Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 11 num. 2 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<b>Comment on “Equilibrium sorption isotherm studies of Cd(II), Pb(II) and Zn (II) ions detoxification from waste water using unmodified and EDTA-modified maize husk”</b>]]> The citing and using of correct version of mathematical equations are of great importance in the writing of a scientific paper. This paper presents the possible errors related to Dubinin-Radushkevich Equation in an article by Igwe and Abia (2007) which was published in Electronic Journal of Biotechnology, 10(4); 536-548. <![CDATA[<b>Can owners afford humanitarian donations in agbiotech - The case of genetically engineered eggplant in India</b>]]> Are humanitarian donations in agbiotech economically feasible for the donor? We address this question by conducting an ex ante analysis of genetically engineered (GE) eggplant in India. Our analysis indicates that it is economically viable for a firm to donate the technology for poor farmers’ use by restricting use to open pollinated varieties while selling hybrid verities. By extension, this means of segmenting markets would likely apply in cases where different levels of production technologies are used based on access to market, irrigation, and credit, at least for perishable crops. <![CDATA[<b>Batch culture growth of <i>Chlorella zofingiensis</i> on effluent derived from two-stage anaerobic digestion of two-phase olive mill solid waste</b>]]> This paper presents the use of an effluent derived from two-stage anaerobic digestion of two-phase olive mill solid waste (OMSW) as a substrate for the production of Chlorella zofingiensis in batch mode. Chlorella zofingiensis when grown autotrophycally can accumulate significant quantities of valuable carotenoids which are used as an additive in fish and poultry farming, as colorants in foods and in health care products. It was found that two-phase OMSW previously treated by two-stage anaerobic digestion and further sterilized may be used as a culture medium for the microalgae Chlorella zofingiensis. Typical growth curves were obtained using both the above-mentioned anaerobic effluent and a synthetic medium. Total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (SCOD) removals of 37% and 45% respectively were achieved in batch experiments after 11 days’ operation time. The specific growth rate was lower when the treated effluent was used as the feed substrate (0.02 h-1) in comparison to the synthetic medium (0.03 h-1). The specific growth rates of the exponential phases were determined by using a first-order kinetic model applied to chlorophyll a (Ca) and total chlorophyll (TC) concentrations, as indirect measurements of the microalgae concentration. It was concluded that the effluent from two-stage anaerobic digestion of two-phase OMSW constituted an appropriate culture medium for the growth of Chlorella zofingiensis, providing a simple technology feasible for producing a very useful product for animal feeding. <![CDATA[<b>Bt protein rhizosecreted from transgenic maize does not accumulate in soil</b>]]> The persistence of CryIAb protein rhizosecreted in soil is important in the assessment of its environmental risk. Here we report that CryIAb protein from transgenic maize does not accumulate at high levels in soils. Levels of CryIAb protein rhizosecreted by three maize transgenic events (BT11, MON810 and 176) were studied in hydroponic cultures and found only in the MON810 and BT11 events but not in event 176 or control plants. Under field conditions, the cryIAb gene and a basal level of CryIAb protein was detected in soils from plots cultivated with transgenic and non-transgenic maize, possibly from Bacillus thuringiensis present in the soils. <![CDATA[<b>Changes in morpho-physiological attributes of <i>Eucalyptus globulus </i>plants in response to different drought hardening treatments</b>]]> Morpho-physiological attributes exhibited in response to drought hardening at the end of the growing season of Eucalyptus globulus Labill under nursery conditions were studied to evaluate the effect of three drought hardening treatments in morpho-physiological traits used as suitable indicators of drought hardiness, such as, plant growth, root growth potential, plant water relationships and survival. Freezing resistance of drought hardened plants was also studied in order to evaluate cross hardening effects in cuttings of Eucalyptus globulus Labill. Drought hardening consisted in induced water stress by watering restriction, until plant stem xylem water potentials (Ψpd) reached to-0.2, -1.3 and -2.4 MPa. Two water stress-rewatering cycles were applied during 54 days of treatment. The hardening treatments caused a significant reduction in plant height, leaf area, specific leaf area, plant, leaf, stem and root biomass. However, stem diameter was not affected. Root growth potential increased with the exposure to moderate water stress (-1.3 MPa). Drought hardening treatments have not effect on water relationship parameters such as saturation osmotic potential (Ψπsat), volumetric module of elasticity (e), relative water content (RWCtlp) and osmotic potential (Ψπtlp) at the turgor loss point. Only 1.7% and 6% of dehydrated dead plants were observed on treatments at -1.3 and -2.4 MPa respectively. Finally, the freezing damage index of leaves (LT50) was not significantly affected by drought hardening treatments. Furthermore, a reduction of 1.1ºC of supercooling capacity was observed at -2.4 MPa. As a conclusion, drought hardening is an important step of plants production programs during the final phase of nursery, because changes in morphological attributes caused by exposure to moderate drought, enable the plants to maintain the balance between transpiration and absorption areas and increase the capacity of plants to generate new roots. <![CDATA[<b>Design and expression of a retro doublet of cecropin with enhanced activity</b>]]> Novel doublet molecules of cecropin A from Drosophila melanogaster were designed and constructed combining the regular (CECdir) with the inverted (CECret) coding sequence of the standard CEC A1 gene resulting in the following configurations: CECdir-CECret and CECret-CECdir. These two recombinant molecules were generated using a three-primer driven PCR reaction yielding composite single functional aminoacidic molecules with the coding sequences of CECdir linked in frame with the coding sequence of CECret and vice versa. In order to obtain these constructions, a retropeptide DNA-coding sequence was chemically synthesized to match the expected polarity of the newly generated CECret sequence. Both doublet antimicrobial peptides (drAMPs) were cloned in the T7 promoter driven expression plasmid pET27b+ and expressed in E. coli BL21 without any fusion protein. Only the former recombinant peptide was expressed and purified from cell extracts and its specific activity against two different bacteria showed to be higher than those displayed by their monomer parental counterparts. <![CDATA[<b>Expression of a <i>Haemonchus contortus </i>cysteine protease in the baculovirus system</b>]]> A Haemonchus contortus recombinant Cysteine Protease (CP) was expressed in the baculovirus system. The CP gene was isolated by PCR from H. contortus cDNA, the PCR amplicon was cloned downstream to the polihedrin promoter within a bacterial expression vector, Sf9 insect cells were used for simultaneous co-transfection with the CP-vector and baculovirus naked DNA, which originated recombinant viruses by homologous recombination capable to express recombinant CP in an insect cell culture. A recombinant protease was identified as a fusion protein with a Ni lithium affinity 6XHis group. Recombinant CP was purified by affinity chromatography to obtain active recombinant protease identified by H. contortus experimentally infested ovine sera on a western blot as a 37 kDa protein, as well as by enzyme activity on PAGE-gelatin. Cysteine protease activity was assayed against synthetic substrates including the dipeptides: Phe-Arg, cathepsin B substrate: Arg-Arg, the caspase tetrapeptide substrate: Tyr-Val-Ala-Asp. Maximum CP activity was detected at pH 6.0 for all synthetic substrates and total inhibition was achieved by E-64 but not by EDTA, pepstatin or PMSF. Recombinant H. contortus CP can be obtained in large amounts from transfected insect cell culture and may be applied to control experiments of ruminant Haemonchosis. <![CDATA[<b>Generation and analysis of an <i>Eucalyptus globulus</i> cDNA library constructed from seedlings subjected to low temperature conditions</b>]]> Eucalyptus globulus is the most important commercial temperate hardwood in the world because of its wood properties and due to its characteristics for biofuel production. However, only a very low number of expressed sequence tags (ESTs) are publicly available for this tree species. We constructed a cDNA from E. globulus seedlings subjected to low temperature and sequenced 9,913 randomly selected clones, generating 8,737 curated ESTs. The assembly produced 1,062 contigs and 3,879 singletons forming a Eucalyptus unigene set. Based on BLASTX analysis, 89.3% of the contigs and 88.5% of the singletons had significant similarity to known genes in the non-redundant database of GenBank. The Eucalyptus unigene set generated is a valuable public resource that provides an initial model for genes and regulatory pathways involved in cell wall biosynthesis at low temperature. <![CDATA[<b>Genetic transformation studies and scale up of hairy root culture of <i>Glycyrrhiza glabra</i> in bioreactor </b>]]> The study was undertaken to induce hairy roots in Glycyrrhiza glabra in leaf explants and to optimize the nutritional requirement for its growth kinetics at shake flask and bioreactor level. Pathogenecity of Agrobacterium depends upon transformation ability of strain and age, type, and physiological state of explants. Agrobacterium rhizogenes strain K599 was used to infect leaf explants of G. glabra. Explants of different age groups were obtained from 2 to 5 weeks old in vitro grown cultures. Bacterial strain K599 could induce hairy roots in 3 and 4 weeks old leaf explants cultured on B5, MS, NB and WP basal semi-solid medium. Leaf explants of 2 and 5 weeks old culture were not responsive to bacterial infection in terms of hairy root induction. Maximum transformation frequency (TF) of tested bacterial strain was 47% obtained in 3 weeks old explants after 25 days of incubation on MS basal semi solid medium. NB and B5 both media composition showed 20% of transformation frequency after 28 and 38 days respectively. WP medium did not support induction of roots in cultured leaf explants infected with A. rhizogenes strain K599even after 50 days of incubation. Further, when all the four media combinations were tested for root growth it was found that though WP was not responsive for hairy root induction, yet all four basal media supported hairy root growth and a gradual increase in fresh weight biomass was observed with an increase in culture duration. However amongst all, the NB medium composition supported best growth of hairy roots followed by MS, B5 and WP media. About 20 times increase in root biomass on fresh weight basis was recorded after 45days of culture in NB medium. Initial inoculum of roots (0.18 g. F.wt./ flask) containing 50 ml of liquid culture medium produced 3.59 g (F. wt.) biomass. A fast growing hairy root clone G6 was grown in a 5 l capacity mechanically agitated bioreactor provided with a nylon mesh septum. After 30 days of sterile run, 310 g of root biomass was harvested from the bioreactor culture vessel, recording about 20 times increase over initial inoculum (16.0 g). <![CDATA[<strong>Identification of unknown genetically modified material admixed in conventional cotton seed and development of an event-specific detection </strong>method]]> Entering the second decade of commercialization of biotech crops, the global area cultivated with transgenic plants constantly expands and national legislations in many countries, particularly in the European Union, require identification and labeling of genetically modified material in food and feed. We describe here a procedure for characterizing transgenic material of unknown origin present in conventional seed lots using a genome walking strategy for isolation and characterization of the junction between the inserted transgene construct and the host plant genomic DNA. The procedure was applied to transgenic cotton detected as adventitious or technically unavoidable presence in a conventional commercial cultivar. The structure of the isolated region revealed that the transgenic material derived from Monsanto’s event 1445 transgenic cotton. Due to the random incorporation of the transgene into the host plant’s genome, the sequence of the junction region obtained using the genome walking strategy, provided the means to develop an event-specific identification method without prior knowledge for the nature of the transformation event. Thus, we documented a methodology for developing an event-specific detection protocol even without prior knowledge of the genetic modification event. <![CDATA[<b>Induction of <i>in vitro </i>flowering in <i>Capsicum frutescens </i>under the influence of silver nitrate and cobalt chloride and pollen transformation</b>]]> The influence of silver nitrate (AgNO3) and cobalt chloride (CoCl2) on shoot multiplication and in vitro flowering in Capsicum frutescens Mill. was investigated. Exogenous administration of AgNO3 and CoCl2 at a concentration of 30 µM resulted in the maximum tissue response in terms of shoot length and number of shoots after 45 days culturing on MS medium. Both silver nitrate (40 µM) and cobalt chloride (30 µM) influenced in vitro flowering after 25 and 45 days respectively. This is the first report on in vitro flowering in C. frutescens. The study also demonstrated successful transformation of pollen obtained from the in vitro flowers. Since capsicum is highly recalcitrant to in vitro plant regeneration, the results of the study may be highly useful in transformation of capsicum using germ free in vitro flowers. <![CDATA[<b>Physiological-enzymatic characteristics and inoculation of mycelial strains of <i>Descolea antarctica</i> Sing. in <i>Nothofagus</i> seedlings</b>]]> At present, reforestation has focused on native forests with anthropogenic intervention and eroded soils. There is interest in producing Nothofagus seedlings which can overcome adverse conditions encountered on reforestation sites. It is necessary to find new fungi that can be utilized as mycorrhizal inoculants and that enable the seedlings to increase their tolerance to adverse conditions. Two ectomycorrhizal strains of the fungus Descolea antarctica (D1 and D2) were cultured at different temperatures, pH levels and the activities of amylases, cellulases, and phosphatases were determined. In greenhouse and nursery trials, the growth responses of inoculated Nothofagus obliqua seedlings were evaluated. D1 and D2 exhibited the highest growth rates at 23ºC. Both strains grew at pH levels from 4 to 11. The highest enzymatic activities were registered for amylase (57.2 mg glucose/ml * g of mycelium * hr) and acid phosphatases (58.1 mg p-nitrophenol/ml * g of mycelium * hr) at 37ºC, and acid phospatases (1.720 mg p-nitrophenol/ml * g of mycelium * hr) and alkaline phosphatases (1.360 mg p-nitrophenol/ml * g of mycelium * hr) at pH 4 and pH 11, respectively. We conclude that suitable N. obliqua seedlings for use in reforestation were obtained using D2 as inoculant. <![CDATA[<b>Selection of sulfur oxidizing bacterium for sulfide removal in sulfate rich wastewater to enhance biogas production</b>]]> Sulfur oxidizing bacteria (SOB) were isolated and tested in order to remove sulfide from high sulfate wastewater to reduce the amount of hydrogen sulfide (H2S) in the produced biogas. A promising SOB isolate, designated as isolate T307, was selected due to its best sulfide removal (86.7%) in the effluent of a sulfate reduction reactor (SRR) over a 24 hrs incubation. The bacterium was able to grow better as a mixotroph (yeast extract as a carbon source) than as a chemolithoautotroph. In addition, as a heterotroph, the bacterium grew well with yeast extract and peptone. Based on partial 16S rRNA gene sequence, the isolated T307 was an Alcaligenes sp. and was able to convert most of sulfide species (total sulfide: TS; dissolved sulfide: DS and H2S) into elemental sulfur or sulfate over a 20 hrs period of cultivation by controlling the speed of shaking. In a biogas reactor set, after pre-treating a sulfide medium with Alcaligenes sp. T307 there was a much higher specific yield of CH4 (238 ml CH4 g-1COD removed) and more biogas (154 ml L-1 d-1) was produced with the biogas containing more methane (48.1% CH4, 51.5% CO2 and 0.41% H2S) in comparison to a control with a specific yield of CH4, (72 ml CH4 g-1COD removed) 86 ml L-1 d-1 biogas produced with a composition of 35.5% CH4, 63.7% CO2 and 0.86% H2S. <![CDATA[<b>Visualisation of the microbial colonisation of a slow sand filter using an Environmental Scanning Electron Microscope</b>]]> The removal of contaminants in slow sand filters occurs mainly in the colmation layer or schmutzdecke - a biologically active layer consisting of algae, bacteria, diatoms and zooplankton. A ripening period of 6 - 8 weeks is required for this layer to form, during which time filter performance is sub-optimal. In the current study, an environmental scanning electron microscope was used to visualise the ripening process of a pilot-scale slow sand filter over a period of eight weeks. To achieve this, sand particles were removed at weekly intervals and observed for biofilm development. Biological mechanisms of removal in slow sand filtration are not fully understood. A visualisation of the colonisation process would enhance the knowledge and understanding of these mechanisms. Colonisation of sand particles and increase in biomass was clearly seen during the ripening period. The mature, ripened filter exhibited a dense extracellular matrix consisting of a wide variety of microorganisms and their extracellular and breakdown products. This research demonstrated the successful use of an environmental scanning electron microscope to visualise the complex, heterogeneous nature of the schmutzdecke in a slow sand filter. Such knowledge could possibly lead to an increase in the application of slow sand filtration, especially for rural communities. <![CDATA[<b>Universal protocol for generating 100bp size standard for endless usage</b>]]> Developing countries are facing severe bottlenecks in the technological advancement in biotechnology, due to restrictions imposed by patent protected products and protocols. This calls for designing of simple and cost-effective alternatives for the indispensable products like DNA molecular weight markers. We demonstrate a novel, rapid and cost-effective method of making in-house 100bp ladder for routine use. In our method we use a single forward primer and five reverse primers designed on the backbone sequence of a commonly used vector template. These primers are used at a universal annealing temperature to amplify ten DNA fragments of accurate size ranging from 100bp to 1000bp. Our PCR-based method can provide size standards for an endless usage.