Molecular Characterization and Potential of Fungal Species Associated with Cassava Waste.

Aims: Efficient utilization of cassava waste for value addition depends largely on proper understanding of its true microbial diversity. The aim of this study was to characterize using molecular methods, fungal species associated with cassava waste and to highlight their industrial potential. Study Design: Cassava peel (CP) waste from CP waste dumpsites and cassava waste water from cassava wastewater discharge outlets were collected from major cassava processing centres in Abeokuta, Ogun State, Nigeria, for the study. Place and Duration of Study: Biotechnology Centre, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria; between June 2011 and March 2012. Methodology: Two molecular methods namely, total fungal community DNA and isolates DNA sequence analysis were employed to characterize and identify the fungal species. Total fungal community DNA was extracted directly from CP waste and cassava wastewater, using the Soil DNA isolation kit (Norgen, Canada), while total genomic DNA was extracted from fungal isolates, using the same kit. The fungal ITS2 (Internal transcribed spacer) gene sequence of total fungal community and genomic DNA was amplified by Polymerase Chain Reaction (PCR) using ITS2 primers. Total fungal community DNA amplicons were spliced into PCR-TRAP Cloning Vector, used to transform competent cells of Escherichia coli and sequenced. Sequences were identified by aligning with sequences in the GenBank. Results: Results showed that 17 fungal species including Eurotiomycetes – Eurotiales (6 species), Mucormycotina – Mucorales (1 species), Sordariomycetes - Hypocreales (1 species), Saccharomycetes Saccharomycetales (8 species), and unidentified fungi (1 species) were present in cassava peel (CP). The dominant species was Aspergillus niger (15.2%). However, cassava wastewater had 27 fungal species including Eurotiomycetes – Eurotiales (2 species), Saccharomycetes Saccharomycetales (24 species) Tremellomycetes-Tremellales (1 species); the dominant species being Saccharomyces cerevisiae and Candida krusei each with 8.7% relative abundance. Conclusion: This study shows that cassava waste, on account of its rich fungal diversity, is an important microbial resource.

Diversity of Bacterial Community in Fermentation of African Oil Bean Seeds (Pentaciethra macrophylla Benth) by comparison of 16S rRNA Gene Fragments.

Aims: The microbial diversity, fermentation dynamic and the predominant microorganisms involved in the fermentation of African oil bean (Pentaciethra macrophylla Benth) seeds to “Ugba” traditional African food in Eastern Nigeria were investigated by analyzing the microbial community DNA of the food using sequences of their 16S rRNA genes fragment analysis. Study Design: Universal bacterial conserved 16S rRNA gene region was used to study bacterial dynamics as well as the diversity during fermentation stages. Predominant microorganisms were investigated with the view to establishing the best possible starter culture for the production of high flavoured “Ugba”. Place and Duration of Study: Biotechnology Centre of Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between January 2007 and May 2009. Methodology: Raw seeds were boiled for two hours for easy removal of the seed coats. Peeled seed cotyledons were sliced, cooked for 4hrs until softened. Sliced cotyledons were washed, wrapped in local leafs for fermentation for a period of 96hrs. Sampling for analysis was performed, at every 24 hours interval. Bacterial Community of freshly fermenting “Ugba” was obtained by washing seeds at room temperature in 0.40% NaCl salt solution for 15 minutes. The supernatant was used for streaking on both Nutrient agar and “Ugba” agar plates and for Community DNA extraction. DNA extraction was carried out from community DNA extracts and culture isolates grown in LB (Luria – Bertani) broth at 37°C for 24 hours using Promega DNA extraction kit. Partial 16S rRNA genes of isolates DNA and entire microbial community DNA were amplified using 16S rRNA primers. Amplified fragments were cloned using the PCRTRAP. The transformed clones were sequenced and aligned with reference sequences in the NCBI data base for identification. Results: This analysis indicated that from community DNA, seventeen clones were identified as Bacillus subtilis, Nine as Bacillus pumilus, four as Bacillus licheniformis, two as Bacillaceae bacterium, two as Bacillus sp Van 22, and two as Staphylococcus spp. Also, of the ten sequenced cloned isolates from the cultural technique, eight were identified as Bacillus subtilis, while two sequences were identified as Bacillus pumilus. The percentage abundance revealed that Bacillus subtilis had the highest abundance of 47.2% followed by Bacillus pumilus with 25%. Conclusion: Bacillus subtilis is the predominant species in Ugba fermentation as it had high percentage abundance throughout the fermentation period. This study indicated that molecular analysis of community DNA provides a more accurate picture of diversity and dynamics of microbial communities.

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs / Caracterização da diversidade bacteriana em amostras de petróleo provenientes de reservatórios brasileiros

This study aimed at evaluating potential differences among the bacterial communities from formation water and oil samples originated from biodegraded and non-biodegraded Brazilian petroleum reservoirs by using a PCR-DGGE based approach. Environmental DNA was isolated and used in PCR reactions with bacterial primers, followed by separation of 16S rDNA fragments in the DGGE. PCR products were also cloned and sequenced, aiming at the taxonomic affiliation of the community members. The fingerprints obtained allowed the direct comparison among the bacterial communities from oil samples presenting distinct degrees of biodegradation, as well as between the communities of formation water and oil sample from the non-biodegraded reservoir. Very similar DGGE band profiles were observed for all samples, and the diversity of the predominant bacterial phylotypes was shown to be low. Cloning and sequencing results revealed major differences between formation water and oil samples from the non-biodegraded reservoir. Bacillus sp. and Halanaerobium sp. were shown to be the predominant components of the bacterial community from the formation water sample, whereas the oil sample also included Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. and Acidithiobacillus ferrooxidans. The PCR-DGGE technique, combined with cloning and sequencing of PCR products, revealed the presence of taxonomic groups not found previously in these samples when using cultivation-based methods and 16S rRNA gene library assembly, confirming the need of a polyphasic study in order to improve the knowledge of the extent of microbial diversity in such extreme environments.

Este estudo teve como objetivo comparar as comunidades bacterianas de amostras de água de formação e de óleo de reservatórios de petróleo brasileiros com diferentes graus de biodegradação usando a técnica de PCR-DGGE. O DNA ambiental foi isolado e empregado em reações de PCR com primers bacterianos, com subseqüente separação dos fragmentos de DNAr 16S em DGGE. Os produtos de PCR foram também clonados e seqüenciados, visando à afiliação taxonômica dos membros da comunidade. Os fingerprints obtidos permitiram a comparação direta entre as comunidades bacterianas das amostras de óleo com diferentes graus de biodegradação, assim como entre as comunidades da água de formação e do óleo do reservatório não biodegradado. Perfis de DGGE muito similares foram observados para todas as amostras, e a diversidade dos filotipos bacterianos predominantes mostrou-se baixa. Os resultados de clonagem e seqüenciamento revelaram diferenças mais significativas entre as amostras de água de formação e de óleo do reservatório não biodegradado. Bacillus sp. e Halanaerobium sp. mostraram-se os componentes predominantes da comunidade bacteriana da presente na amostra de água de formação, ao passo que a amostra de óleo incluiu também Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. e Acidithiobacillus ferrooxidans. A técnica de PCR-DGGE, combinada com clonagem e seqüenciamento dos produtos de PCR, revelou a presença de grupos taxonômicos não encontrados anteriormente nestas amostras quando métodos baseados em cultivo e na construção de bibliotecas de genes RNAr 16S foram utilizados, evidenciando a necessidade de um estudo polifásico a fim de contribuir para o conhecimento da diversidade microbiana em ambientes extremos como reservatórios de petróleo.