Cloning and characterization of an acidic lipase from a lipolytic bacterium in tempeh.

BACKGROUND: Lipases have emerged as essential biocatalysts, having the ability to contribute to a wide range of industrial applications. Microbial lipases have garnered significant industrial attention due to their stability, selectivity, and broad substrate specificity. In the previous study, a unique lipolytic bacterium (Micrococcus luteus EMP48-D) was isolated from tempeh. It turns out the bacteria produce an acidic lipase, which is important in biodiesel production. Our main objectives were to clone the acidic lipase and investigate its potential in biodiesel production. RESULT: In this study, the gene encoding a lipase from M. luteus EMP48-D was cloned and expressed heterologously in Escherichia coli. To our knowledge, this is the first attempt at the cloning and expression of the lipase gene from Micrococcus luteus. The amino acid sequence was deduced from the nucleotide sequence (1356 bp) corresponded to a protein of 451 amino acid residues with a molecular weight of about 40 kDa. The presence of a signal peptide suggested that the protein was extracellular. A sequence analysis revealed that the protein had a lipase-specific Gly-X-Ser-X-Gly motif. The enzyme was identified as an acidic lipase with a pH preference of 5.0. Fatty acid preferences for enzyme activities were C8 and C12 (p-nitrophenyl esters), with optimum temperatures at 30-40 °C and still remaining active at 80°C. The enzyme was also shown to convert up to 70% of the substrate into fatty acid methyl ester. CONCLUSION: The enzyme was a novel acidic lipase that demonstrated both hydrolytic and transesterification reactions. It appeared particularly promising for the synthesis of biodiesel as this enzyme's catalytic reaction was optimum at low temperatures and was still active at high temperatures.

Evaluation of Caffeine Ingested Timing on Endurance Performance based on CYP1A2 rs762551 Profiling in Healthy Sedentary Young Adults.

Background: Caffeine is generally suggested to increase VO2max in endurance performance. Nevertheless, the response to caffeine ingestion does not seem to be uniform across individuals. Therefore, caffeine ingested timing on endurance performance based on the type of CYP1A2 single nucleotide polymorphism rs762551, that were classified as fast and slow metabolizers, need to be evaluated. Methods: Thirty participants participated in this study. DNA was obtained from saliva samples and genotyped using polymerase chain reaction-restriction fragment length polymorphism. Each respondent completed beep tests under three treatments blindly: placebo, 4 mg/kg body mass of caffeine one hour, and two hours before test. Results: Caffeine increased estimated VO2max in fast metabolizers (caffeine=29.39±4.79, placebo=27.33±4.02, p<0.05) and slow metabolizers (caffeine=31.25±6.19, placebo=29.17±5.32, p<0.05) in one hour before test. Caffeine also increased estimated VO2max in fast metabolizers (caffeine=28.91±4.65, placebo=27.33±4.02, p<0.05) and slow metabolizers (caffeine=32.53±6.68, placebo=29.17±5.32, p<0.05) in two hour before test. However, for slow metabolizers, the increasing was greater when caffeine was administered two hours before test (slow=3.37±2.07, fast=1.57±1.62, p<0.05). Conclusion: Genetic variance may affect the optimal caffeine ingestion timing, sedentary individuals who want to enhance their endurance performance may ingest caffeine 1 hour before exercise for fast metabolizers and 2 hours before exercise for slow metabolizers.

A New Approach for Controlling Agrobacterium tumefaciens Post Transformation Using Lytic Bacteriophage.

Overgrowth of Agrobacterium tumefaciens has frequently been found in Agrobacterium-mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate Agrobacterium after transformation using a bacteriophage. To develop this alternative method, we conducted effectiveness studies of two lytic bacteriophages (ΦK2 and ΦK4) and performed an application test to control Agrobacterium growth after transformation. According to plaque morphological characterization and molecular analysis, the two bacteriophages used in this experiment were distinct. Moreover, some stability physicochemical and growth kinetics, such as adsorption time and susceptibility test, also showed that both bacteriophages differed. On the other hand, the optimum temperature and pH of both phages were the same at 28-30 °C and pH 7. Further investigation showed that both ΦK2 and ΦK4 were able to reduce the overgrowth of A. tumefaciens post transformation. Moreover, applying the cocktail (mixture of ΦK2 and ΦK4) with antibiotic application eradicated A. tumefaciens (0% overgrowth percentage). This result indicates that the application of bacteriophage could be used as an alternative way to eradicate the overgrowth of A. tumefaciens subsequent to transformation.

A Chromosome-level Reference Genome of African Oil Palm Provides Insights into Its Divergence and Stress Adaptation.

The palm family (Arecaceae), consisting of ∼ 2600 species, is the third most economically important family of plants. The African oil palm (Elaeis guineensis) is one of the most important palms. However, the genome sequences of palms that are currently available are still limited and fragmented. Here, we report a high-quality chromosome-level reference genome of an oil palm, Dura, assembled by integrating long reads with ∼ 150נgenome coverage. The assembled genome was 1.7 Gb in size, covering 94.5% of the estimated genome, of which 91.6% were assigned into 16 pseudochromosomes and 73.7% were repetitive sequences. Relying on the conserved synteny with oil palm, the existing draft genome sequences of both date palm and coconut were further assembled into chromosomal level. Transposon burst, particularly long terminal repeat retrotransposons, following the last whole-genome duplication, likely explains the genome size variation across palms. Sequence analysis of the VIRESCENS gene in palms suggests that DNA variations in this gene are related to fruit colors. Recent duplications of highly tandemly repeated pathogenesis-related proteins from the same tandem arrays play an important role in defense responses to Ganoderma. Whole-genome resequencing of both ancestral African and introduced oil palms in Southeast Asia reveals that genes under putative selection are notably associated with stress responses, suggesting adaptation to stresses in the new habitat. The genomic resources and insights gained in this study could be exploited for accelerating genetic improvement and understanding the evolution of palms.

Effects of Tempeh Probiotics on Elderly With Cognitive Impairment.

Introduction: Oral consumption of probiotics can alter Gut Microbiota by causing changes in the production of probiotic derivatives. Therefore, by utilizing Gut-Brain-Axis (GBA), probiotics could provide an opportunity for central nervous system (CNS) modulation, including cognitive function. Tempeh is a traditional Indonesian food rich in probiotics and beneficial for cognitive function. However, the type of probiotics that play a role in cognitive improvement and the number of probiotics needed for the benefits of increasing cognitive function was unknown. Method: This experimental study involved a total of 93 subjects, divided into 3 groups: A, B and C/control (n: 33, 32, and 28), who were provided with probiotic supplementation isolated from tempeh for 12 weeks intervention. Inclusion criteria were age > 60 years, and memory impairment with the third repetition value of Word List Memory Immediate Recall (WLMIR) < 7. Subjects with diabetes were excluded. Cognitive function examinations were carried out before and after treatment. The tempeh-derived probiotics were prepared trough several processes. Genomic isolation, detection of GABA-encoding genes, and species identification using the 16S-rRNA gene encoding were performed. Results: The probiotics isolate used in the intervention was identified as Limosilactobacillus fermentum. We assigned this isolate as L. fermentum A2.8. The presence of the gene encoding GABA was found on this isolate. There was an increase in the cognitive domains of memory, learning process, and verbal fluency (p < 0.05) in group A (probiotics at concentration of 108 CFU/mL). Memory function, visuospatial, and verbal fluency improved (p < 0.05) in group B (probiotics at concentration of 107 CFU/mL). Only an increase in the memory domain was observed in the control group. Improvement of the learning process occurred only in group A (p = 0.006). Conclusion: Administration of probiotics derived from L. fermentum A2.8 increased the cognitive domains of memory, language and visuospatial function. However, probiotic supplementation at a concentration of 108 CFU/mL was better in improving the learning process. This study succeeded in detecting Lactic Acid Bacterial isolates L. fermentum A2.8 that enclosed gene encoding glutamate decarboxylase (gad) which is involved in the synthesis of -aminobutyric acid (GABA), a neurotransmitter vital for cognitive function.

Insight into the symbiotic lifestyle of DPANN archaea revealed by cultivation and genome analyses.

Decades of culture-independent analyses have resulted in proposals of many tentative archaeal phyla with no cultivable representative. Members of DPANN (an acronym of the names of the first included phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota), an archaeal superphylum composed of at least 10 of these tentative phyla, are generally considered obligate symbionts dependent on other microorganisms. While many draft/complete genome sequences of DPANN archaea are available and their biological functions have been considerably predicted, only a few examples of their successful laboratory cultivation have been reported, limiting our knowledge of their symbiotic lifestyles. Here, we investigated physiology, morphology, and host specificity of an archaeon of the phylum "Candidatus Micrarchaeota" (ARM-1) belonging to the DPANN superphylum by cultivation. We constructed a stable coculture system composed of ARM-1 and its original host Metallosphaera sp. AS-7 belonging to the order Sulfolobales Further host-switching experiments confirmed that ARM-1 grew on five different archaeal species from three genera-Metallosphaera, Acidianus, and Saccharolobus-originating from geologically distinct hot, acidic environments. The results suggested the existence of DPANN archaea that can grow by relying on a range of hosts. Genomic analyses showed inferred metabolic capabilities, common/unique genetic contents of ARM-1 among cultivated micrarchaeal representatives, and the possibility of horizontal gene transfer between ARM-1 and members of the order Sulfolobales Our report sheds light on the symbiotic lifestyles of DPANN archaea and will contribute to the elucidation of their biological/ecological functions.

Microbiological quality of tempeh with different wraps: banana leaf versus plastic.

Tempeh is a traditional Indonesian fermented food widely consumed and became staple food in some Indonesian diet. Commercially, tempeh is available in banana leaf or plastic wraps. The wraps are not only important for fermentation and giving final form of tempeh,  but also potential source of food microbiome. This study aimed to investigate the effect of different packaging materials on the lactic acid bacteria (LAB) quantity and community composition of tempeh from three manufacturers with different production environments. Each tempeh wrapped with banana leaf or plastic during the fermentation process and then LAB from every tempeh sample were quantified using qPCR and plate count method. Terminal Restriction Fragment Length Polymorphism analysis was carried out to assign bacterial community composition from these samples. The LAB population quantity of fresh tempeh from three manufacturers were essentially similar around 10 log CFU/g. However, there is a difference in the proportion of cultured and uncultured bacteria. Principal Coordinate Analyses based on Bray-Curtis similarity matrices showed an apparent clustering pattern for tempeh samples according to tempeh manufacturers. Meanwhile, packaging materials did not significantly influence changes in  bacterial community composition. The result of this work could be used for determination, authentication, and improvement of tempeh quality.

Antifungal Peptides from a Burkholderia Strain Suppress Basal Stem Rot Disease of Oil Palm.

Basal stem rot (BSR) is the most common disease of oil palm (Elaeis guineensis) in Southeast Asia. BSR is caused by a white-rot fungus Ganoderma boninense. The disease is difficult to manage. Therefore, development of novel and environmentally safe approaches to control the disease is important. Species of Burkholderia are known to have diverse lifestyles, some of which can benefit plants by suppressing diseases or increasing plant growth. In the present study, antifungal peptides produced by a bacterial strain isolated from the rhizosphere of an oil palm tree, Burkholderia sp. strain CP01, exhibited strong growth inhibition on G. boninense. A loss-of-function mutant of CP01 was generated, and it has enabled the identification of a 1.2-kDa peptide and its variants as the active antifungal compounds. High-resolution mass spectrometry revealed six analogous compounds with monoisotopic masses similar to the previously reported cyclic lipopeptides occidiofungin and burkholdine. The antifungal compounds of CP01 were secreted into media, and we sought to use CP01 culture extract without living cells to control BSR disease. Glasshouse experiments showed that CP01 culture extract suppressed BSR disease in oil palm seedlings. The ability of CP01 to produce an antifungal substance and suppress plant disease suggests its potential applications as a biofungicide in agriculture.

Dominant Enterobacteriaceae in tempeh were primarily originated from soybean.

During tempeh production, boiling was considered as heat treatment that could significantly reduce or eliminate bacterial population in soybean before fungal inoculation. The objective of this study was to enumerate and trace Enterobacteriaceae communities in pre-boiling soybean, post-boiling soybean, and fresh tempeh designated as RTI and EMP. Standard plate count and qRT-PCR were employed to determine the culturable and non-culturable bacteria, while Enterobacterial Repetitive Intragenic Consensus PCR was conducted to determine the intraspecies genomic variations. Fresh tempeh from both RTI and EMP contained approximately 107 and 108 CFU/g of Enterobacteriaceae respectively. The number of bacteria in pre-boiling soybean were 10,000 times lower than in fresh tempeh. Our study showed that most Enterobacteriaceae were severely injured or quiescent during boiling process and quickly recovered up to 109 CFU/g in fresh tempeh. Some Klebsiella isolates found in tempeh were genetically identical to isolates in soybean, but different from those of medical isolates. This study suggested that soybean could be the main origin of Klebsiella in fresh tempeh.

Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification.

BACKGROUND: Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. RESULTS: LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0-5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. CONCLUSIONS: This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

Shotgun metagenomic analysis reveals new insights into bacterial community profiles in tempeh.

OBJECTIVE: Amplicon sequencing targeting 16S ribosomal RNA (rRNA) has been widely used to profile the microbial community from fermented food samples. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in estimating microbial community composition. As potential paraprobiotics sources, a comprehensive profiling study of tempeh microbial ecology could contribute to tempeh product development. This study employed a shotgun metagenomic approach, where metagenome fragments from tempeh samples were sequenced directly for taxonomic and functional profiling analysis. RESULTS: Taxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from the shotgun metagenomic analysis in all tempeh samples. In terms of composition, this shotgun metagenomic study revealed that Proteobacteria was the most abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed gene based on this metagenomic analysis. The metagenome-assembled genomes (MAGs) results from the binning pipeline could reveal almost complete whole genome sequence of Lactobacillus fermentum, Enterococcus cecorum, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

Tempeh Consumption and Cognitive Improvement in Mild Cognitive Impairment.

INTRODUCTION: Tempeh consumption has been linked to the improvement of cognitive function in older people. However, to what extent the amount of microorganism or the size of tempeh serving consumed per day influences the benefit to cognitive functions has not yet been studied. METHODS: This experimental study involved a total of 90 respondents, who were divided into 3 groups: group A (consuming 100 g of Tempeh A/day), group B (consuming 100 g of Tempeh B/day), and group C (control). Intervention was given for 6 months. Cognitive assessments were done before and after the intervention. Blood uric acid level was checked at the end of intervention to examine the effect of tempeh consumption on this. The inclusion criteria were respondents aged 60 years or over with mild cognitive impairment (MCI) who agreed not to consume other fermented food during the study period. Respondents with diabetes were excluded. RESULTS: There were 84 subjects at the end of the study, majority being female (71.4%) and aged over 65 years (72.6%). An increase in global cognitive scores was found in both groups A and B. The increase in language domain scores was found only in group A. CONCLUSION: Both Tempeh A or Tempeh B consumption for 6 months appeared to be beneficial in improving global cognitive function of older people with MCI. Consuming Tempeh A, which had a lower number of microorganisms, was also associated with an improvement in the language domain.

SIRT1 as a Potential Biomarker for Obesity.

Asian people generally have thin figures; yet, they often suffer from hidden alarming metabolic conditions due to high visceral fat area (VFA). Therefore, it is crucial to have a biomarker to predict visceral obesity to prevent further complications. SIRT1, a NAD-dependent deacetylase gene, is responsible for upregulating lipolysis genes and is downregulated after acute high-fat meal consumption. However, the chronic exposure effect remains unknown. The purpose of this study was to assess the association between SIRT1 mRNA expression, fat intake, and visceral obesity in Indonesian population. METHODS: This cross-sectional study involved 38 healthy subjects (20-30 y old, not suffering any chronic diseases or fever, not taking any medication or treatment, not smoking, not drinking alcohol frequently, not being pregnant, and not breastfeeding). Dietary patterns from 24-h food recall, physical activities fom international physical activity questionnaire (IPAQ), medical data from annual medical check-up, and body compositions were measured using InBody720 and compared with SIRT1 expression from peripheral blood mononuclear cells (PBMCs) samples. RESULTS: Subjects with excessive percentage of body fat (PBF) had a significantly higher body mass index (BMI) (normal: 20.28±2.09, excessive: 23.86±3.71, p=0.023) and VFA (normal: 48.00±9.38, excessive: 79.17±16.14, p=5×1025). The SIRT1 mRNA expression was significantly higher in subjects with excessive PBF (normal: 1±0.43, excessive: 3.68±2.62, p=0.018) and positively correlated with PBF (ρ=0.376, p=0.045). CONCLUSION: SIRT1 acted as a potential marker for obesity in the evaluated population.

Association of FTO rs9939609 and CD36 rs1761667 with Visceral Obesity.

In 2016, more than 1.9 billion adults were overweight; of which, over 650 million of adults were obese. Genetics and lifestyle play important roles in the development of obesity. Studies have shown that genetic variants contribute in developing obesity; such as FTO and CD36, which regulate metabolism and food preferences. Many researches have also emphasized the importance of lifestyle in obesity prevention. However, the interactions of both factors were still underexplored. Therefore, this study was aimed to assess the interaction between FTO-CD36 variants and fat consumption on the metabolic status of healthy Indonesians. Twenty-one females and seventeen males were involved in this cross-sectional study. CD36 rs1761667 and FTO rs9939609 genotypes were identified from blood samples using PCR-RFLP. Data were compared with dietary patterns (24-h food recall), physical activities (IPAQ), medical records, and body compositions (InBody720). Results: CD36 rs1761667 AA and AG group showed higher -but not significant- fat consumption, WHR, and VFA compared to GG. The trend persisted after gender and physical activity adjustment. Meanwhile, FTO rs9939609 AT group showed significant higher WC, WHR and VFA in male subjects after gender and energy balance adjustment: WC (TT: 74.40±3.85, AT: 85.50±5.92, p=0.011), WHR (TT: 0.85±0.02, AT: 0.92±0.04, p=0.010), and VFA (TT: 48.65±10.61, AT: 78.48±15.18, p=0.010). CD36 rs1761667 might be correlated with higher fat consumption and visceral obesity; while FTO rs9939609 showed a significant association with male visceral obesity. These results indicates that both genetic variants were potential as visceral obesity markers.

Isolation, Cloning and Co-Expression of Lipase and Foldase Genes of Burkholderia territorii GP3 from Mount Papandayan Soil.

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of 80°C at pH 11.0. The optimum substrate for enzyme activity was C10, which is highly stable in methanol solvent. The enzyme was strongly activated by Ca2+, Mg2+, and strongly inhibited by Fe2+ and Zn2+. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

Author Correction: Transcriptome and functional analysis reveals hybrid vigor for oil biosynthesis in oil palm.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has been fixed in the paper.

Cloning and characterization of EgGDSL, a gene associated with oil content in oil palm.

Oil palm (Elaeis guineensis, Jacq.) is a key tropical oil crop, which provides over one third of the global vegetable oil production, but few genes related to oil yield have been characterized. In this study, a GDSL esterase/lipase gene, which was significantly associated with oil content, was isolated from oil palm and designated as EgGDSL. Its functional characterization was carried out through ectopic expression in Arabidopsis ecotype Col-0. It was shown that expression of EgGDSL in Arabidopsis led to the increased total fatty acid content by 9.5% compared with the wild type. Further analysis of the fatty acid composition revealed that stearic acid (18:0) increased in the seeds of the transgenic lines, but the levels of linoleic acid (18:2) plus 11-eicosenoic acid drastically declined. Quantitative real-time PCR (qPCR) revealed that in oil palm, EgGDSL was highly expressed in mesocarp followed by leaf, and the expression level was very low in the root. The expression level of EgGDSL gene began to increase at two months after flowering (MAF) and reached its peak by four MAF, then declined rapidly, and reached its lowest level during the mature period (6 MAF). The EgGDSL gene was more highly expressed in oil palm trees with high oil content than that with low oil content, demonstrating that the transcription level of EgGDSL correlated with the amount of oil accumulation. The gene may be valuable for engineering fatty acid metabolism in crop improvement programmes and for marker-assisted breeding.

Sulfurisphaera javensis sp. nov., a hyperthermophilic and acidophilic archaeon isolated from Indonesian hot spring, and reclassification of Sulfolobus tokodaii Suzuki et al. 2002 as Sulfurisphaera tokodaii comb. nov.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KD-1T, was isolated from an acidic hot spring in Indonesia and characterized with the phylogenetically related species Sulfurisphaera ohwakuensis Kurosawa et al. 1998, Sulfolobus tokodaii Suzuki et al., 2002 and Sulfolobus yangmingensis Jan et al. 1999. Cells of KD-1T were irregular cocci with diameters of 0.9-1.3 µm. The strain grew at 60-90 °C (optimum 80-85 °C), pH 2.5-6.0 (optimum pH 3.5-4.0) and 0-1.0 % (w/v) NaCl concentration. KD-1T grew anaerobically in the presence of S0 (headspace: H2/CO2) and FeCl3 (headspace: N2). Under aerobic conditions, chemolithoautotrophic growth occurred on S0, pyrite, K2S4O6, Na2S2O3 and H2. This strain utilized various complex substrates, such as yeast extract, but did not grow on sugars and amino acids as the sole carbon source. The main core lipids were calditoglycerocaldarchaeol and caldarchaeol. The DNA G+C content was 30.6 mol%. Analyses of phylogenetic trees based on 16S rRNA and 23S rRNA genes indicated that KD-1T formed an independent lineage near Sulfurisphaera ohwakuensis TA-1T, Sulfolobus tokodaii 7T and Sulfolobus yangmingensis YM1T. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, KD-1T represents a novel species of the genus Sulfurisphaera Kurosawa et al. 1998, for which the name Sulfurisphaera javensis sp. nov. is proposed. The type strain is KD-1T (=JCM 32117T=InaCC Ar81T). Based on the data, we also propose the reclassification of Sulfolobus tokodaii Suzuki et al., 2002 as Sulfurisphaera tokodaii comb. nov. (type strain 7T=JCM 10545T=DSM 16993T).

Developing genome-wide SNPs and constructing an ultrahigh-density linkage map in oil palm.

Oil palm (Elaeis guineensis Jacq.) is the leading oil-producing crops and the most important edible oil resource worldwide. DNA markers and genetic linkage maps are essential resources for marker-assisted selection to accelerate genetic improvement. We conducted RAD-seq on an Illumina NextSeq500 to discover genome-wide SNPs, and used the SNPs to construct a linkage map for an oil palm (Tenera) population derived from a cross between a Deli Dura and an AVROS Pisifera. The RAD-seq produced 1,076 million single-end reads across the breeding population containing 155 trees. Mining this dataset detected 510,251 loci. After filtering out loci with low accuracy and more than 20% missing data, 11,394 SNPs were retained. Using these SNPs, in combination with 188 anchor SNPs and 123 microsatellites, we constructed a linkage map containing 10,023 markers covering 16 chromosomes. The map length is 2,938.2 cM with an average marker space of 0.29 cM. The large number of SNPs will supply ample choices of DNA markers in analysing the genetic diversity, population structure and evolution of oil palm. This high-density linkage map will contribute to mapping quantitative trait loci (QTL) for important traits, thus accelerating oil palm genetic improvement.

Diversity of lactic acid bacterial in inasua fermentation.

BACKGROUND AND OBJECTIVES: Inasua is one of the traditional fermented fish products in Maluku, Indonesia. There are two types of inasua, i.e. with and without sap. The research aimed to study the succession of lactic acid bacteria (LAB) during fermentation and microbial composition in inasua. MATERIALS AND METHODS: The sample of inasua was taken from two traditional producers in Layeni village, Ceram Island. The diversity of lactic acid bacteria was analyzed based on the 16S rRNA gene sequence. RESULTS: The succession of lactic acid bacteria was strongly influenced by the physicochemical characteristics during fermentation. Lactobacillus plantarum was found dominant in both inasuas fermentation processes. At end of fermentation, L. plantarum was still found dominant in inasua with sap while inasua without sap was dominated by Leuconostoc mesenteroides. In addition, Lactobacillus paracasei (LAB) was found only in inasua with sap. The result of Denaturing Gradient Gel Electrophoresis (DGGE) revealed that Lactobacillus was the dominant bacteria in inasua with sap while Staphylococcus was dominant in inasua without sap. CONCLUSION: Inasua with sap was found with higher bacterial diversity index and lower evenness and dominance indices, as well as more complex LAB succession pattern during fermentation and bacterial composition, as opposed to inasua without sap.