Latest Microbiology Research Month: April-2019

Molecular Analysis of Genetic Diversity of Pectobacterium carotovorum Subsp Carotovorum Isolated in Morocco by PCR Amplification of the 16S-23S Intergenic Spacer Region

Aims: Pectobacterium carotovorum is a ubiquitous bacterium that causes soft rot in different crop plants throughout the world. In Morocco, approximately 95% of the Strains isolates from potato plants with tuber soft rot are P. carotovorum. In this study, we test whether PCR ribotyping can be used to distinguish strains of Pectobacterium carotovorum isolated from soft rot potato and to differentiate among strains from different geographic regions.

Place and Duration of Study: Laboratory of Virology, Microbiology and Quality / Ecotoxicology and Biodiversity, department Biology, Faculty of Sciences and Techniques, University Hassan-II Mohammedia Casablanca.

Methodology: Eighty-three pectolytic enterobacteria were collected from potatoes rotten in Morocco, the strains were isolated in the Cristal Violet Pectate (CVP) medium and were purified in LPGA agar (yeast extract, peptone, glucose and agar). After purification, strains were identified by physiological and biochemical tests. The confirmation of species was performed by PCR using primers Y1 and Y2. The genetic diversity of Pectobacterium carotovoum was investigated by PCR ribotyping using primers G1/L1, which are complementary to conserved regions of the rRNA operon. Furthermore, the profiles obtained were compared by the Unweighted Pair Group Method.

Results: The biochemical and physiological analysis demonstrated that the predominant pectolytic enterobacterium present in Morocco is Pectobacterium carotovorum subsp carotovorum. The specific confirmation of species P. carotovorum by PCR has yielded a 434 bp DNA fragment of the pelY gene with all isolates. Further, PCR amplification of the 16S-23S Intergenic spacer Region (ITS-PCR) has presented a specific pattern made of 2-6 fragments ranging from 300 bp to 800 bp. The UPGMA tree has shown that there is considerable genetic diversity in P. carotovorum strains, which can be divided into four distinct groups. [1]

Decolorization of Different Azo Dyes and Detoxification of Dyeing Wastewater by Pseudomonas stutzeri (SB_13) Isolated from Textile Dyes Effluent

Aims: The present study aimed to estimate the decolorization of three individual azo dyes or in mixture, as well the decolorization of dyeing wastewater was evaluated using bacterial strain of Pseudomonas stutzeri (SB_13) or bacterial consortium isolated from textile dyes effluent. The cytotoxicity effect of dyeing wastewater and its biodegraded metabolites, as well the detoxification efficacy were evaluated.

Study Design: Soil and water samples were collected from the textile dyeing industrial area for bacterial isolation. Effect of different parameters on the dye decolorization by bacterial strains was optimized.

Place and Duration of Study: The study was performed in Botany & Microbiology Department, Faculty of Science, Al-azhar University, from July 2014 until January 2016.

Methodology: Pseudomonas stutzeri strain (SB_13) was isolated from textile dyes effluent and its ability for decolorization of different azo dyes and detoxification of dyeing wastewater samples was investigated. Comparison the decolorization effectiveness of bacterial strain (SB_13) which was used individually and in a bacterial consortium which contains two previously studied Klebsiella strains of (Klebsiella pneumoniae (Kp) and Klebsiella variicola (Kv)) was also observed. Decolorization of Disperse Blue (R16), Disperse Yellow (D4), and Reactive Red Synozol (R4) dyes which were used singly and in mixture was estimated under different concentrations and incubation conditions.

Results: The highest decolorization rates of single or mixtures of azo dyes were observed with 2% glucose or sucrose, 2% ammonium sulfate, and 3% (v/v) bacterial inoculum size, at pH of 5-7, temperature of 35ºC, and after 72-96 hrs. Mixed cultures of (SB_13 &Kp), (SB_13 &Kv), and (SB_13& Kp& Kv) significantly decolorized 59.5% of Disperse Blue (R16), 52% of Disperse Yellow (D4), and (18.3%) of Reactive Red Synozol (R4) dyes, respectively more than those found by individual strain (SB_13). Individual strain of (SB_13) showed the highest decolorization 61% capacity of azo dyes mixture compared to those observed by bacterial consortiums. The treatment of dyeing wastewater with SB_13 strain significantly reduced the phytotoxicity of wastewater (from 100% of abnormal mitosis to 23.6%) as compared with other treatments. [2]

Cellulase Producing Potential of Aspergillus terreus Uv2 on Cellulosic Wastes Pretreated with Acid and Alkali

Aims: Cellulases offer very wide applications in biotechnology and enzymes from microbial origins present inexpensive source. Production of value added chemicals from wastes will be an exciting translation from waste to wealth and an eco-friendly initiative instead of the incineration option often given to cellulosic wastes.

Study Design: Sulphuric acid and Sodium hydroxide solutions were prepared at 0.5 M and 2 M concentrations to pretreat three cellulosic wastes that had been made neutral prior to fermentation with a known cellulase producing mold

Place and Duration of Study: All experiments were conducted in the laboratory of the Department of Microbiology, Federal University of Technology, Minna, Nigeria for a period of six weeks.

Methodology: Hypercellulase producing Aspergillus terreus UV2 strain was used to ferment pretreated cellulosic wastes: Corn cob, corn straw and bagasse, using submerged fermentation in Mandel basal medium. The crystalline lignocelluloses were milled and fractionated into 850 μ particle size and pretreated in two concentrations (0.5 M and 2 M) of both acid (sulphuric acid) and alkali (sodium hydroxide) independently and were left for varying residence time of one hour or three hours in the digester at ambient temperature, Optimum spore concentration of 1.0 x 106 spores/ml and pH of 4.8. Supernatants of crude enzyme were taken and assayed at 24 hours interval.

Results: Cellulase activity peaked at 96 hours. Enzyme secretion in the cellulosic wastes was highest in sugarcane bagasse, followed by the corn cob and then the corn straw corresponding to 51%, 40% and 16% respectively. Alkali pretreated cellulosics gave higher yield of cellulase than its counterpart acid. Non-pretreated residues gave only low enzyme titers. Bagasse produced optimum cellulase yield of 0.068 IU/ml/min within 120 hours when subjected to 2 M NaOH digestion for one hour before fermentation. This translated to 39% increase in enzyme expression when compared with non-treated bagasse of 0.049 IU/ml/min.

Conclusion: Sugarcane bagasse therefore when digested with mild alkali (2 M NaOH) for a pretreatment period of one hour holds a great possibility for cellulase production using a mutant mold, Aspergillus terreus UV2. Production of value added chemicals from cellulosic wastes will be an exciting translation from waste to wealth. [3]

Reference

[1] Amdan, M., Faquihi, H., Terta, M., M. Ennaji, M. and Ait Mhand, R. (2015) “Molecular Analysis of Genetic Diversity of Pectobacterium carotovorum Subsp Carotovorum Isolated in Morocco by PCR Amplification of the 16S-23S Intergenic Spacer Region”, Biotechnology Journal International, 7(3), pp. 102-110. doi: 10.9734/BBJ/2015/16045. (Web link)

[2] Fouda, A., El-Din Hassan, S., Salah Azab, M. and Saied, E. (2016) “Decolorization of Different Azo Dyes and Detoxification of Dyeing Wastewater by Pseudomonas stutzeri (SB_13) Isolated from Textile Dyes Effluent”, Biotechnology Journal International, 15(4), pp. 1-18. doi: 10.9734/BBJ/2016/28363. (Web link)

[3] Damisa, D., A. Kuta, F., P. Abioye, O., D. Bala, J. and E. Egbe, N. (2015) “Cellulase Producing Potential of Aspergillus terreus Uv2 on Cellulosic Wastes Pretreated with Acid and Alkali”, Biotechnology Journal International, 9(2), pp. 1-10. doi: 10.9734/BBJ/2015/18773. (Web link)