Clemente Michael Vui Ling Wong

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

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.

Polymerization Degrees, Molecular Weights and Protein-Binding Affinities of Condensed Tannin Fractions from a Leucaena leucocephala Hybrid

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.

Effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on in vitro methane production and rumen fermentation

BACKGROUND Molecular weights (MWs) and their chemical structures are the primary factors determining the influence of condensed tannins (CTs) on animal nutrition and methane (CH4 ) production in ruminants. In this study the MWs of five CT fractions from Leucaena leucocephala hybrid-Rendang (LLR) were determined and the CT fractions were investigated for their effects on CH4 production and rumen fermentation. RESULTS The number-average molecular weight (Mn ) of fraction F1 (1265.8 Da), which was eluted first, was the highest, followed by those of fractions F2 (1028.6 Da), F3 (652.2 Da), F4 (562.2 Da) and F5 (469.6 Da). The total gas (mL g(-1) dry matter (DM)) and CH4 production decreased significantly (P < 0.05) with increasing MWs of the CT fractions, but there were no significant (P > 0.05) differences between the CT fractions and control on DM degradation. However, the in vitro N disappearance decreased significantly (P < 0.05) with the inclusion of CT fraction F1 (highest MW) compared with the control and other fractions (F2-F5). The inclusion of CT fraction F1 also significantly decreased (P < 0.05) total volatile fatty acid and acetic acid concentrations compared with the control. The acetic/propionic acid ratio was significantly decreased (P < 0.05) by fraction F1 but not by the control and other fractions (F2-F5). CONCLUSION The CT fractions of different MWs from LLR could affect rumen fermentation and CH4 production, and the impact was more pronounced for the CT fraction with a higher MW.

Sequence analysis of 16S rRNA gene and 16S–23S rRNA gene intergenic spacer region for differentiation of probioticsLactobacillus strains isolated from the gastrointestinal tract of chicken

Twelve probioticLactobacillus 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.

Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro.

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.

Characterisation of a cryptic plasmid from an Antarctic bacterium Pedobacter cryoconitis strain BG5.

A cryptic plasmid, pMWHK1 recovered from an Antarctic bacterium Pedobacter cryoconitis BG5 was sequenced and characterised. The plasmid is a circular 6206bp molecule with eight putative open reading frames designated as orf1, orf2, orf3, orf4, orf5, orf6, orf7 and orf8. All the putative open reading frames of pMWHK1 are found to be actively transcribed. Proteins encoded by orf2 and orf4 are predicted to be responsible for the mobilization and replication of the plasmid respectively. orf4 shares 55% and 61% identities with the theta-type Rep proteins from two strains of Riemerella anatipestifer. This suggests that pMWHK1 could be a member of the theta-type replicating plasmid. The origin of replication is located within the AT-rich region upstream of orf4. orf5 and orf6 encode bacterial toxin-antitoxin proteins predicted to maintain plasmid stability. orf3 encodes an entry exclusion protein that is hypothetically involved in reducing the frequency of DNA transfer through conjugation. orf1, orf7 and orf8 encode proteins with unknown functions. Plasmid, pMWHK1 is stably maintained in P. cryoconitis BG5 at 20°C.

Isolation and characterization of Antarctic psychrotroph Streptomyces sp. strain INACH3013

Abstract An actinobacterial strain with antimicrobial activity, INACH3013, was isolated from soil collected from Antarctica. The taxonomic status of the isolate was established using a polyphasic approach. The strain was identified as belonging to the genus Streptomyces based on the scanning electron microscopic observation and partial 16S rRNA gene sequence analysis. The sequence analysis revealed that strain INACH3013 is closely related to Streptomyces fildesensis (99.8%), S. beijiangensis (98.1%) and S. purpureus (97.2%). A phylogenetic tree constructed using the partial 16S rRNA gene sequences of strain INACH3013 and closely related strains revealed that INACH3013 fell into the same subclade as S. fildesensis and S. purpureus. Strain INACH3013 was observed to be psychrotolerant, slightly halotolerant (up to 5% NaCl) and capable of inhibiting the growth of seven Gram-negative and eight Gram-positive foodborne pathogens. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of the strain is KJ624755.

Development of a semidefined growth medium for Pedobacter cryoconitis BG5 using statistical experimental design

ABSTRACT Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K2HPO4 along with three additional nutrients (yeast extract, MgSO4, and NH4Cl) were identified to form the basal medium which was further analyzed by Plackett–Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH4Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600 nm) cell density of 7.5 using 23.7 g/L of tryptone, 8.8 g/L of yeast extract, and 0.7 g/L of NH4Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.

Changes in rumen protozoal community by condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid in vitro

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.

The trade-off between antimicrobial production and growth of an Antarctic psychrotroph Streptomyces sp. strain INACH3013

The trade-off between antimicrobial production and growth of an Antarctic psychrotroph Streptomyces sp. strain INACH3013 PARIS LEONARDO LAVIN, SHEAU TING YONG, CLEMENTE MICHAEL V.L. WONG, ALEX RICARDO GONZALEZ PEREZ and CRISTINA DORADOR Laboratorio de Complejidad Microbiana y Ecología Funcional, Instituto Antofagasta, Universidad de Antofagasta, Chile Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia National Antarctic Research Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia Universidad de los Lagos, Region de los Lagos, Chile palavin@gmail.com