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The human movement to and from Antarctica has increased significantly in recent decades, particularly to the South Shetland Islands, King George Island (KGI), and Deception Island (DCI). Such movements may result in unintentional soil transfer to other warmer regions, such as tropical countries. However, the ability of Antarctic bacteria to survive in tropical climates remained unknown. Hence, the objectives of this work were (i) to determine the bacterial diversity of the soils at the study sites on the two islands, and (ii) to determine if simulated tropical-like growth climate conditions would impact overall diversity and increase the abundance of potentially harmful bacteria in the Antarctic soils. KGI and DCI soils were incubated for 12 months under simulated tropical conditions. After 6 and 12-months, samples were collected and subjected to metagenomic DNA extraction, 16S rDNA amplification, sequencing, and alignment analysis. The 12-month denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in fingerprinting patterns and bacterial diversity indices. Following that, bacterial diversity analyses for KGI and DCI soils were undertaken using V3-V4 16S rDNA amplicon sequencing. Major bacterial phyla in KGI and DCI soils comprised Actinobacteria, Proteobacteria, and Verrucomicrobia. Except for Proteobacteria in KGI soils and Acidobacteria and Chloroflexi in DCI soils, most phyla in both soils did not acclimate to simulated tropical conditions. Changes in diversity were also observed at the genus level, with Methylobacterium spp. predominating in both soils after incubation. After the 12-month incubation, the abundance of potentially pathogenic bacteria such as Mycobacterium, Massilia, and Williamsia spp. increased. Overall, there was a loss of bacterial diversity in both Antarctic soils after 12 months, indicating that most bacteria from both islands sampling sites cannot survive well if the soils were accidentally transported into warmer climate

Rice wine is an alcoholic drink produced by fermentation of glutinous rice. It is a famous traditional drink in East Malaysia. The starter culture origins, which consisted of a yeast mixture determines the wine taste and alcohol content percentage. Most of the rice wine producers relied on the yeast starter culture sold in the market or from the leftover stock from the previous rice wine preparation which may not have proper quality control. Very often the content and composition of microbes in the yeast starter culture are unknown. Hence, rice wine produced is not consistent. Sometimes it is tasty and sweet, and sometimes it is sour, and this can be an issue if one wishes to market it as a product. Therefore, there is a need to formulate good quality yeast starter cultures to address the issues of product consistency and product quality. There is a long history of using grape yeasts to ferment grapes for wine production. Nevertheless, information on the fermentation of glutinous rice or starch using grape yeasts is sparse. Hence, the objectives of this project were to isolate yeast present during the grape must spontaneous fermentation, for the formulation of starter cultures. Different types of growth medium such as Yeast Potato Dextrose (YPD) and potato dextrose agar media were used to isolate the yeast. Fifteen yeast isolates, GY1 to GY15 were successfully isolated and purified. The fifteen isolates were combined and freeze-dried to form the starter culture for batch fermentation of glutinous rice. The colony-forming units (cfu) of the starter culture were 1 x105which formed a good starter culture.

Thermophilic microorganisms have always been an important part of the ecosystem, particularly in a hot environment, as they play a key role in nutrient recycling at high temperatures where most microorganisms cannot cope. While most of the thermophiles are archaea, thermophiles can also be found among some species of bacteria. These bacteria are very useful in the fundamental study of heat adaptation, and they are also important as potential sources of thermostable enzymes and metabolites. Recently, we have isolated a Gram-positive thermophilic bacterium, Geobacillus sp. TFV3 from a volcanic soil sample from Deception Island, Antarctica. This project was undertaken to analyze the genes of this thermophilic Antarctic bacterium and to determine the presence of thermal-stress adaptation proteins in its genome. The genome of Geobacillus sp. TFV3 was first purified, sequenced, assembled, and annotated. The complete genome was found to harbor genes encoding for useful thermal-stress adaptation proteins. The majority of these proteins were categorized under the family of molecular chaperone and heat shock protein. This genomic information could eventually provide insights on how the bacterium adapts itself towards high growth temperatures.

Glaciozyma antarctica PI12, a psychrophilic yeast from Antarctica, grows well at low temperatures. However, it is not clear how it responds and adapts to cold and freeze stresses. Hence, this project was set out to determine the cold-adaptation strategies and mechanisms of G. antarctica PI12 using a transcriptomic analysis approach. G. antarctica PI12 cells, grown in rich medium at 12 °C, were exposed to freeze stress at 0 and − 12 °C for 6 h and 24 h. Their transcriptomes were sequenced and analyzed. A hundred and sixty-eight genes were differentially expressed. The yeast gene expression patterns were found to be dependent on the severity of the cold with more genes being differentially expressed at − 12 °C than at 0 °C. Glaciozyma antarctica PI12 was found to share some common adaptation strategies with other yeasts, Saccharomyces cerevisiae and Mrakia spp., but at the same time, found to have some of its own unique strategies and mechanisms. Among the unique mechanisms was the production of antifreeze protein to prevent ice-crystallization inside and outside the cell. In addition, several molecular chaperones, detoxifiers of reactive oxygen species (ROS), and transcription and translation genes were constitutively expressed in G. antarctica PI12 to enable the cells to endure the fluctuating freezing temperatures. Interestingly, G. antarctica PI12 used nitrite as an alternative terminal acceptor of electrons when the oxygen level was low to minimize disruption of energy production in the cell. These mechanisms coupled with several other common mechanisms ensured that G. antarctica PI12 adapted well to the cold temperatures.

Psychrophilic and psychrotrophic bacteria play important roles in nutrient cycling in cold environments. These bacteria are suitable as model organisms for studying cold-adaptation, and sources of cold-active enzymes and metabolites for industrial applications. Here, we report the genome sequences of two Cryobacterium sp. strains SO1 and SO2. Genes coding major proteins related to cold- or thermal-stress adaptations and those with industrial applications found in their genomes are described.

King George Island (KGI) and Deception Island (DCI) are members of the South Shetland Islands in Antarctica, each with their own landscape and local environmental factors. Both sites are suitable for longterm monitoring of bacterial diversity shift due to warming, as temperature rises relatively faster than East Antarctica. This study was conducted to determine and compare the baseline diversity of soil bacteria in KGI and DCI. 16S rDNA amplicons of bacteria from both sites were sequenced using Illumina next generation sequencer. Results showed that major phyla in KGI and DCI were Actinobacteria, Proteobacteria, Chloroflexi, Verrucomicrobia, Bacteriodetes and Acidobacteria. The distribution and evenness of the soil bacterial communities varied at genus level. The genera Sphingomonas sp. was predominant at both sites while the subsequent six major genera differed. Two bacterial genera, Legionella and Clostridium were also found in low abundance in both sites, both of which may contain pathogenic members. Further verification will be required to determine whether the pathogenic members of these genera are present in both sites.

Global warming is the main concern in today’s century as it comes with numerous side effects to the natural environment. Open Top Chambers (OTC) consist of metal constructions with transparent vertical side-walls and a frustum on top. An opening in the middle of the frustum allows an air exchange to reduce temperature and humidity effects in the chamber. The size of the open top chamber which is located in Universiti Putra Malaysia is slanted 60o , 50cm tall, 2.08m basal diameter hexagon chamber. The Open Top Chamber experiments were carried out to determine how much global warming has affected and is still affecting the temperature, pH, the moisture and the growth of the microbes in the tropical soil. The aim of this study is to elucidate the effects of temperature increase on the soil microbes’ population and on the pH of the soil. The study was conducted to observe the effect of heat on the population of soil microbes and the pH of the soil which was collected on the same day for 6 consecutive months. The microbes from the samples were grown on agar plates. The population of microbes on the plates were used as values were for Colony Forming Unit (CFU) value calculations. The effects of OTCs on mean temperature showed a large range of CFU values throughout the 6 months but did not differ significantly between studies. Increases in mean monthly and diurnal temperature were strongly related, indicating that the presence of warming effect by the OTCs. Such predictive power allows a better mechanistic understanding of observed biotic response to experimental warming. This study will be useful for the understanding of the global warming effect on microbes. The Open Top Chamber experiment has proven to be one of the effective model for global warming research and data collected especially on the growth of soil microbial obtained would be of great use for further experiments.

Aims: To evaluate the effects of condensed tannins (CTs) fractions of differing molecular weights (MWs) from a Leucaena leucocephala hybrid-Rendang on the rumen protozoal community in vitro. Methods and Results: The effects of unfractionated CTs (F0) and CT fractions of different MWs (F1 > F2 > F3 > F4 > F5) on protozoal population and community were evaluated in vitro using rumen microbes and ground guinea grass as the substrate. Higher-MW CT fractions F1 and F2 significantly (P < 0·05) decrease the number of ciliate protozoa. The real-time PCR analysis showed that the total protozoa was significantly (P < 0·05) lower in F0 and all CTs with fractions F1 and F2 having the lowest value. High-throughput sequencing of the partial 18S rRNA gene showed that the genus Entodinium significantly (P < 0·05) decreased with increasing MWs of CT, whereas Anoplodinium-Diplodinium were significantly (P < 0·05) increased. Inclusion of the highest MW CT fraction F1 decreased the relative abundance of the minor genera such as Eudiplodinium and Polyplastron compared to the control and CT fractions F2–F5. Conclusion: CTs of differing MWs could reduce and alter the rumen protozoa population in vitro. This effect was more pronounced for higher-MW CTs. Significance and Impact of the Study: The high MW CTs should be considered as a feed supplement in the ruminant diet to reduce the protozoal population which are known to be associated with methanogens as a means to mitigate methane production in the rumen.

In this paper we report the first characterization of cultivable bacteria obtained from the Antarctic sea urchin Sterechinus neumayeri. The coelomic fluid was obtained from a pool of sea urchins which was plated onto different media to isolate the bacteria. A total of 42 isolates of psychrotrophic and aerobic γ-Proteobacteria (59.5%), Flavobacteria (33.3%) and Actinomycetes (7.2%) were isolated and sequenced. These bacteria were exposed to heavy metals and antibiotics, where 38 strains were analysed by the minimal inhibitory concentration method. Antibiotic resistance was detected in 44% of cultivable strains, and a further 13% presented co-resistance to antibiotics and heavy metals. The genera of bacteria that showed an increased resistance and co-resistance to metals and antibiotics were Flavobacterium, Psychrobacter and Pseudomonas. Additionally, 30.9% of isolated bacterial strains contained plasmids, which are probably related to resistance and co-resistance to metals. These results indicate that sea urchin-associated bacteria could be reservoirs for antibiotic resistance genes.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8Da; F2, 1028.6Da; F3, 652.2Da; F4, 562.2Da; F5, 469.6Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P<0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3-F5. Although inclusion of F0 and CT fractions (F1-F5) significantly (P<0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P<0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P<0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1–F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3–F5. Although inclusion of F0 and CT fractions (F1–F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW.

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimized and characterized. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.

Trichoderma species have the potential for application in composting as biological control agents in controlling disease and increasing yield of production in the agricultural industry. The prevalent soil fungus of Trichoderma produces lignocellulolytic enzymes that assist the degradation of woody lignocellulose materials. The aim of the experimental work was to check the potential of lignocellulolytic Trichoderma fungi for the use of rapid composting of oil palm empty fruit bunches fibers. Fifty-two of Trichoderma isolates from Sabah and seven isolates from Antarctic were examined for in-vitro lignocellulolytic activity by assaying their ability to develop dark brown pigments, yellow halo zone, and clear white zone on tannic acid media (TAM) for lignin; Jensen Media (JM) for cellulose; and modified Melin-Nokrans media (MMNM) for starch. The best six Sabah Trichoderma isolates (5D, 10L2, 10P, 5E, 10X, and 10E2) were found to be potential lignocellulolytic agents based on their diameter of halo zone formed on amended media for further testing of in vitro bioconversion of oil palm empty fruit bunches. The diameters of halo zones were measured for the analysis of their ability in degrading lignin, cellulose, and starch. In contrast, Antarctic Trichoderma isolates consistently exhibited lower lignocellulolytic activities based on their smaller diameter of halo zone formed on TAM, JM, and MMNM. Most of the Trichoderma isolates are found to synthesize polyphenol oxidase, endoglucanases, and are able to hydrolyze starch to glucose in the three different media. Thus, the finding shows the potential of these isolates for use in large-scale composting of oil palm empty fruit bunches.

Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1–F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight—from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

Methane is the second most important greenhouse gas that contributes to global warming and climate change. It has a heat trapping potential 23 times that of carbon dioxide. Globally, ruminant livestock produce about 80 million metric tonnes of enteric methane annually, accounting to about 30% of global anthropogenic methane production. Methane produced during enteric fermentation also contributes to a loss of energy of up to 15% for the animal. Thus, mitigation of methane production by ruminants not only reduces greenhouse gas emission but also improves feed efficiency and reduces production cost. There has been considerable interest in use of plant extracts to mitigate enteric ruminal methane emissions. Condensed tannins are secondary plant metabolites that have been considered for mitigating methane production in ruminants, but they may also decrease digestibility of feed in ruminants. Leucaena leucocephala is a tree legume that has been used as a feed supplement for ruminants because of its high protein content. However, it also contains condensed tannins. The L. leucocephala hybrids in Malaysia have been found to have higher condensed tannin contents than the parent L. leucocephala. The effectiveness of condensed tannins from these L. leucocephala hybrids on reduction of methane has not been studied. Thus, this study was conducted to investigate the effects of pure condensed tannins, extracted from young leaves and shoots of L. leucocephala hybrid-Rendang on methane mitigation, rumen fermentation parameters such as pH, dry matter (DM) digestibility, nitrogen degradability and volatile fatty acids production, as well as populations of methanogens and protozoa. The “in vitro gas production test” was used in the investigation as it provides data on fermentation parameters of feed with a high correlation to its in vivo base. The results showed that the condensed tannin extract, at a low level of 30 mg/g DM could reduce methane production by 39% as compared to the co

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.

This report describes the isolation and characterization of bacterial isolates that produce anti-microbial compounds from one of the South Shetland Islands, King George Island, Antarctica. Of a total 2465 bacterial isolates recovered from the soil samples, six (BG5, MTC3, WEK1, WEA1, MA2 and CG21) demonstrated inhibitory effects on the growth of one or more Gram-negative or Gram-positive indicator foodborne pathogens (i.e. Escherichia coli 0157: H7, Salmonella spp., Klebsiella pneumoniae, Enterobacter cloacae, Vibrio parahaemolyticus and Bacillus cereus). Upon examination of their 16S rRNA sequences and biochemical profiles, the six Antarctic bacterial isolates were identified as Gram-negative Pedobacter cryoconitis (BG5), Pseudomonas migulae (WEK1), P. corrugata (WEA1) and Pseudomonas spp. (MTC3, MA2, and CG21). While inhibitors produced by strains BG5, MTC3 and CG21 were sensitive to protease treatment, those produced by strains WEK1, WEA1, and MA2 were insensitive to catalase, lipase, alpha-amylase, and protease enzymes. In addtion, the six Antarctic bacterial isolates appeared to be resistant to multiple antibiotics.

Basal stem rot (BSR) of oil palm (Elaeis guineensis) is caused by Ganoderma boninense, and, commercially, is one of the most devastating diseases in South East Asia. Losses of more than 80% of stands by the time they are halfway through their normal economic life have been reported. High incidence of BSR results in economic losses due to zero yields from dead palms and significantly reduced weight and number of fruit bunches in infected but living palms. Due to the importance of oil palm industry to Malaysia's economy, the transfer of any materials that are related to Ganoderma is strictly prohibited from Peninsular Malaysia to Sabah and Sarawak. No basic identification method using molecular techniques for the identification of G. boninense exist in this study for G. boninense isolates from Sabah. The only report on this pathogens isolate in Sabah was based on their morphology and pathogencity. In conjunction with the morphological similarities among the different isolates, there are numerous opinions on the aggressiveness of the pathogen in Sabah. The isolates of G. boninense from Sabah were claimed to be less aggressive compared to those from Peninsular Malaysia. This may be due to lower incidence of BSR in Sabah and Sarawak compared to Peninsular Malaysia. Since the oil palm industry is a fast income-generating tool, data related to it may be highly sensitive. Researchers, plantation managers and entrepreneurs are not forthcoming with the information on oil palm genetic materials or isolates of G. boninense with others. The current speculations on the Sabah isolates need further investigation. In this study, we report the identity of isolates Ganoderma from Langkon Oil Palm Estate in Sabah, Malaysia. The identity of these isolates was confirmed using DNA sequence analysis after PCR amplification. The latter method shows that the Sabah isolates were very similar to aggressive G. boninense strains FA5017 or FA5035 from West Malaysia, with a maximum similarity of 9

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.

There is little information on the bacterial diversity of the Fildes Peninsula, King George Island. Hence, this study was conducted to determine the bacterial population of sediments and soils from the lakes, river, glacier and an abandoned oil tank area in the Fildes Peninsula, using a metagenomic approach. DNA was extracted from the sediment and soil samples, and analyzed using the 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 299 DNA fragments resolved using the DGGE were sequenced. The results of the analysis provided an overview of the predominant groups of bacteria and the diversity of the bacterial communities. The most abundant phyla of bacteria in Fildes Peninsula were Bacteroidetes, Proteobacteria, Acidobacteria, Gemmatimonadetes, Nitrospira, Firmicutes, Actinobacteria, Chloroflexi, Cyanobacteria, Spirochaetes, Deinococcus-Thermus, WS3 and BRC1. All of the sediment samples from the lakes had different representatives of dominant bacterial species. Interestingly, 15% of the operational taxonomic units (OTUs) did not group into any of the existing phyla in the Ribosomal Database Project (RDP). One of the OTUs had a similarity of <0.90 when compared to the GenBank sequences and probably was a novel bacterium specific to that location. The majority of the bacterial 16S rDNA sequences were found to be closely related to those found elsewhere. © 2010 Elsevier B.V.

BACKGROUND: Bile salt deconjugation by Lactobacillus strains is often closely linked to bile tolerance and survival of the strains in the gut and lowering of cholesterol in the host. The present study investigated the deconjugation of bile salts and removal of cholesterol by 12 Lactobacillus strains in vitro. The 12 strains were previously isolated from the gastrointestineal tract of chickens. RESULTS: The 12 Lactobacillus strains could deconjugate sodium glycocholate (GCA, 16.87-100%) and sodium taurocholate (TCA, 1.69-57.43%) bile salts to varying degrees, with all strains except L. salivarius I 24 having a higher affinity for GCA. The 12 Lactobacillus strains also showed significant (P < 0.05) differences in their ability to remove cholesterol from the growth medium (26.74-85.41%). Significant (P < 0.05) correlations were observed between cholesterol removal and deconjugation of TCA (r = 0.83) among the L. reuteri strains (C1, C10 and C16) and between cholesterol removal and deconjugation of TCA (r = 0.38) and GCA (r = 0.70) among the L. brevis strains (I 12, I 23, I 25, I 211 and I 218). In contrast, although L. gallinarum I 16 and I 26 and L. panis C 17 showed high deconjugating activity, there was no correlation between cholesterol removal and deconjugation of bile salts in these strains. CONCLUSION: The results showed that the 12 Lactobacillus strains were able to deconjugate bile salts and remove cholesterol in vitro, but not all strains with high deconjugating activity removed cholesterol effectively. © 2009 Society of Chemical Industry.

Sabah with its mega-biodiversity is believed to harbour various chitinolytic microorganisms which exhibit optimal chitinase activities at the local ambiance. Bacterial strain BRI 1 was recovered from mangrove soil in Kota Belud, Sabah using Chitinase Detection Agar, pH6.5. The strain BRI 1 was placed under the genus Streptomyces as its physical morphology showed typical streptomycete appearance on solid medium. This was further supported with the analysis of its amplified ~1.5kb 16S rDNA fragment in which it showed close relation to Streptomyces sp.Amplification of family 18 chitinase gene generated an amplicon of 397 bp. Similarly, amplification of family 19 chitinase gene resulted in 342 bp amplicon. Chitinase identity of both amplicons were confirmed in which they showed similarity to chitinase genes from Streptomyces sp.. Crude chitinase activity of BRI 1 showed 8.61 Unit, a three-fold higher than the activity exhibited by Streptomyces griseus which only showed 2.54 U in a triplicate assay using chitin azure as enzymatic substrate. The data resulted in this report serve as a platform for further investigations involving characterization of the chitinases and manipulation of the chitinase genes.

BACKGROUND: Bile salt deconjugation by Lactobacillus strains is often closely linked to bile tolerance and survival of the strains in the gut and lowering of cholesterol in the host. The present study investigated the deconjugation of bile salts and removal of cholesterol by 12 Lactobacillus strains in vitro. The 12 strains were previously isolated from the gastrointestinal tract of chickens. RESULTS: The 12 Lactobacillus strains could deconjugate sodium glycocholate (GCA, 16.87-100%) and sodium taurocholate (TCA, 1.69-57.43%) bile salts to varying degrees, with all strains except L. salivarius I 24 having a higher affinity for GCA. The 12 Lactobacillus strains also showed significant (P < 0.05) differences in their ability to remove cholesterol from the growth medium (26.74-85.41%). Significant (P < 0.05) correlations were observed between cholesterol removal and deconjugation of TCA (r = 0.83) among the L. reuteri strains (C1, C10 and C16) and between cholesterol removal and deconjugation of TCA (r = 0.38) and GCA (r = 0.70) among the L. brevis strains (I 12, I 23, I 25, I 211 and I 218). In contrast, although L. gallinarum I 16 and I 26 and L. panis C 17 showed high deconjugating activity, there was no correlation between cholesterol removal and deconjugation of bile salts in these strains. CONCLUSION: The results showed that the 12 Lactobacillus strains were able to deconjugate bile salts and remove cholesterol in vitro, but not all strains with high deconjugating activity removed cholesterol effectively.

The ability to detect the presence of transgenes in crop-derived foods depends on the quantity and quality of DNA obtained from a product to be analyzed. The efficiency of DNA extraction protocols differs due to the nature of each food product. In this paper, we described two main DNA extraction protocols and their modifications that have been applied and evaluated for DNA extraction from raw and processed food as well as animal feed. The yield and quality for five categories of food and feed samples namely, raw soybean, raw maize, animal feed, smooth tofu and soymilk are discussed. The statistical interaction analyses showed that the cetyltrimethyl ammonium bromide (CTAB) method was proven to be the best method to extract DNA from raw soybean, maize and animal feed samples which not only obtained high DNA yield of 32.7, 28.4 and 33.4 ng DNA/mg sample respectively, but also produced high quality DNA with the absorbance A260/A280 ratio of 1.9, 1.9 and 2.0, respectively. These DNA were suitable for PCR amplification which produced a 164 bp DNA fragment of the lectin gene from soybean, and a 277 bp DNA fragment of the zein gene from maize. In the processed food category, the Wizard isolation method was found to be the best for the extraction of DNA from smooth tofu and soymilk with the yield of 13.2 and 3.4 ng DNA/mg sample, and the quality of the DNA at the absorbance A260/A280 ratio ranged from 1.9 to 1.7. These DNA were successfully amplified using primers specific to the lectin gene of soybean. © All Right Reserved.

BACKGROUND: The use of antibiotic growth promoters in animal feeds is not approved for laying hens in many countries, and economically feasible biological measures which include probiotics are developed to improve hen performance. The present study investigated the effects of probiotics on hen performance for a 48-week period and the cholesterol, total lipid and fatty acid composition of egg yolk at 24, 28 and 32 weeks of age. RESULTS: Egg weight of Lactobacillus culture (LC)-fed hens was significantly (P < 0.05) greater than that of control hens throughout the laying period. From 20 to 44 weeks of age, LC-fed hens produced a significantly (P < 0.05) lower percentage of small eggs and a higher percentage of large eggs, and from 45 to 68 weeks of age a significantly (P < 0.05) lower percentage of medium eggs and a higher percentage of large and extra-large eggs than control hens. Significantly (P < 0.05) less cholesterol was found in egg yolks of hens fed LC at 24 and 28 weeks of age, but not at 32 weeks of age. The total lipid content and the fatty acid composition of egg yolks were similar between the treatments at 24, 28 and 32 weeks of age, except for stearic acid (C18:0), which was significantly reduced in the egg yolk of LC-fed hens at 28-32 weeks of age. CONCLUSION: The greatest benefit of LC was in increasing egg weight and improving egg size by influencing a shift from small and medium to large and extra-large eggs. © 2008 Society of Chemical Industry.

The ability to detect the presence of transgenes in crop-derived foods depends on the quantity and quality of DNA obtained from a product to be analyzed. The efficiency of DNA extraction protocols differs due to the nature of each food product. In this paper, we described two main DNA extraction protocols and their modifications that have been applied and evaluated for DNA extraction from raw and processed food as well as animal feed. The yield and quality for five categories of food and feed samples namely, raw soybean, raw maize, animal feed, smooth tofu and soymilk are discussed. The statistical interaction analyses showed that the cetyltrimethyl ammonium bromide (CTAB) method was proven to be the best method to extract DNA from raw soybean, maize and animal feed samples which not only obtained high DNA yield of 32.7, 28.4 and 33.4 ng DNA/mg sample respectively, but also produced high quality DNA with the absorbance A260/A280 ratio of 1.9, 1.9 and 2.0, respectively. These DNA were suitable for PCR amplification which produced a 164 bp DNA fragment of the lectin gene from soybean, and a 277 bp DNA fragment of the zein gene from maize. In the processed food category, the Wizard isolation method was found to be the best for the extraction of DNA from smooth tofu and soymilk with the yield of 13.2 and 3.4 ng DNA/mg sample, and the quality of the DNA at the absorbance A260/A280 ratio ranged from 1.9 to 1.7. These DNA were successfully amplified using primers specific to the lectin gene of soybean.

Twelve probiotic Lactobacillus strains which were previously identified with classical biochemical tests were re-identified using molecular methods. Comparative sequence analyses of the 16S rRNA gene and 16S-23S rRNA gene intergenic spacer region (ISR) were applied. Results of the study showed that mis-identification at species level occurred at high rate when classical biochemical tests were used. Nine of the strains showed discrepancy in their identity. These nine strains which were previously identified through biochemical tests as L. brevis C1, L. brevis C10, L. fermentum C16, L. brevis C17, L. crispatus I12, L. acidophilus I16, L. fermentum I24, L. fermentum I25 and L. acidophilus I26 were re-identified as L. reuteri C1, L. reuteri C10, L. reuteri C16, L. panis C17, L. brevis I12, L. gallinarum I16, L. salivarius I24, L. brevis I25 and L. gallinarum I26, respectively, using 16S rRNA gene and 16S-23S rRNA gene ISR analysis. Lactobacillus strains I16 and I26 initially could not be classified into a single taxon by 16S rRNA gene sequencing but the identities of these two strains were eventually resolved by 16S-23S rRNA gene ISR sequence analysis as L. gallinarum. Sequence analysis of 16S rRNA gene in complementary with 16S-23S rRNA gene ISR could be potentially useful for rapid and reliable identification of bacteria.

A feeding experiment was carried out for 42 days to evaluate the effects of a mixture of 12 Lactobacillus cultures (LC) or oxytetracycline (OTC) on growth performance and serum lipids of broiler chicks (Hubbard). Two hundred and seventy 1-day-old chicks were assigned randomly to three dietary treatments. The treatments were (I) a basal diet (control) (ii) basal diet+1 g kg-1 LC and (iii) basal diet+50 mg kg-1 OTC. Each dietary treatment had six replicate cages with 15 chicks per cage. Body weights and feed to gain ratios of broilers were determined at 21 and 42 days of age while serum lipids were determined at 42 days of age. From 1 to 42 days of age, broilers fed LC or OTC had significantly (p<0.05) better growth than the control broilers. The feed conversion ratios were improved significantly (p<0.05) during the growing (1 to 21 days of age) and finishing (22 to 42 days of age) periods in broilers fed LC or OTC, but the best feed to gain ratio was obtained in the LC-fed broilers. Serum total cholesterol was significantly (p<0.05) reduced in broilers supplemented with LC as compared to broilers receiving OTC or control diet. Low density lipoprotein cholesterol and triglycerides were significantly (p<0.05) lower in broilers fed LC but significantly (p<0.05) higher in broilers fed OTC when compared to the control broilers. The results indicated that LC had a hypocholesterolaemic effect on broilers in contrast to OTC. © Asian Network for Scientific Information, 2008.

Food labeling in accordance with Novel Food Regulation has been enforced in the European Community since 1997 with a series of updated legislations namely, EC/258/97, EC/1139/98, EC/49/2000, EC/50/2000 and EC/1829/2003. Guidelines and labeling regulations for the use of GMOs materials in food and feed products has also been introduced in Malaysia and Vietnam. Therefore, the demand for the establishment and development of a robust and rapid operation procedure for GMO detection has increased recently in both countries. The procedure of GMO detection emphasizes not only on detection tests but also on confirmation assays. This study employed PCR technology for detection and direct DNA sequencing for confirmation procedures respectively. The results demonstrated for the first time the presence of GM plants with glyphosate-resistant trait led by the control of P35S promoter and NOS terminator in either Malaysian or Vietnamese feed with high frequency (20 positive samples out of 24 analyzed samples). The P35S promoter, EPSPS gene and NOS terminator sequences obtained showed some mutations on single-stranded and double-stranded targeted sequences caused by single nucleotide insertion or single nucleotide changes. These results reinforce the need for development of detection procedures to comply with food/feed labeling system.

Twelve probiotic Lactobacillus strains which were previously identified with classical biochemical tests were re-identified using molecular methods. Comparative sequence analyses of the 16S rRNA gene and 16S–23S rRNA gene intergenic spacer region (ISR) were applied. Results of the study showed that mis-identification at species level occurred at high rate when classical biochemical tests were used. Nine of the strains showed discrepancy in their identity. These nine strains which were previously identified through biochemical tests asL. brevis C1,L. brevis C10,L. fermentum C16,L. brevis C17,L. crispatus I12,L. acidophilus I16,L. fermentum I24,L. fermentum I25 andL. acidophilus I26 were re-identified asL. reuteri C1,L. reuteri C10,L. reuteri C16,L. panis C17,L. brevis I12,L. gallinarum I16,L. salivarius I24,L. brevis I25 andL. gallinarum I26, respectively, using 16S rRNA gene and 16S–23S rRNA gene ISR analysis. Lactobacillus strains I16 and I26 initially could not be classified into a single taxon by 16S rRNA gene sequencing but the identities of these two strains were eventually resolved by 16S–23S rRNA gene ISR sequence analysis asL. gallinarum. Sequence analysis of 16S rRNA gene in complementary with 16S–23S rRNA gene ISR could be potentially useful for rapid and reliable identification of bacteria.

Food labeling in accordance with Novel Food Regulation has been enforced in the European Community since 1997 with a series of updated legislations namely, EC/258/97, EC/1139/98, EC/49/2000, EC/50/2000 and EC/1829/2003. Guidelines and labeling regulations for the use of GMOs materials in food and feed products has also been introduced in Malaysia and Vietnam. Therefore, the demand for the establishment and development of a robust and rapid operation procedure for GMO detection has increased recently in both countries. The procedure of GMO detection emphasizes not only on detection tests but also on confirmation assays. This study employed PCR technology for detection and direct DNA sequencing for confirmation procedures respectively. The results demonstrated for the first time the presence of GM plants with glyphosate-resistant trait led by the control of P35S promoter and NOS terminator in either Malaysian or Vietnamese feed with high frequency (20 positive samples out of 24 analyzed samples). The P35S promoter, EPSPS gene and NOS terminator sequences obtained showed some mutations on single-stranded and double-stranded targeted sequences caused by single nucleotide insertion or single nucleotide changes. These results reinforce the need for development of detection procedures to comply with food/feed labeling system.

Salinity reduces plant growth and crop production globally. The discovery of genes in salinity tolerant plants will provide the basis for effective genetic engineering strategies, leading to greater stress tolerance in economically important crops. In this study, we have identified and isolated 107 salinity tolerant candidate genes from a mangrove plant, Acanthus ebracteatus Vahl by using bacterial functional assay. Sequence analysis of these putative salinity tolerant cDNA candidates revealed that 65% of them have not been reported to be stress related and may have great potential for the elucidation of unique salinity tolerant mechanisms in mangrove. Among the genes identified were also genes that had previously been linked to stress response including salinity tolerance, verifying the reliability of this method in isolating salinity tolerant genes by using E. coli as a host. © 2007 Springer-Verlag.

In this study, we have identified and isolated 126 salinity tolerant cDNAs from the root of a mangrove plant, Bruguiera cylindrica (L.) Blume by using suppression subtractive hybridization (SSH) and bacterial functional screening. Sequencing of 51 subtracted cDNA clones that were differentially expressed in the root of B. cylindrica exposed to 20 parts per thousand (ppt) NaCl water revealed 10 tentative unique genes (TUGs) with putative functions in protein synthesis, storage and destination, metabolism, intracellular trafficking and other functions; and 9 unknown proteins. Meanwhile, the 75 cDNA sequences of B. cylindrica that conferred salinity tolerance to Escherichia coli consisted of 29 TUGs with putative functions in transportation, metabolism and other functions; and 33 with unknown functions. Both approaches yielded 42 unique sequencess that have not been reported else where to be stress related and might provide further understanding of adaptations of this plant to salinity stress.

Salinity is a major abiotic stress that greatly affects plant growth and crop production. Sodium ions in saline soil are toxic to plants because of their adverse effects on potassium nutrition, cytosolic enzyme Activities, photosynthesis, and metabolism. It is important to identify genes involved in salinity tolerance from mangrove plants that survive under saline conditions. In this study, a total of 864 randomly selected cDNA clones were isolated and sequenced from the primary cDNA library of Acanthusebracteatus. Among the 521 readable sequences, 138 of them were assembled into 43 contigs, whereas 383 were singletons. Sequence analyses demonstrated that 349 of these expressed sequence tags showed significant homology to functional proteins, of which 18% are particularly interesting as they correspond to genes involved in stress response. Some of these clones, including putative mannitol dehydrogenase, plastidic aldolase, secretory peroxidase, ascorbate peroxidase, and vacuolar H+-ATPase, may be related to osmotic homeostasis, ionic homeostasis, and detoxification.

An experiment was conducted to study the effects of feed supplementation with a mixture of Lactobacillus cultures ( LC) on cholesterol, fat and fatty acid composition in the liver, muscle and carcass of broiler chickens. One hundred and thirty- six, one- day- old male broiler chicks ( Avian- 43) were assigned randomly to two dietary treatments: ( i) a basal diet ( control), and ( ii) a basal diet + 0.1% LC. The cholesterol contents of the carcass and liver but not the muscle, were significantly ( P < 0.05) lower in LC- fed broilers. The fat contents of the liver, muscle and carcass were also significantly ( P < 0.05) lower in the LC- fed broilers when compared to the control broilers. Supplementation of LC in the broiler diets significantly ( P < 0.05) reduced the oleic acid ( C-18: 1) levels of the liver, muscle and carcass but the arachidonic acid ( C-20:4) level was significantly ( P < 0.05) increased in the liver only. Supplementation of LC also increased the total polyunsaturated fatty acids ( PUFA) in the liver. The results of the present study indicate that LC reduces the fat content of the liver, muscle and carcass of broiler chickens, but it has very little potential to modify the fatty acid composition.