitory to the fermenting organism. In the present study, the acid hydrolysate the lignocellulosic substrate (Saccharum spontaneum) was subjected to different detoxification methods. Treatment with alkali (sodium hydroxide and calcium hydroxide), treatment with reducing agent i.e. sodium sulfite, the use of ammonium hydroxide for simultaneous detoxification and addition of nutrients was experimented. Analysis of reducing sugars, furans and total phenolic compounds was performed before and after different treatments. Treatment with calcium hydroxide followed by active charcoal was the most efficient detoxification method, which resulted in 80% reduction in total phenolics and 90% reduction in furans with 10% sugar loss. Treatment with sodium hydroxide and ammonia resulted in substantial decrease in the concentrations of inhibitors but the sugar loss was more when compared to calcium hydroxide treatment, treatment with reducing agent has not given considerable results.
Periyasamy Ashokkumar, Vijayaragavan Kannan (India) Optimization and Purification of Extracellular Nuclease from Bacillus firmus VKPACU-1 (pp 58-65)
Original Research Paper: A strain of Bacillus firmus (VKPACU-1) produced high levels of extracellular ribonuclease (RNase) when grown on tryptone, peptone and glucose media. The test strain produced a maximum amount of nuclease at the stationary phase (32 hrs). The crude enzyme was optimally active at pH 6.5 and 35°C. Tryptone and peptone are the superior nitrogen sources. The addition of Mn2+ to the growth medium significantly enhanced enzyme production while glucose and maltose were excellent carbon sources for RNase production. The organism showed good growth and highest nuclease production in cultures grown with 0.5% NaCl. Peptone (0.5%), beef extract (0.5%), tryptone (0.5%), sodium chloride (0.5%) and Mn2+ (2mM) were chosen as the basal medium components as they supported the highest nuclease (RNase) production. There was higher activity with RNA and heat-denatured DNA than with native DNA. The molecular mass of the purified nuclease was about 17.1 kDa.
Grace Ebiega, Ibrahim Hassan Garba (Nigeria) Assessment of the Biodiesel Parameters of the Seed Oil of Water Melon (Citrullus vulgaris) (pp 66-69)
Original Research Paper: Base-catalyzed transesterification of water melon Citrullus vulgaris seed oil (CVSO) was carried out at a methanol/oil ratio of 6:1 at 50°C to produce biodiesel, a fuel comprising mono-alkyl esters of long chain fatty acids derived from vegetable oil or animal fat. The effects of potassium hydroxide (KOH) and sodium hydroxide (NaOH) catalysts were also investigated. The purpose of the transesterification process is to lower the viscosity, density and flash point of the oil. Prior to transesterification, the physico-chemical properties of the CVSO such as specific gravity, viscosity, moisture content, refractive index, density, acid value, saponification value, iodine value, and peroxide value, were determined and the results all fall within the recommended ranges as stated by biodiesel standard quality control organizations. Fuel quality parameters like density, viscosity, cloud point, flash point, pour point, acid value, cetane number, higher heating values and carbon residue were also within the recommended range. The results of the effects of the two catalysts on the yield of the biodiesel showed that KOH catalyst gave a higher yield of biodiesel compared to the NaOH catalyst. The biodiesel yield for KOH catalyzed process was 64.36%, while the yield for NaOH catalyzed process was 41.37%. The fuel properties of CVSO methyl esters were similar to those of rape seed, soya bean and sunflower biodiesel. Hence the refined, chemically processed and degummed CVSO can be used to run compression ignition on engines for longer duration since the major problem of chocking with unmodified vegetable oils has been overcome.
Munawwar Ali Khan, Sultan Mohammed Faheem (United Arab Emirates) Quantum Dots: Application for the Detection of Salmonella typhimurium (pp 70-73)
Original Research Paper: Quantum dots (QDs) or nanocrystals 5 to 50 nm in size have recently emerged as a novel and promising class of fluorophores for cellular imaging. Unlike conventional organic dyes, QDs can be excited by a wide spectrum of wavelengths to give enhanced photostability, and their emission spectra, which differ according to size and material composition, are narrow, symmetrical, and tunable. This novel detection strategy can significantly improve the efficiency of molecular techniques used in the identification of important microbes related to human diseases. The aim of this study was to apply QDs linked antibodies to detect pathogenic Salmonella typhimurium cells. The signal, photostabilty and efficiency of QDs linked antibodies was compared with the organic fluorophore linked antibody for the detection of Salmonella cells. The use of QDs as fluorophores has shown greater enhancement in photostability and brighter signal than conventional organic dyes in detecting S. typhimurium cells. This approach might also be extended for the simultaneous detection of more than one pathogenic microorganism in the clinical, food or environmentalsamplesby using specific antibodies linked with the QDs of different sizes and colors.
Ra’a A. Said, Yousef Haik (United Arab Emirates) Micro-machining of Shaped Array Tip Electrodes(pp 74-77)
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Original Research Paper: This paper reports a spacing-stacking micro-machining technique for realizing shaped array tip micro-electrodes. The technique can provide flexible array elements spacing with unlimited array configurations regardless of their complexity. In the proposed technique, manufacturing of array tip microelectrodes proceeds by shaping conducting wires that will form the array tips. The shaping of tips is achieved by fast grinding and polishing of the wires ends while encapsulated in glass sealing material. The array is then formed by sequencing the shaped wires and glass spacer fibers in layers using grooved moulds prepared using wire electric discharge machining. The spaced wire layers are then stacked in an appropriate order to provide the required array configurations. Every array element wire is bonded to a pad on a PCB connector, thus allowing simultaneous recording or driving of individual array tips. The entire array is then inserted in a glass micro-pipette that is heated and pulled and then sealed by the injection of hardening epoxy. The proposed technique is demonstrated by presenting examples of fabricated array micro-electrodes. The realized array electrodes are intended for applications in localized electrochemical deposition and scanning microscopy; however, electrodes fabricated by the presented technology are also suitable for several applications, including recording neural and biological activity.
Melika Ebrahimpour, Mohsen Jahanshahi, Maryam Khavarpoor (Iran) Purification of Nanoparticle Bioproduct in Integrated Processes: Plasmid DNA Separation and Recovery(pp 78-80)
Short Communication: The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. Pharmaceutical-grade plasmid DNA for use in vaccines requires the development of reproducible and scaleable down stream processes. The aim of this study is investigation and comparison of pDNA separation by aqueous two phase system and anion-exchange chromatography as popular techniques in plasmid DNA purification. In this work anion exchange chromatography carried out in column with 1.3 cm diameter filled with 8 ml streamline DEAE and polymer-salt system (ATPS) consisted of polyethylene glycol (PEG300)-K2HPO4 was used for the purification of plasmid DNA vectors. Results show that 88% of pDNA purified by expanded bed chromatography in contrast with 84% recovery of pDNA in top phase of aqueous two phase system.
Saud Aldajah, Ammar Al-Omari, Yousef Haik (United Arab Emirates) A Continuum-Based Finite Element Model of Carbon Nanotube Polymeric Composite(pp 81-84)
Research Note: The development of a finite element model that is appropriate for the computation of the mechanical properties of nanocomposite materials is the purpose of this research paper. The nanocomposite considered in this research is made of a polymer and aligned carbon nanotubes (CNTs); the applied tensile load is in the same direction of the aligned CNTs. The model development is based on the assumption that carbon nanotubes can be modeled as beam elements using ABAQUS software package. A representative volume element (RVE) method was employed in which it was assumed that the nanocomposite has geometric periodicity with respect to local length scale and that the elastic properties of nanocomposite can be represented by those of the representative volume element. The effective modulus of elasticity predicted by this method is compared with analytical and experimental results available in the literature.
Ihab Obaidat, Bashar Issa, Virendra Mohite (United Arab Emirates), Yousef Haik (United Arab Emirates/USA) Controlling the Curie-temperature of Magnetic Nanoparticles for Hyperthermia (pp 85-88)
Research Note: The control of Curie temperature is very necessary in hyperthermia where cancerous cells are heated up to temperatures of 42-43°C using magnetic nanoparticles (NPs). In this paper we point out that surface spins have a major role in determining the Curie temperature of ferrite Mn1-xZnxFe2O4 nanoparticles, where x = 0.5, 0.6, 0.8, and 1.0. The addition of Zn in Mn1-xZnxFe2O4 is suggested to cause changes in the lattice distances. These changes are expected to be more pronounced near the surface of the NPs. Accordingly, surface disorder occurred which resulted in surface spins. Our magnetization measurements revealed several trends. For each particular x value, the field cooled (FC) magnetization of the NPs remained nearly constant at temperatures below 50 K. Above this temperature, the magnetization either exhibited peaked regions or decreased sharply. The Curie temperature also increased up to x = 0.6, and then decreased for x = 0.8 and 1.0. The existence of the initial constant magnetization and the appearance of peak regions were considered to be signatures of surface spin-glass structures. A core-shell magnetization model of the ferromagnetic surface and the ferrimagnetic core was introduced to account for these results.