Vol. 47, October, 2014.

1/47 USING SOME ORGANIC MATERIALS ON GROWTH IN VITRO CORDYLINE SPECIES PLANTS

Sherif .F. El-Sharabasy, Yasmein.El-Sayed* and Gehan. Safwat*

The Central Laboratory for Date Palm Researches and Development, Agriculture Research Center, Cairo, Egypt. *Modern Sciences and Arts University.

The current study was carried out to investigate Cordyline Species plantlets production by tissue culture technique. Two complex organic additives (Malt and Yeast extract at (0.0, 250, 500, 1000 mgl-1) were added to MS nutrients media to evaluate their effect during shooting and rooting stages. According to our result, the number of shoots and number of roots were significantly highest on (500 mgl-1) at Malt extract, while Yeast extract was more effect rather than Malt extract on number of leaves. Malt extract gave the best result on quantity of the chlorophyll A.

2/47 PHYTOCHEMICAL AND ANTIMICROBIAL STUDIES ON POLYGONUM EQUISETIFORME (FAMILY POLYGONACEAE)

Zeinab A. EL-Swaify

Botany and Microbiology Department, Faculty of Science (Girls Branch), Al-Azhar University, Cairo, Egypt

Polygonum equisetiforme is one of six species belonging to genus Polygonum. This species was subjected to morphological, anatomical, phytochemical and antimicrobial studies. The phytochemical studies revealed that it contains flavonoids, tannins, carbohydrates/ glycosides, saponins, sterols, terpenoids, sulphates, chlorides and alkaloids. Also the free and combined sugars, amino acids, phenolic acids, fatty acids, hydrocarbons, sterols and minerals were analysed quantitatively. Determination of phenolic acids in leaves, stem and root were carried out and the new compound benzene dicarboxylic acid was isolated for the first time from this plant. From the activity of the plant extract against microorganisms the plant can be considered as a promising antifungal and antibacterial agent.

3/47 OPTIMIZATION OF CULTURAL CONDITIONS FOR BIOSURFACTANT PRODUCTION FROM BACILLUS SUBTILIS ISOLATED FROM OIL RESERVOIR

Mamdouh S. El-Gamal, *Saad El-Din M. Desouky, **ElShahawy, M.R.and

Hashim Arafa Bekhit Abd-Rabou

Botany & Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt.

*Production Department, Petroleum Research Institute, Cairo, Egypt.

**National Center for Radiation, Authority of Atomic Energy, Cairo,

Biosurfactants are surface active compounds produced by microorganisms. These molecules reduce surface and interfacial tension. Water samples were collected from El-Wastany Petroleum Company in middle delta. Bacillus subtilis was isolated from the sample and this isolate was screened for biosurfactans activity by Emulsification capacity. Biosurfactant production by the isolated bacterium using different pH values, temperatures, concentrations of carbon and nitrogen sources were studied. The data show the optimum conditions are at pH7, 30˚C, starch 6 g/l and sodium nitrate 1.3 g/l.

4/47 COMPARISON OF WATER AND ACID PULPING FOR GLUCOSE PRODUCTION FROM BAGASSE

Ahmed S. Khera, Waleed K. El-Zawawy, Maha M. Ibrahim, Yasser R. Abdel-Fattah*, Zeinab K. Abd-El Aziz**and Mohamed H. El-Sayed***

Cellulose and Paper Department, National Research Center, El-Tahrir St., Dokki, Giza, Egypt.

*Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, 21934 New Burg El-Arab City, Alexandria, Egypt.

**Microbiology and Botany Department, Faculty of Science, Girls, El-Azhar University, Cairo, Egypt.

***Microbiology and Botany Department, Faculty of Science, Boys, El-Azhar University, Cairo, Egypt

Agricultural residues are renewable resources that can be used to produce ethanol and many other value-added products. Agricultural residues are considered to be a lignocellulosic material, where its three main chemical constituents are cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are polysaccharides of primarily fermentable sugars, i.e. glucose and xylose respectively. Hemicellulose also includes small fractions of arabinose, galactose, and mannose, all of which are fermentable as well. The current research was to investigates the use of bagasse, which is a lignocellulosic material, to be a source for ethanol production. The agricultural biomass was first analyzed and was noticed to contain 78.2% holocellulose, 1.83% ash and 25.13% lignin then a laboratory experimental set-up was designed in order to perform the necessary conversions. Cellulose, which is to be converted to glucose, was achieved from the bagasse by pre-treatment method applying water hydrolysis pulping, hydrothermal pulping, and acid pulping then followed by enzymatic hydrolysis into glucose by Trichoderma reesei cellulases. The produced glucose was then converted into ethanol by fermentation by the action of isolated yeasts from different sources. The total reducing sugars produced from the enzymatic hydrolysis of cellulose was measured by the glucose kit method. The data from the enzyme hydrolysis, time study, were analyzed to provide information on enzyme hydrolysis rates. The results indicated that 11.2 to 27.2 g/L of glucose can be produced from bagasse depending on the pretreatment methods.

5/47 MICROBIAL DECOLOURIZATION OF AZO DYE BY FREE AND IMMOBILIZED CELLS IN WASTEWATERS

Mohamed A. Ramdan, A.M. Hashem, Walaa Al Shareef* and Tamer Essam

Microbiology and Immunology Department and Biotechnology Centre, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo11562, Egypt.

*Microbiology and Immunology Department, Faculty of Pharmacy, October 6 University, Cairo, Egypt

Dyes are used in many industries such as textile, food and pharmaceutical industries. Many dyes are poisonous and since many are water soluble they can be hard to remove from industrial waste water. If these dyes are not removed from the waste water this could lead to damage of ecosystems and become poisonous for humans and animals. To be able to remove unwanted dye surplus from waste water immobilized organisms can be utilized. Immobilization applications seem to be more encouraging than those with free cells. Therefore, the study was conducted to determine whether immobilization could enhance the degradation of Reactive Black 5 (RB5) dye under static conditions. Immobilized cells of Penicilliumroqueforti and Aspergillus fumigatuson powdered activated carbon (PAC) (20g/l) in mineral salt medium (MSM) showed 64 % degradation of RB5 (up to 20 mg/l) and 71 % degradation of RB5 (up to 20 mg/l), respectively, compare to free cells after 7 days of incubation. Moreover, immobilized cells of Candida albicans, Penicilliumroqueforti and Aspergillus fumigatuson kaoline (20 g/l) showed the best extent of degradation of RB5 compared to free cells (61, 56 and 65), respectively. Therefore, fungal isolates were inoculated on different solid support,the best degradation of RB5 was attained. Determination of tyrosinase, lignin peroxidase and laccase activity by tested isolates revealed that optimum enzyme activity was obtained after 7 days of incubation equivalent to the optimum degradation by free cells of isolates. It is likely thatoxidoreductase enzymes (tyrosinase and laccase) were involved in the degradation of BR5 by Candida albicans, Penicilliumroqueforti and Aspergillus fumigates.

 

6/47 IN VITRO PROPAGATION OF SELECTED AGED DATE PALM MALE CLONE VIA DIRECT ADVENTITIOUS BUDS PROLIFERATION

Adel E. Hegazy

Genetic Engineering and Biotechnology Research Institute (GEBRI), Plant Biotechnology Department, Sadat City University, Egypt.

Very young invisible closed male spathes of selected aged date palm (Phoenix dactylifera L) clone was early detected and separated from the tree according to scheduled time previously calculated. Ethyl alcohol (70 %) spraying was used to the totally closed spathes for surface sterilization then, flamed under aseptic conditions followed by cold immersion (5-8 °C) in filter sterilized antioxidant solution containing citric and ascorbic acids 150 mg/L each for 2 hours, prior to dissecting. Results indicated that immature spikelets (comb shape) which individually separated, shortened to single flowers (explants) and cultured on Modified MS (Murashige and Skoog 1962) medium (MMS) supplemented with IAA (0.1 mg/L) + NOA (0.1 mg/L) + Kin (0.5 mg/L) + 2iP (1.0 mg/L) was recorded higher explant survival (100 %), superior in adventitious buds formation (22.22 %) and scored the highest significant numbers of adventitious buds (2.5), buds fresh weight (2.67 g) and growth value (20.23) after 4 months of incubation under dark conditions followed by 2 months under light conditions.   Produced adventitious buds cultured on the same medium in addition to glutathione (20 mg/L) significantly increased fresh weight (9.74 g ) and recorded higher no. of buds (13.33) and growth value (6.10) as compared with the control after 8 weeks of incubation.The polyamine (PAs); Spermine (100 mg/L) obtained the highest significant no. of buds (32.89), higher fresh weight ( 10.50 g )   and growth value (9.56) after 8 weeks of incubation. Individual shoot clump cultured on basal MS medium supplemented with IBA (0.5 mg/L) achieved the highest significant values of root formation (100 %), number of roots (3.66 ) and root length (5.9 cm) after 8 weeks of incubation. Growing mixture containing compost and perlite (1:1, v/v) recorded the highest significant number of leaves (3.77), leaf area (15.23 cm2) and plantlets survival (80 %) after three months in acclimatization. This work could encourage the availability to overcome somaclonal variation in date palm vitroplants since callus production was avoided.      

7/47 SODIUM CHLORIDE ENHANCED SILYMARIN PRODUCTION IN CALLUS CULTURES OF SILYBUM MARIANUM (L)GAERTN.

Hani Moubasher, Shahenaz M. Al-Wakeel and Mohamed R. Ali

Botany Department, Faculty of Science, Uuniversity of Cairo, Giza, Egypt.

Hypocotyl-derived Calli of Silybium marianum established on MS medium containing 0.5 mg/l BA and 1 mg/l NAA, were employed to study the influence of abiotic elicitor Sodium chloride (NaCl). Results of our study have shown that all NaCl concentrations tested (25, 50 and 100 mM) favored the growth, total soluble proteins content and silymarin production at different elicitation periods (7, 14, 21, 28 days). The best silymarin production was 4.3 folds as compared to control, that was obtained from callus treated with 50 mM and after 7 days of treatment.

8/47 THE ANTIMICROBIAL EFFICACY OF VANCOMYCIN OR TEICOPLANIN COMBINATIONS WITH GENTAMICIN OR RIFAMPICIN AGAINST SELECTED EGYPTIAN STAPHYLOCOCCUS CLINICAL ISOLATES

Nourhan H. Fanaki, Hoda M. G. Omar, Nihal K. Moussa and Eva A. Edward

Pharmaceutical Microbiology Department, Faculty of Pharmacy Alexandria University

A comparison was made between different methods for the determination of the antimicrobial efficacy of the two glycopeptides (vancomycin and teicoplanin), and that of gentamicin and rifampicin against selected Egyptian staphylococcus clinical isolates. The effectiveness of the combinations of both glycopeptides with gentamicin or rifampicin, as well as that of the combination of gentamicin(CN) with rifampicin (RA), was tested against these selected isolates. Attempts were done to cure R-plasmids responsible for gentamicin resistance, from some selected isolates, using ethidium bromide, and also to transfer these plasmids by conjugation, using the broth mating technique. All isolates were screened for their susceptibility to the four tested antibiotics using the antibiotic disc diffusion and the agar dilution methods. The broth dilution methods and the standard E-test were carried out for selected isolates. It was noticed that there was almost no considerable difference between the agar and broth dilution methods, compared to the results obtained by the antibiotic disc diffusion method except for the isolates possessing marginal sensitivity or intermediate levels of resistance to vancomycin and/or teicoplanin. Concerning the E-test, our results showed that this method was characterized by a high specificity, but by somewhat low sensitivity. Antibiotic combinations were tested using the checkerboard technique. The most promising combinations were further studied using the time-kill assay, in both the stationary and log phases. Vancomycin and teicoplanin showed the highest activity against all the tested isolates. In general, the studied antibiotic combinations proved an advantage over the single drug treatment when tested using the checkerboard technique. The time-kill assay proved enhanced activity of the combinations during the exponential phase compared to the stationary one. Curing of plasmids conferring gentamicin resistance was successful in 2 out of 9 tested staphylococcus clinical isolates, with curing rates of 1% for each. These 2 staphylococcus isolates were tested for the transferability of gentamicin-resistance plasmids by conjugation, where promising conjugation frequencies, ranging between 5.4 X 10-2 and 7.48 X 10-2 CFU / donor cells, were obtained. We concluded that resistance to the glycopeptides (VA and TEC) was not common in Egypt due to their limited use. Also, the E-test might result in the underestimation of intermediate and complete resistance to glycopeptides. Although in vitro studies emphasized the usefulness of the combination of one of the glycopeptides (VA or TEC) with CN or RA, as well as that of CN with RA, against staphylococci, still in vivo studies are required concerning the safety and effectiveness of such combinations for the treatment of staphylococcal infections.

9/47 IN VITRO GROWTH AND SURVIVAL OF SALMONELLA ENTERICA IN PESTICIDE SOLUTIONS RECOMMENDED FOR USE IN EGYPT

Hoda H. El-Hendawy, Marwa R. Abd El-Salamand Mohammad S. Flefel*

Botany and Microbiology Department, Faculty of Science, Helwan University, Ain Helwan, Cairo, Egypt

*Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt

The ability of 12 commercial pesticide formulations, recommended for use by the Egyptian Ministry of Agriculture, to support survival or enhance growth of three Salmonella enterica serovars were tested. Pesticides were diluted in sterile tap water, inoculated with 4 log CFUml-1 of Salmonella isolates separately and incubated statically in the dark at 30 °C for 96 h. Bacterial survivors were determined during the period of incubation by direct plating on the surface of Tryptic Soy Agar (TSA) plates. Some pesticides such as Chloronil, Kenzban, Aquacide, Cortiano and Lambda Star inhibited Salmonella growth to some extent, whereas others such as Wettasul, Kemazd and Ravo increased bacterial populations by 0.13 to 0.95 log CFU ml-1 after 24 h incubation. Initial pH value of pesticides was changed when diluted with water, and consequently, the bacterial behavior was changed in different pesticide solutions. Reconstitution of pesticides in sterile tap water containing 0.6 µg ml-1 Bovine Serum Albumin (BSA), Maximum Recovery Diluent (MRD) containing 1% peptone (MRD), surface irrigation water and Buffered Peptone Water (BPW) containing 10% peptone, significantly increased bacterial population in the different pesticide solutions. These results indicate that the tested pesticides might represent a threat for human health because they enhance the growth of human bacterial pathogens distributed in soil, irrigation water and plant surface.

10/47 MOLECULAR CHARACTERIZATION OF SOME SELECTED EGYPTIAN STAPHYLOCOCCUS CLINICAL ISOLATES WITH VARYING RESISTANCE TO GENTAMICIN, RIFAMPICIN AND SOME GLYCOPEPTIDES

Nourhan H. Fanaki – Hoda M. G. Omar – Nihal K. Moussa and Eva A. Edward

Pharmaceutical Microbiology Department, Faculty of Pharmacy Alexandria University

Trials for the induction of resistance to the glycopeptides (vancomycin and teicoplanin) among selected Egyptian staphylococcus clinical isolates were performed in the absence and presence of diclofenac sodium as an inducer. This induction was more promising in the presence than in the absence of diclofenac sodium. Transmission electron microscopy was used to study the effect of glycopeptides on the ultrastructure of bacterial cell wall. It revealed the thickening of bacterial cell wall in the presence of glycopeptides, also, it revealed other morphological changes such as defective cell walls. Attempts were done to isolate plasmids mediating gentamicin resistance from selected isolates, as well as to transform such plasmids to chemically prepared competent cells. Prior to the plasmid isolation procedure, trials for the lysis of bacterial cell walls were done using a combination of chemical and mechanical methods (a combination of lysozyme, 30mg/ml, with glass beads"Ø 0.75-1mm"). It was found that, only one isolate S30, out of two tested isolates, was lysed using this combination. Consequently, the plasmid conferring gentamicin resistance was isolated and transformed to chemically competent cells Escherichia coli DH5α, with a transformation efficiency equal to 59.7 transformants/µg plasmid DNA. The detection of genes conferring resistance to vancomycin, teicoplanin and rifampicin among selected isolates, was carried out using polymerase chain reaction. Concerning the distribution of constitutive and inducible glycopeptide resistance genes, the van genes tested (van A and van B) were not detected at all. However, one or more of the autolysis genes such as arlR, lytR and lrgB, which are negative regulators of autolysis, were detected in some cases. Moreover, the detection of cell-wall associated genes, pbp4 and/or pbp2, in other cases, emphasized that the increased production of PBPs could allow a better competition of PBPs with glycopeptides for the peptidoglycan precursor. The gene rpoBstaph was detected in 75% of the tested staphylococcus clinical isolates showing reduced susceptibility to RA, thus, indicating that mutations in the rpoB gene, encoding the ß-subunit of RNA polymerase, could be responsible for such resistance. In general, it could be concluded that diclofenac sodium can act as an inducer of glycopeptide resistance in staphylococci. In the absence of detectable van genes, cell-wall thickness can act as a major contributor to glycopeptide resistance among staphylococci. Finally, multiple and various mutations in the rpoB gene could mediate resistance to RA in staphylococci.

11/47 PRODUCTION OF PECTINASES FROM THERMOPHILIC ANOXYBACILLUS SPECIES ISOLATED FROM EL-WAHAT DESERT SOIL, EGYPT.

Amr, f. Mahmoud, Mamdouh. S. Elgamal, Essam, H. Abdel-Shakour and Salem, S. Salem.

    

Botany & Microbiology Department, Faculty of Science, Al-Azhar University,Cairo,Egypt

Thermophilic microorganisms are of special interest as a source of novel thermostable enzymes. A total of 62 bacterial strains, isolated from water hot springs and desert soil in Egypt were screened for the extracellular enzyme, pectinase. The results showed that, the two bacterial isolates: Wh20 and Wh24 were found to exhibit a higher pectinase production. The two most potent bacterial isolates were identified based on morphological and biochemical characters as a species belonging to the genus Bacillus and identified as Anoxybacillus contaminans Wh20 & Anoxybacillus flavithermus Wh24. Identification was confirmed by 16S r RNA sequencing. Maximum pectinases productivities for Anoxybacillus contaminans Wh20 & Anoxybacillus flavithermus Wh24 were (43.54 and 46.72 U/ml respectively) at pH 8.0 incubated for 18h at 55ºC under static condition. In presence of lactose and peptone as additional carbon and nitrogen source respectively and 0.4% pectin substrate concentration. Pectinolytic activity exhibited high thermostability over broad range of pH 6.0-10 and temperature 50-90 ºC. The crude pectinase was purified by salt precipitation, dialysis and gel filtration chromatography. The specific activities of the purified pectinases for Anoxybacillus contaminans Wh20 & Anoxybacillus flavithermus Wh24 were estimated to be 2250 and 2087.46 U/mg of enzyme protein respectively corresponding to (19.27 and 16.93 time) purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),for Anoxybacillus contaminans Wh20 showed that, the molecular weight of purified enzyme was determined at two bands, 30 kDa and 35 kDa, while Anoxybacillus flavithermus Wh24, showed that one band at 32 kDa. This pectinase may be industrially used in extraction and clarification processes.

12/47 EFFECT OF INTERACTION BETWEEN NaCl AND CALCIUM SALTS ON GERMINATION OF ALFALFA SEEDS

Safia M. Ghazy, Ibrahim M. Zeid and Zeinab A. Shedeed

Botany Department, Faculty of Science, Helwan University.   

Inorganic fertilizers as calcium based fertilizers are used to improve growth, production and yield of crops so it is significantly supported globalpopulation growth. These fertilizers supplied plants with essential nutrients for their growth. The purpose of this study is to use calcium fertilizers to improve the germinability of alfalfa seeds under salt stress conditions. Sodium chloride (NaCl) was supplied at four concentrations 25, 50, 75 and 100 mM with and/or without treatment with Ca(NO3)2, CaCl2, CaSO4 and CaHPO4 in different three concentrations 5, 7.5 and 10 mM for each calcium salt included at half strength Hogaland's solution (pH range 6.0-6.5). Seeds of alfalfa were germinated at 25°C in the darkness for 4 days. Germination percentage, total soluble sugar and protein contents and α-amylase activity in alfalfa seeds were decreased, while protease activity increased with NaCl stress. Calcium treatments enhanced the germination percentage at 50 and 75 mM NaCl stress, while the same seeds showed high sensitivity at 100 mM NaCl.

13/47 CONTROLLING OF EXTRA-GENES EXPRESSION LOCATED IN PLASMIDS

Hanan H. Ahmed and E. M. Ammar

Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Egypt

The aim of this study was to investigate the effect of media and temperature on maximize plasmids patterns (profiles), plasmid elimination and cured bacteria (competent cells) for biotechnology without any risk of inducing mutation in bacteria, biological control and control the genes on plasmids. It was known that genetic information on these plasmids were eliminate spontaneously when plasmid curing. This study were carried out on Bacillus thuringiensis kurstaki. The effect of minimal and complete media and exposed to different temperature was investigated. The results showed that there was no significant difference in bacterial cell number or plasmids pattern, when the bacteria were grown in Luria agar and nutrient agar media at 30°C under aerobic condition, as well as the bacteria showed significant different in bacterial cell number and the bacteria showed change in the plasmid profiles of some colonies exposed to different temperature degrees. The cell lost most of plasmids after exposure to very low and very high temperature at 5°C and 45°C respectively.


14/47 ALLEVIATION OF THE ADVERSE EFFECTS OF SALINITY STRESS BY CALCIUM SALTS

Safia M. Ghazy, Ibrahim M. Zeid and Zeinab A. Shedeed

Botany Department, Faculty of Science, Helwan University.

The interactive effects of salinity and calcium salts on growth and metabolism traits of alfalfa were studied in a pot experiment. Treatments included two levels of NaCl (50 and 75 mM) and different calcium salts (5 mM CaCl2, 7.5 mM Ca(NO3)2 and 7.5 mM CaHPO4). Salinity significantly reduced the plant growth as reflected by a decrease in the shoot, root length and weight (fresh and dry). Nodules fresh weight decreased at low and high salinity but dry weight decreased only at high salinity. Carotenoids, total soluble sugar, RNA, ammonia and proline accumulated under low (25 mM) or high (50 mM) or both NaCl stress, while chlorophyll a, photosynthetic activity, total soluble protein and DNA decreased. Application of supplemental calcium as calcium salts resulted in partially restoring the adverse effects of high salinity on plant growth. Calcium treatments improved the shoot and root lengths, fresh and dry weight especially at high salinity level (75 mM). Calcium treatments enhanced sugar, total soluble protein, DNA, RNA and shoot proline content, while decreased ammonia and nodule proline under salinity stress. Thus, calcium treatments especially calcium nitrate showed ameliorative effect on alfalfa growth under stress.
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