Mining the genome of Bacillus velezensis FS26 for probiotic markers and secondary metabolites with antimicrobial properties against aquaculture pathogens.

Bacillus velezensis FS26 is a bacterium from the genus Bacillus that has been proven as a potential probiotic in aquaculture with a good antagonistic effect on Aeromonas spp. and Vibrio spp. Whole-genome sequencing (WGS) allows a comprehensive and in-depth analysis at the molecular level, and it is becoming an increasingly significant technique in aquaculture research. Although numerous probiotic genomes have been sequenced and investigated recently, there are minimal data on in silico analysis of B. velezensis as a probiotic bacterium isolated from aquaculture sources. Thus, this study aims to analyse the general genome characteristics and probiotic markers from the B. velezensis FS26 genome with secondary metabolites predicted against aquaculture pathogens. The B. velezensis FS26 genome (GenBank Accession: JAOPEO000000000) assembly proved to be of high quality, with eight contigs containing 3,926,371 bp and an average G + C content of 46.5%. According to antiSMASH analysis, five clusters of secondary metabolites from the B. velezensis FS26 genome showed 100% similarity. These clusters include Cluster 2 (bacilysin), Cluster 6 (bacillibactin), Cluster 7 (fengycin), Cluster 8 (bacillaene), and Cluster 9 (macrolactin H), which signify promising antibacterial, antifungal, and anticyanobacterial agents against pathogens in aquaculture. The probiotic markers of B. velezensis FS26 genome for adhesion capability in the hosts' intestine, as well as the acid and bile salt-tolerant genes, were also detected through the Prokaryotic Genome Annotation System (Prokka) annotation pipeline. These results are in agreement with our previous in vitro data, suggesting that the in silico investigation facilitates establishing B. velezensis FS26 as a beneficial probiotic for use in aquaculture.

Application of High-Throughput Sequencing (HTS) to Enhance the Well-Being of an Endangered Species (Malayan Tapir): Characterization of Gut Microbiome Using MG-RAST.

The Tapirus indicus, also known as Malayan tapir, has been listed as a rapidly declining animal species in the past decades, along with being declared and categorized as an endangered species by the International Union for Conservation of Nature (IUCN) 2016. This tapir species is geographically distributed across several countries in Southeast Asia such as Peninsular Malaysia, Indonesia (Sumatra), South Thailand, and Myanmar. Amongst these countries, the Peninsula Malaysia forest is recorded to contain the highest number of Malayan tapir population. Unfortunately, in the past decades, the population of Malayan tapirs has declined swiftly due to serious deforestation, habitat fragmentation, and heavy vehicle accidents during road crossings at forest routes. Concerned by this predicament, the Department of Wildlife and National Parks (DWNP) Peninsular Malaysia collaborated with a few local universities to conduct various studies aimed at increasing the population number of tapirs in Malaysia. Several studies were conducted with the aim of enhancing the well-being of tapirs in captivity. Veterinarians face problems when it comes to selecting healthy and suitable tapirs for breeding programs at conservation centers. Conventional molecular methods using high-throughput sequencing provides a solution in determining the health condition of Malayan tapirs using the Next-Generation Sequencing (NGS) technology. Unaware by most, gut microbiome plays an important role in determining the health condition of an organism by various aspects: (1) digestion control; (2) benefiting the immune system; and (3) playing a role as a "second brain." Commensal gut bacterial communities (microbiomes) are predicted to influence organism health and disease. Imbalance of unhealthy and healthy microbes in the gut may contribute to weight gain, high blood sugar, high cholesterol, and other disorders. In infancy, neonatal gut microbiomes are colonized with maternal and environmental flora, and mature toward a stable composition in two to three years. Interactions between the microorganism communities and the host allow for the establishment of microbiological roles. Identifying the core microbiome(s) are essential in the prediction of diseases and changes in environmental behavior of microorganisms. The dataset of 16S rRNA amplicon sequencing of Malayan tapir was deposited in the MG-RAST portal. Parameters such as quality control, taxonomic prediction (unknown and predicted), diversity (rarefaction), and diversity (alpha) were analyzed using sequencing approaches (Amplicon sequencing). Comparisons of parameters, according to the type of sequencing, showed significant differences, except for the prediction variable. In the Amplicon sequencing datasets, the parameters Rarefaction and Unknown had the highest correlation, while Alpha and Predicted had the lowest. Firmicutes, Bacteroidetes, Proteobacteria, Bacilli, and Bacteroidia were the most representative genera in Malayan tapir amplicon sequences, which indicated that most of the tapirs were healthy. However, continuous assessment to maintain the well-being of tapir for long term is still required. This chapter focuses on the introduction of 16S rRNA amplicon metagenomics in analyzing Malayan tapir gut microbiome dataset.

Application of Index of Atmospheric Purity (IAP) along elevation gradients in Gunung Jerai, Kedah, Malaysia.

The utilisation of biological organisms, especially lichens in the environmental biomonitoring approach, has been proven to be an effective and low-cost technique suitable for developing countries like Malaysia. Index of Atmospheric Purity (IAP) tracked compositional changes in lichen communities which correlate with changes in levels of atmospheric pollution. Gunung Jerai was formed during the Cambrian Period; thus, it is a biodiversity hotspot ideal for a diverse range of lichens. In the present work, a total of 44 corticolous lichen species were sampled and identified to evaluate the pollution status of Gunung Jerai using IAP, starting from 80 to 1200 m with 300 m intervals. The samples were collected within 10 × 50 cm sampling grids attached to 60 trees, bringing a total of 120 000 cm2 of the sampling area. The air quality of the sampling area was determined by IAP score, a low score indicated by high levels of pollution. Results showed that the lowest IAP score was recorded at 300 m; meanwhile, the highest IAP score was recorded at 900 m elevation. Elevational gradient and pollution have a significant effect on the IAP score of Gunung Jerai. On average, Gunung Jerai is indicated as having a low pollution status. However, several elevations of the rainforest showed high and moderate pollution status. The IAP method is best to assess environmental pollution and provide quicker results than chemical monitoring methods. Further research could be done to evaluate the other sampling sites adjacent to other areas of Gunung Jerai.

Use of Ulva reticulata as a growth supplement for tomato (Solanum lycopersicum).

Mass proliferation and accumulation of the green macroalga Ulva reticulata are problems in coastal areas and affect other ecosystems, such as those involving seagrasses. In the absence of any intervention, the decomposition of these macroalgae over time can disrupt the balance of recipient ecosystems. Attention has been given to the potential use of U. reticulata as a supplier of nutrients for crop species such as tomatoes as a possible solution to the buildup of this unusable seaweed species, which is usually left to decompose in affected seagrass ecosystems; this is the case in the Merambong seagrass meadow in the Sungai Pulai estuary in Gelang Patah, southwestern Johor, Malaysia. We analyzed the macro- and micronutrient contents in U. reticulata to determine nutrient availability. We also performed greenhouse studies to test the effects of crude extracts from dried U. reticulata-Extract "A" and fresh U. reticulata-Extract "B" on plant growth, total yield, and quality vine-ripened fruits. Compared to other seaweed extracts used as plant growth promoters, U. reticulata extracts have higher nitrogen (N), manganese (Mn), zinc (Zn), and iron (Fe) contents. The application of 30% Extracts "A" and "B" and 50% Extracts "A" and "B" significantly affected tomato plant height. However, extract concentrations that promoted plant height and hastened flowering and fruiting did not increase total fruit yields. Both treatments that positively affected tomato plant height and hastened flowering and fruiting resulted in increased contents of total soluble solids (TSS), beta-carotene, lycopene, ascorbic acid and total titratable acidity (TTA) in the vine-ripened fruits. Agronomically, the application of 5% Extracts "A" and "B", 10%-20% Extracts "A" and "B", and 50% Extract "A" doubled the total yield compared to those of the control, and 40% Extract "A" resulted in the highest total fruit yield. In general, tomato plants responded well to Extract "A" than Extract "B" and presented good total fruit yield and quality.

Evaluation of three Bacillus spp. isolated from the gut of giant freshwater prawn as potential probiotics against pathogens causing Vibriosis and Aeromonosis.

Pathogens from the Vibrio and Aeromonas genera often cause detrimental effects to the aquaculture sector. Previously, antibiotics was used to resolve the infections, but this caused the spread of antibiotic resistant bacteria and antibiotic resistance genes into the environment. As an approach to address this issue, probiotic bacteria were introduced to improve the hosts' microbiome, disease protection, health condition, growth efficiency, feed consumption, stress response and general vigour. However, reports showed that some commercially available probiotics were restricted to a small number of microbial species and there are inconsistencies concerning its effectiveness. Hence, the aim of this study was to isolate and evaluate new Bacillus spp. from the gut of giant freshwater prawn as potential probiotics. Three Bacillus spp. isolates, Bacillus subtilis FS6 (MZ960135), Bacillus velezensis FS26 (MZ960133) and Bacillus pumilus FS97 (MZ960136) were characterised, and in vitro testing showed good probiotic properties which can help in dealing with diseases in aquaculture. Among the Bacillus spp., Bacillus velezensis FS26 showed higher antimicrobial activity towards Aeromonas hydrophila LMG 13658 and Aeromonas veronii clone DK-A. veronii-27 at 23.7 mm and 25 mm, respectively. Bacillus subtilis FS6 and Bacillus velezensis FS26 resulted in good adherence to both xylene and chloroform hydrocarbons. The Bacillus spp. isolated displayed high survivability towards 0.3% bile salt and exhibited amylase, protease, and lipase activities. Thus, the isolated Bacillus spp. are considered safe based on the sensitivity analysis towards antibiotics and γ-haemolytic activity.

Microsatellite-Based Genetic Characterization of the Indigenous Katjang Goat in Peninsular Malaysia.

The Katjang goat is the only indigenous domestic goat breed in Malaysia. Following a national baseline survey from 2001 to 2002, this breed was reported to the FAO as being at risk of extinction. In this study, 36 microsatellite markers were screened, and 25 polymorphic markers were used to analyze the genetic structure of the Katjang goat breed in Peninsular Malaysia. A sample set of data derived from another 10 populations from three published research studies was used as an outgroup for an inter-population genetic study. The analysis showed that the mean value of the observed heterozygosity was 0.29 ± 0.14, and the expected heterozygosity was 0.72 ± 0.14, which indicated low genetic diversity. The inbreeding coefficient, FIS, was high, at 0.46. Significant (p < 0.01) deviations from the Hardy Weinberg equilibrium were noted for all loci. The bottleneck analysis using the Wilcoxon Rank test under the two-phase model of mutation was significant (p < 0.01) for heterozygosity excess, which suggested that the Katjang breed had undergone significant population reduction in the past. Through combined analysis of data from publicly available research, almost the entire population of Katjang goats represent the centroid and are grouped together on a multidimensional scaling plot, except for the Terengganu population. Network analysis revealed that the goat population from Pahang formed the centrality of the network.

A metagenome for lacustrine Cladophora (Cladophorales) reveals remarkable diversity of eukaryotic epibionts and genes relevant to materials cycling.

Periphyton dominated by the cellulose-rich filamentous green alga Cladophora forms conspicuous growths along rocky marine and freshwater shorelines worldwide, providing habitat for diverse epibionts. Bacterial epibionts have been inferred to display diverse functions of biogeochemical significance: N-fixation and other redox reactions, phosphorus accumulation, and organic degradation. Here, we report taxonomic diversity of eukaryotic and prokaryotic epibionts and diversity of genes associated with materials cycling in a Cladophora metagenome sampled from Lake Mendota, Dane Co., WI, USA, during the growing season of 2012. A total of 1,060 distinct 16S, 173 18S, and 351 28S rRNA operational taxonomic units, from which >220 genera or species of bacteria (~60), protists (~80), fungi (6), and microscopic metazoa (~80), were distinguished with the use of reference databases. We inferred the presence of several algal taxa generally associated with marine systems and detected Jaoa, a freshwater periphytic ulvophyte previously thought endemic to China. We identified six distinct nifH gene sequences marking nitrogen fixation, >25 bacterial and eukaryotic cellulases relevant to sedimentary C-cycling and technological applications, and genes encoding enzymes in aerobic and anaerobic pathways for vitamin B12 biosynthesis. These results emphasize the importance of Cladophora in providing habitat for microscopic metazoa, fungi, protists, and bacteria that are often inconspicuous, yet play important roles in ecosystem biogeochemistry.

The Genus Cladophora Kützing (Ulvophyceae) as a Globally Distributed Ecological Engineer.

The green algal genus Cladophora forms conspicuous nearshore populations in marine and freshwaters worldwide, commonly dominating peri-phyton communities. As the result of human activities, including the nutrient pollution of nearshore waters, Cladophora-dominated periphyton can form nuisance blooms. On the other hand, Cladophora has ecological functions that are beneficial, but less well appreciated. For example, Cladophora has previously been characterized as an ecological engineer because its complex structure fosters functional and taxonomic diversity of benthic microfauna. Here, we review classic and recent literature concerning taxonomy, cell biology, morphology, reproductive biology, and ecology of the genus Cladophora, to examine how this alga functions to modify habitats and influence littoral biogeochemistry. We review the evidence that Cladophora supports large, diverse populations of microalgal and bacterial epiphytes that influence the cycling of carbon and other key elements, and that the high production of cellulose and hydrocarbons by Cladophora-dominated periphyton has the potential for diverse technological applications, including wastewater remediation coupled to renewable biofuel production. We postulate that well-known aspects of Cladophora morphology, hydrodynamically stable and perennial holdfasts, distinctively branched architecture, unusually large cell and sporangial size and robust cell wall construction, are major factors contributing to the multiple roles of this organism as an ecological engineer.

The epiphytic microbiota of the globally widespread macroalga Cladophora glomerata (Chlorophyta, Cladophorales).

PREMISE OF THE STUDY: The filamentous chlorophyte Cladophora produces abundant nearshore populations in marine and freshwaters worldwide, often dominating periphyton communities and producing nuisance growths under eutrophic conditions. High surface area and environmental persistence foster such high functional and taxonomic diversity of epiphytic microfauna and microalgae that Cladophora has been labeled an ecological engineer. We tested the hypotheses that (1) Cladophora supports a structurally and functionally diverse epiphytic prokaryotic microbiota that influences materials cycling and (2) mutualistic host-microbe interactions occur. Because previous molecular sequencing-based analyses of the microbiota of C. glomerata found as western Lake Michigan beach drift had identified pathogenic associates such as Escherichia coli, we also asked if actively growing lentic C. glomerata harbors known pathogens. METHODS: We used 16S rRNA gene amplicon pyrosequencing to examine the microbiota of C. glomerata of Lake Mendota, Dane, Wisconsin, United States, during the growing season of 2011, at the genus- or species-level to infer functional phenotypes. We used correlative scanning electron and fluorescence microscopy to describe major prokaryotic morphotypes. KEY RESULTS: We found microscopic evidence for diverse bacterial morphotypes, and molecular evidence for ca. 100 distinct sequence types classifiable to genus at the 80% confidence level or species at the 96-97% level within nine bacterial phyla, but not E. coli or related human pathogens. CONCLUSIONS: We inferred that bacterial epiphytes of lentic C. glomerata have diverse functions in materials cycling, with traits that indicate the occurrence of mutualistic interactions with the algal host.

Freshwater diatoms as a source of lipids for biofuels.

Until recently, biodiesel production has been derived from terrestrial plants such as soybean and canola, leading to competition between biodiesel production and agricultural production for source materials. Microalgae have the potential to synthesize 30 times more oil per hectare than terrestrial plants without competing for agricultural land. We examined four genera (Cyclotella, Aulacoseira, Fragilaria, Synedra) of common freshwater diatoms (Bacillariophyceae) for growth and lipid content in defined medium (sD11) that replicates hypereutrophic conditions in lakes and wastewater treatment plant effluents and optimized the medium for silicon content. Cyclotella and Aulacoseira produced the highest levels of total lipids, 60 and 43 µg total lipids/ml, respectively. Both diatoms are rich in fatty acids C14, C16, C16:1, C16:2,7,10, and C22:5n3. Of the diatoms examined, Cyclotella reached the highest population density (>2.5 × 10(6) cells/ml) in stationary phase when many of the cells appeared to be filled entirely with oil. Silicon enrichment studies indicated that for optimal utilization of phosphorus and nitrogen by diatoms growing in wastewater effluent, the amount of silicon present or added to the effluent should be 17.5 times the mass of phosphorus in the effluent. With high growth rates, high lipid contents, and rapid settling rates, Cyclotella and Aulacoseira are candidates for biodiesel production.

Bacterial production of free fatty acids from freshwater macroalgal cellulose.

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 µM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 µg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.