Kenneth Francis Rodrigues

Yeasts in sustainable bioethanol production: a review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.

Homogeneous nature of Malaysian Marine fish Epinephelus fuscoguttatus (Perciformes; Serranidae): Evidence based on molecular markers, morphology and fourier transform infrared analysis

Taxonomic confusion exists within the genus Epinephelus due to the lack of morphological specializations and the overwhelming number of species reported in several studies. The homogenous nature of the morphology has created confusion in the Malaysian Marine fish species Epinephelus fuscoguttatus and Epinephelus hexagonatus. In this study, the partial DNA sequence of the 16S gene and mitochondrial nucleotide sequences of two gene regions, Cytochrome Oxidase Subunit I and III were used to investigate the phylogenetic relationship between them. In the phylogenetic trees, E. fuscoguttatus was monophyletic with E. hexagonatus species and morphology examination shows that no significant differences were found in the morphometric features between these two taxa. This suggests that E. fuscoguttatus is not distinguishable from E. hexagonatus species, and that E. fuscoguttatus have been identified to be E. hexagonatus species is likely attributed to differences in environment and ability to camouflage themselves under certain conditions. Interestingly, this finding was also supported by Principal Component Analysis on Attenuated Total Reflectance–Fourier-transform Infrared (ATR-FTIR) data analysis. Molecular, morphological and meristic characteristics were combined with ATR-FTIR analysis used in this study offer new perspectives in fish species identification. To our knowledge, this is the first report of an extensive genetic population study of E. fuscoguttatus in Malaysia and this understanding will play an important role in informing genetic stock-specific strategies for the management and conservation of this highly valued fish.

MOLECULAR MARKERS FOR PARENTAGE ANALYSIS IN THE GROUPER F1 HYBRID EPINEPHELUS COIOIDES × EPINEPHELUS LANCEOLATUS (ACTINOPTERYGII: PERCIFORMES: SERRANIDAE)

Background. The aquaculture industry is driven by the need to develop novel interspecific hybrids with improved culture characteristics, however currently no molecular markers are available for the validation of F1 hybrids in the case of groupers. The presently reported study was directed toward the development of molecular markers for F1 hybrids obtained by artificial spawning of eggs derived from orange-spotted grouper, Epinephelus coioides (Hamilton, 1822), with sperm from giant grouper, E. lanceolatus (Bloch, 1790). Materials and methods. DNA was extracted and purified from the fin clip of a single F1 hybrid of E. coioides A— E. lanceolatus. Small insert shotgun genomics libraries were constructed, 40 positive transformants were sequenced, and the sequences deposited at the GenBank. Thirty locus-specific primer pairs were designed and tested across 64 F1 hybrid specimens as well as DNA samples extracted from coral grouper, Epinephelus corallicola (Valenciennes, 1828), and brown-marbled grouper, E. fuscoguttatus (ForsskA¥l, 1775). Results. A panel of thirty molecular markers was developed to verify parentage, of these eleven and six of the markers were inherited from the sire and dam respectively. Two markers were specific to the F1 hybrid and none of the markers could be assigned to the closely related groupers E. corallicola and E. fuscoguttatus. Conclusion. The markers developed can be applied to assign parentage and determine the degree of introgression in interspecific grouper hybrids developed from E. coioides and E. lanceolatus. The methods developed in this study can be extended to characterize Quantitative Trait Loci (QTLs) and to identify interspecific F1 hybrids in the wild.

Consolidated Checklist of Hard Corals of the Genus Acropora Oken, 1815 (Scleractinia: Acroporidae) in North Borneo, East Malaysia

Acropora is the most biologically diverse group of reef-building coral, and its richness peaks at the Indo-Malay-Philippine Archipelago, the centre of global coral reef biodiversity. In this paper, we describe the species richness of Acropora fauna of North Borneo, East Malaysia, based on review of literature and as corroborated by voucher specimens. Eighty-three species of Acropora are reported here; four species are literature based and 79 are supported by voucher specimens that were subsequently photographed. New records for North Borneo were recorded for 12 species, including Acropora suharsonoi Wallace 1994 that was previously thought to be confined to a few islands along Lombok Strait, Indonesia. The diversity of Acropora in North Borneo is comparable to that of Indonesia and the Philippines, despite the areas smaller reef areas. This further reinforces its inclusion as part the global hotspot of coral biodiversity.

Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia.

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB) that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF) of a patient diagnosed with tuberculous meningitis (TBM). The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503.

Quantitative real-time PCR for determination of transgene in callus of Jatropha curcas.

Jatropha curcas is an important plant belonging to the family Euphorbiaceae which is a potential candidate for biofuel production. Genetic transformation protocol for J. curcas callus mediated by Agrobacterium tumefaciens were optimized using a pCAMBIA1303 plasmid which carries green fluorescent protein (GFP) gene as a reporter. Results obtained were based on the highest percentage of GFP expression which was observed three days post-transformation. Immersion of callus into 1×105 cfu ml-1 (OD600nm 0.6) of A. tumefaciens LBA4404 with addition of 300 µM of acetosyringone for 45 min, two days of pre-culture and three days of co-cultivation periods were determined to be ideal for J. curcas callus transformation. Putative transformants were selected in the presence of 25 mg/l hygromycin. Surviving calli were transferred into proliferation media (MS with 1 mg/l NAA and 1 mg/l BAP) to proliferate the callus for further molecular analyses and to confirm the presence of the target GFP transgene in the putative transformants. Polymerase chain reaction (PCR) was carried out using a 35S specific primer pair confirmed the presence of the 454 bp of 35S promoter region from the transformed callus. Quantitative real-time PCR (qRT-PCR) was carried out to demonstrate the integration and copy number of the 35S promoter in the putative tranformants. The 35S promoter gene (178 bp) as a target gene and J. curcas actin gene (179 bp) which functions as reference gene was designed to detect the positive transformants and control sample in real-time PCR reaction analysis. The results indicated that the actin specific PCR product was present in both the control and transformed calli, however the 35S PCR product was found only in the positive transformants. The similarity in CT values confirmed that both the genes were present as single copy thus confirming a single integration event.

Genomic Molecular Markers for the DNA Fingerprinting of Sperm Derived from Epinephelus lanceolatus and E. fuscoguttatus

Groupers belong to the subfamily Epinephelinae of the family Serranidae. They are an economically important marine fisheries resource and are commercially cultivated throughout the tropical and temperate regions of the world. The aquaculture industry relies on artificial breeding of groupers in order to obtain fingerlings which are free of pathogens and demonstrate a uniform growth rate. Rapid validation of sperm and eggs is a major challenge to breeders. Single locus DNA markers are ideal for the authentication of germplasm as they generate single PCR amplicons which do not require further sequencing. This study focused on the development of single locus DNA markers for genotyping of sperm samples derived from two species of grouper, the Giant grouper (Epinephelus lanceolatus) and the Tiger grouper (E. fuscoguttatus). Single locus molecular markers were developed using DNA sequences obtained from shotgun genomic libraries and tested against sperm samples derived from each of the species and the closely related groupers E. coioides and E. corallicola. A total of 54 molecular markers were developed of Research Article Annual Research & Review in Biology, 4(1): 93-104, 2014 94 which six were found to be specific to E. fuscoguttatus and seven to E. lanceolatus. The remaining markers generated PCR products in all of the four species and were rejected as suitable candidates for genotyping. The markers developed as a result of this study are relevant to fish breeders and fish farmers as they species specific, inexpensive and augment traditional methods of identification based on phenotypic characterization.

Comparative Genomic Analysis of Bacillus thuringiensis Reveals Molecular Adaptation to Copper Tolerance

Bacillus thuringiensis is a type of Gram positive and rod shaped bacterium that is found in a wide range of habitats. Despite the intensive studies conducted on this bacterium, most of the information available are related to its pathogenic characteristics, with only a limited number of publications mentioning its ability to survive in extreme environments. Recently, a B. thuringiensis MCMY1 strain was successfully isolated from a copper contaminated site in Mamut Copper Mine, Sabah. This study aimed to conduct a comparative genomic analysis by using the genome sequence of MCMY1 strain published in GenBank (PRJNA374601) as a target genome for comparison with other available B. thuringiensis genomes at the GenBank. Whole genome alignment, Fragment all-against-all comparison analysis, phylogenetic reconstruction and specific copper genes comparison were applied to all forty-five B. thuringiensis genomes to reveal the molecular adaptation to copper tolerance. The comparative results indicated that B. thuringiensis MCMY1 strain is closely related to strain Bt407 and strain IS5056. This strain harbors almost all available copper genes annotated from the forty-five B. thuringiensis genomes, except for the gene for Magnesium and cobalt efflux protein (CorC) which plays an indirect role in reducing the oxidative stress that caused by copper and other metal ions. Furthermore, the findings also showed that the Copper resistance gene family, CopABCDZ and its repressor (CsoR) are conserved in almost all sequenced genomes but the presence of the genes for Cytoplasmic copper homeostasis protein (CutC) and CorC across the sample genomes are highly inconsonant. The variation of these genes across the B. thuringiensis genomes suggests that each strain may have adapted to their specific ecological niche. However, further investigations will be need to support this preliminary hypothesis.

Complete mitochondrial genome of six Cheilinus undulatus (Napoleon Wrasse): an endangered marine fish species from Sabah, Malaysia

Abstract We report here the complete mitochondrial (mt) genomes of six individuals of Cheilinus undulatus (Napoleon Wrasse), an endangered marine fish species. The six mt DNA sequences had an average size of 17,000 kb and encoded 22 tRNA, two sRNA, 13 highly conserved protein coding genes and a control region. The polymorphic variation (control region) in these six individuals suggests their potential use as a specific marker for phylogeographic conservation. Moreover, the sequence polymorphism within the control region (D-loop) suggests that this locus can be applied for phylogenetic studies.

Extensive sharing of mitochondrial COI and CYB haplotypes among reef-building staghorn corals (Acropora spp.) in Sabah, North Borneo

Abstract This study is aimed at establishing a baseline on the genetic diversity of the Acropora corals of Sabah, North Borneo based on variations in the partial COI and CYB nucleotide sequences. Comparison across 50 shallow-water Acropora morphospecies indicated that the low substitution rates in the two genes were due to negative selection and that rate heterogeneity between them was asymmetric. CYB appeared to have evolved faster than COI in the Acropora as indicated by differences in the rate of pairwise genetic distance, degrees of transition bias (Ts/Tv), synonymous-to-nonsynonymous rate ratio (dN/dS), and substitution patterns at the three codon positions. Despite the relatively high haplotype diversity (Hd), nucleotide diversity (π) of the haplotype datasets was low due to stringent purifying selection operating on the genes. Subsequently, we identified individual COI and CYB haplotypes that were each extensively shared across sympatrically and allopatrically distributed Indo-Pacific Acropora. These reciprocally common mtDNA types were suspected to be ancestral forms of the genes whereas other haplotypes have mostly evolved from autoapomorphic mutations which have not been fixed within the species even though they are selectively neutral. To our knowledge, this is the first report on DNA barcodes of Acropora species in North Borneo and this understanding will play an important role in the management and conservation of these important reef-building corals.