Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 11 num. 1 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<b>Diversity of bacterial communities in acid mine drainage from the Shen-bu copper mine, Gansu province, China</b>]]> This study presents bacterial population analyses of microbial communities inhabiting three sites of acid mine drainage (AMD) in the Shen-bu copper mine, Gansu Province, China. These sites were located next to acid-leached chalcopyrite slagheaps that had been abandoned since 1995. The pH values of these samples with high concentrations of metals ranged from 2.0 to 3.5. Amplified ribosomal DNA restriction analysis (ARDRA) was used to characterize the bacterial population by amplifying the 16S rRNA gene of microorganisms. A total of 39 operational taxonomic units (OTUs) were obtained from the three samples and sequenced from 384 clones. Sequence data and phylogenetic analyses showed that two dominant clones (JYC-1B, JYC-1D) in sample JYC-1 represented 69.5% of the total clones affiliated with Acidithiobacillus ferrooxidans (γ-Proteobacteria), and the most dominant clones of JYC-2 and JYC-3 were affiliated with Caulobacter crescentus (α-Protebacteria). At the level of bacterial divisions, differences in the relative incidence of particular phylogenetic groups among the three samples and discrepancies in physicochemical characteristics suggested that the physico-chemical characteristics had an influence on phylogenetic diversity. Furthermore, the relationships between the discrepancies of physicochemical characteristics and the diversity of the bacteria communities in the three samples suggested that the biogeochemical properties, pH and concentration of soluble metal, could be key factors in controlling the structure of the bacterial population <![CDATA[<strong>Effect of methyl branching of C<sub>8</sub>H<sub>18</sub> alkanes and water activity on lipase-catalyzed enantioselective esterification of ibuprofen</strong>]]> The purpose of this research was to study the effect of the methyl branching of a high log P alkane solvent and the water activity in the organic medium on the initial rate and the enantioselectivity of ibuprofen esterification catalyzed by Candida rugosa lipase. Resolution of ibuprofen is important because S-(+)-ibuprofen has the desired pharmacological activity, whereas the R-(-)-enantiomer causes much of the side effects. The Candida rugosa lipase-catalyzed reaction in isooctane at 40ºC and 0.73 water activity gave the best results, both in terms of the initial reaction rate and the enantioselectivity of the reaction. An increase in water activity allowed a higher reaction rate and enantiomeric excess in each of the four solvents. An increase in methyl branching did not necessarily increase the initial reaction rate, but it allowed a higher enantioselectivity, evidenced by an increase in the substrate enantiomeric excess <![CDATA[<b>Evaluation of the uniformity and stability of T-DNA integration and gene expression in transgenic apple plants</b>]]> The generation of transgenic apple plants relies on the molecular analysis of transgene integration and expression based on polymerase chain reaction (PCR) analysis, blotting techniques and enzymatic assays on vitro leaves of putative transgenic regenerates. In order to assess the uniformity and the stability of transfer DNA (T-DNA) integration and gene expression, we studied 26 transgenic apple lines carrying the attacin E gene from Hyalophora cecropia, the β-glucuronidase gene, and the nptII gene. Plants were evaluated using standard molecular techniques, such as PCR, Southern blot, reverse transcription PCR (RT-PCR) and Enzyme Linked Immunosorbent Assay (ELISA), and propagated in vitro on non-selective antibiotic-free media for four years to mimic natural conditions in the field. In some T-lines transgene integration and expression did not remain stable; differences were also found between distinct plants of a single T-line. Individual plants with partially or completely silenced transgenes were identified as well as plants with non-detectable T-DNA. Several lines appeared chimeric or partially silenced. Although most molecular techniques can reliably detect the presence of transgenic cells, they often fail to detect mixtures of transformed and non-transformed cells, or cells with silenced transgenes. This should be taken into consideration, especially in the case of vegetatively propagated trees, where non-transformed or silenced plant parts could mistakenly be used as propagation material. <![CDATA[<b>Heterozygosity following half-sib recurrent selection in popcorn using isoenzyme markers</b>]]> Isozyme biochemical marker may be useful tool for genomic analysis of maize populations undergoing recurrent selection. Thus, isozymes markers was utilized for assess the changes in the genetic variability and distance in a Brazilian composite population of popcorn following four cycles of recurrent selection for yield. One hundred and ninety-six half-sib families were evaluated from each cycle and the ten highest-yielding families (5.2%) were recombined to produce the next cycle. Isozyme analysis considered 80 seedlings per cycle. Simple linear regression equations were estimated among the allele frequencies in each locus in function of the selection cycles, the genetic distances among the cycles and the average heterozygosity per locus for each cycle. Regression analysis did not reveal any common trend for changes in allele frequencies presumably due to selection. The estimates of the number of polymorphic locus, of the mean of allele per locus and the mean heterozygosity did not reveal any reduction in variability. It was concluded that four selection cycles did not cause relevant changes in the variability or genetic distance among the selection cycles of CMS-43 popcorn population. Isozymes markers analysis showed that the number of recombined half-sib families in recurrent selection was suitable. <![CDATA[<strong>Identification of differentially expressed genes in developing cotton fibers (<i>Gossypium hirsutum</i> L) through differential display</strong>]]> Cotton fibers are differentiated, non-dividing cells that originate from the epidermal layer of developing ovules. To identify genes involved in cotton fiber development, we performed non-radioactive differential display reverse transcriptase PCR (DDRT-PCR) on the purified mRNA. This technique was tested on mRNA isolated from five different developmental stages of cotton fiber including 0, 5, 10, 15 and 20 DPA (days after pollination). The mRNA purified from total RNA was reversibly transcribed using three anchored oligo-dT primers. Polymerase chain reaction (PCR) amplification of each cDNA preparation was carried out in combination with seven arbitrary primers. The amplified products were resolved on 1% agarose gel containing ethidium bromide. DNA was extracted from seventeen differentially expressed bands and cloned in pTZ57R/T vector. The sequencing and BLAST search analysis indicated that 12 of the differentially expressed genes matched the previously characterized genes, while 3 of them matched the uncharacterized sequences of cotton fiber expressed sequence tags (ESTs) reported previously to be associated with cotton fiber and 2 of the clones had homology with putative proteins. The technique can be used to efficiently identify differentially expressed genes and can be expanded to large scale studies by increasing the number of random decamers. <![CDATA[<b>Inhibition of attachment of some fouling diatoms and settlement of <em>Ulva lactuca</em> zoospores by film-forming bacterium and their extracellular products isolated from biofouled substrata in Northern Chile</b>]]> The biofouling of surfaces submerged in the marine environment includes primary colonization of the substrate by microorganisms including bacteria, microalgae, and microscopic reproductive propagules of macroorganisms such as algal zoospores. The present study reports the evaluation of the inhibitory potential of biofilms and extracellular products (EP) of the indigenous bacterium Alteromonas sp strain Ni1-LEM on the settlement of marine biofouling such as: (i) eight marine benthic diatoms and (ii) zoospores of the alga Ulva lactuca, as well as the germination of these zoospores and was compared with reference strains with proven antifouling properties, Halomonas marina (ATCC 25374) and Pseudoalteromonas tunicata. Highest antifouling activity was found for the indigenous strain. In attempts to better define the chemical nature of the antifouling substance in the EP of the Alteromonas sp strain Ni1-LEM, the culture filtrates were tested for activity after heat treatment, enzymatic treatments, dialysis through semipermeable membranes, and separation into polar (aqueous) and non-polar (organic) fractions. The results suggested that the antifouling substance in the culture filtrates to be protein or peptide in nature, thermostable, hydrophilic, and equal to or greater than 3500 daltons in molecular size. Antifouling substances from bacteria may lead to the development of novel antifouling agents in the future. <![CDATA[<b>Overexpression of the pineapple fruit bromelain gene (BAA) in transgenic Chinese cabbage (<i>Brassica rapa</i>) results in enhanced resistance to bacterial soft rot</b>]]> Bromelain is a crude protein extract obtained from pineapple stems, which comprises a variety of proteolytic enzymes. It exhibits potential therapeutic activities against trauma, inflammation, autoimmune diseases and malignant disorders. In this study, we cloned BAA1 (the gene encoding fruit bromelain) into a plant expression vector that was then used to transform Brassica rapa and overexpress BAA1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. We demonstrate that constitutive overexpression of BAA1 in B. rapa confers enhanced resistance to the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum. These results suggest that it could be utilized for protecting plants from attack by bacterial pathogens <![CDATA[<b>Oxygen mass transfer studies on batch cultivation of <i>P. aeruginosa</i> in a biocalorimeter</b>]]> In the present work volumetric mass transfer coefficient (kLa) was investigated during batch cultivations of Pseudomonas aeruginosa on a nutrient media. The effects of process variables (viz. impeller speed, oxygen flow and geometry of impeller) on the volumetric mass transfer coefficient of oxygen, kLa, in a biocalorimeter (Bio-RC1) was investigated and reported in this research work. The experimental data have been analyzed employing MATLAB to obtain the influences of the process parameters on kLa. An attempt was made to correlate volumetric mass transfer coefficient with metabolic heat production rate at optimized process conditions. The correlation reported in this work would be useful to control and scale up of bioprocesses. <![CDATA[<strong>Performance of biofilm carriers in anaerobic digestion of sisal leaf waste leachate</strong>]]> Three methanogenic biofilm bioreactors were studied to evaluate the performance of three types of carriers. The carrier material were consisted of sisal fibre waste, pumice stone and porous glass beads, and the bioprocess evaluated was the methanogenesis anaerobic digestion of sisal leaf waste leachate. Process performance was investigated by increasing the organic loading rate (OLR) step-wise. The best results were obtained from the bioreactor packed with sisal fibre waste. It had the highest chemical oxygen demand (COD) removal efficiencies in the range of 80-93% at OLRs in the range of 2.4-25 g COD L-1d-1. The degradation pattern of volatile fatty acids (VFAs) showed that the degradation of propionate was limiting at higher OLRs. The stable pH and higher partial alkalinity (PA) of the outflow illustrated that packed-bed bioreactors have a good ability to withstand the variations in load and volatile fatty acid concentrations that can occur in a two-stage anaerobic process. In conclusion, sisal fibre waste was shown to be a novel promising biofilm carrier and would work very well in methanogenic biofilm bioreactors treating sisal leaf tissue waste leachate. Furthermore both sisal wastes are available in the neighbourhood of sisal industries, which makes anaerobic digestion scale up at sisal factory level feasible and cost-effective. <![CDATA[<b>Plant regeneration via indirect somatic embryogenesis and optimisation of genetic transformation in C<i>offea arabica</i> L. cvs. Caturra and Catuaí</b>]]> A protocol for Coffea arabica L. cvs. Caturra and Catuaí plant regeneration via indirect somatic embryogenesis (ISE) was established. Furthermore, a biolistic mediated genetic transformation protocol was optimized for Catuaí callus aggregates. Maximum callus induction was obtained when Caturra (87%) and Catuaí (67%) leaves were cultured on Murashige and Skoog medium with 18.56 µM kinetin and 4.52 µM2,4-dichlorophenoxyacetic acid (2,4-D). Catuaí suspension cultures were established from embryogenic callus using liquid proliferation CP and Sli media and diffused light and darkness. The higher suspension cultures fresh weight was obtained using Erlenmeyer (1425.4 ± 354.9 mg) than Recipient for Automated Temporary Immersion System (RITA®) (518.6 ± 55.1 mg), whereas the dry weight of suspension cultures was not significantly affected by the culture system used. Higher number of embryos per vessel (307.6 ± 49.0) and their fresh weight (9.6 ± 1.5 mg) were obtained with semisolid R medium than S3 medium. The highest somatic embryo development (25.0 ± 2.7) and fresh weight (780.0 ± 85.4 mg) were obtained with 1 min of immersion every 8 hrs. Higher fresh weight of regenerated plantlets was obtained with liquid Yasuda medium in RITA® (124.6 ± 16.3 mg) than semisolid media (36.3 ± 11.3 mg). For genetic transformation, the effect of helium pressure (900 and 1550 psi), and target distance (9 and 12 cm) and plasmid (pCAMBIA 1301, pCAMBIA 1305.2 and pCAMBIA 1301-BAR) on transient uidA expression Catuaí suspension cultures were evaluated. The highest number of blue spots was obtained using 900 psi and 9 cm (125.8 ± 17.3). Stable uidA expression was observed on Catuaí callus aggregates transformed with pCAMBIA 2301 and cultured on 100 mg l-1 of kanamycin. <![CDATA[<b>Ranking agro-technical methods and environmental parameters in the biodegradation of petroleum-contaminated soils in Nigeria</b>]]> A combination of experimental cells consisting of some agro-technical methods aimed at accelerating the biodegradation of petroleum contaminated soils were evaluated in order to ascertain the relevance of these methods and the relative attention due necessary soil environmental parameters. The methods of treatment involved the variation of tilling, watering and nutrient application, plus biopile and phytoremediation treatments. In the experiments described, petroleum contamination of soils was simulated under field conditions, the remedial treatments were then utilized for clean up. Analysis of soil parameters after a six-week study period showed an increase in total heterotrophic bacteria (THB) counts across all the treatments, with THB counts increasing with increment in soil nutrient level and initial concentration of the contaminant. The total hydrocarbon content (THC) analysis, based on a performance index introduced in this study, indicated that on the average, the variation of nutrient application, tilling and watering facilitated the attenuation of THC at the rate of 429.4 mg/kg day, 653.2 mg/kg day, and 327.5 mg/kg day respectively. While the combined effect of various levels of nutrients, tiling and watering performed at the rate of 558.7 mg/kg day, biopile and phytoremediation treatments recorded 427.9 mg/kg day and 489.3 mg/kg day respectively. These results imply that though nutrient application, watering and other factors affect the biodegradation process, frequent tilling for maximum oxygen exposure is the most important factor that affects the biodegradation of petroleum-hydrocarbons in tropical soils. <![CDATA[<b>Role of PPase-SE in <i>Geotrichum klebahnii</i>, a yeast-like fungus able to solubilize pectin</b>]]> Protopectinases (PPases) constitute a heterogeneous group of extracellular enzymes able to release soluble pectin from insoluble protopectin in plant tissues. Geotrichum klebahnii (ATCC 42397) produces PPase-SE with endopolygalacturonase activity. PPase-SE has been used for pectin extraction and maceration of plant tissues. Here, the capacity of G. klebahnii to use different pectins as carbon and energy sources (CES) was studied, in addition to PPase-SE capacity to release pectin from lemon peel. The strain was unable to use pectin from different origins as CES. When G. klebahnii was cultivated with mixtures of different amounts of glucose and citrus pectin as CES, the biomass obtained was proportional to the initial concentration of glucose, which was completely consumed. In addition, it produced PPase-SE in a glucose-containing medium. A culture was used for the extraction of pectin from lemon peels. Pectin was enzymatically extracted simultaneously with tissue maceration, yielding 3.7 g of (dry) pectin per 100 g of (wet) lemon peel. Extracted pectin was not metabolized by the strain. It was concluded that G. klebahnii uses PPase-SE to macerate, invade and colonize plant tissues, thus releasing soluble sugars to be used as CES without metabolizing solubilized pectin. <![CDATA[<b><i>Agrobacterium</i></b><b>-mediated transient transformation of Mexican prickly poppy (<i>Argemone mexicana</i> L.)</b>]]> Shoot apex, leaf primordia, leaf sections and roots from Mexican prickly poppy seedlings, were inoculated with Agrobacterium tumefaciens harboring the binary vector pCAMBIA2301, which contained the &beta;-glucuronidase (uid A) gene. Histochemical &beta;-glucuronidase (GUS) assay in infected explants showed transient gus gene expression between 3 and 12 days after inoculation. To our knowledge, this is the first report of A. mexicana susceptibility to A. tumefaciens-mediated genetic transformation <![CDATA[<b>Analysis of genetic diversity among Indian niger [<i>Guizotia abyssinica</i> (L. f.) Cass.] cultivars based on randomly amplified polymorphic DNA markers</b>]]> Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 18 cultivars of niger from India. Total genomic DNA was extracted and subjected to RAPD analysis using 80 arbitrary 10-mer primers; 17 primers were selected, which yielded a total of 124 bands, 41.20% of them polymorphic. None of the primers produced unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidean Distance matrix which revealed a minimum genetic distance between cultivars JNC-6 and N-48 and a maximum distance between IGP-76 and JN-30. Based on the distance matrix, a cluster analysis was done using a minimum variance algorithm. The dendrogram generated, based on Ward’s method, grouped 18 niger cultivars into two major clusters. The first cluster consisted of early maturing cultivars (e.g. N-129 and N-134; 80-90 days), and the second of late maturing cultivars (e.g. GA-8 and GA-9; 135-145 days). The present study shows that there is high diversity among the niger cultivars tested and indicates the potential of RAPD markers for identification and maintenance of niger germplasm for crop improvement purposes. <![CDATA[<b>Multiple pulses improve electroporation efficiency in <i>Agrobacterium tumefaciens</i></b>]]> Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency.