Metagenome analysis of tempeh production: Where did the bacterial community in tempeh come from?

Aims: Tempeh is a soy-based traditional food fermented by Rhizopus oligosporus. Although this mold is the main microorganism responsible for tempeh fermentation, various unknown bacteria presence in tempeh could enhance tempeh’s nutritional value. This study is aimed to examine the identity of bacteria in tempeh bacterial community by combining metagenomics analysis and culturable technique. Methodology and results: Samples were obtained from a tempeh producer which consists of raw soybeans, fresh water used to soak the beans, soaking water after the beans were soaked for 18 h, dehulled-soybean before inoculation, starter culture, and fresh tempeh. All samples were plated onto Enterobacteriaceae and Lactic Acid Bacteria agar media, and the total DNA was extracted for metagenomics analysis based on 16S rRNA gene cloning and High-Throughput Sequencing (HTS). Metagenomic analysis indicated that Firmicutes and Proteobacteria were the predominant and subdominant bacteria, respectively, while the culturable technique showed Proteobacteria were the predominant bacteria. Firmicutes species detected in tempeh were similar to the ones in the soaking water, which were populated by Lactobacillus. However, another predominant bacteria from tempeh, Enterococcus, was similar to minor population of Enterococcus detected in dehulled-soybean before inoculation. Based on the cloned 16S rRNA genes, we observed L. agilis, L. fermentum, and E. cecorum as the predominant bacteria in tempeh. The starter culture, which was dominated by Clostridium, did not alter bacterial community in tempeh, since its proportion was only 2.7% in tempeh clean reads. Conclusion, significance and impact of study: The dominant bacteria in tempeh was Lactobacillus from Firmicutes. The bacterial community in tempeh was not affected by the starter culture used, but mainly because of the soybean soaking process.

Whole genome analysis of Klebsiella: Unique genes associated with isolates from Indonesian tempeh

Aims: Our previous study demonstrated that Klebsiella IIEMP-3 associated with tempeh was genetically different from those of medical isolates. In addition to the whole genome sequence of Klebsiella IIEMP-3, the draft genome sequence of another isolate, i.e. IWJB-6 was employed for comparison. In this study, the details of the virulence genes and unique gene in both Klebsiella isolates were compared employing in silico and in vitro analysis. Methodology and results: Whole genome of Klebsiella IIEMP-3 and IWJB-6 were annotated to investigate the virulence factor. Klebsiella IIEMP-3 and IWJB-6 were obtained from tempeh producers in Bogor, West Java - Indonesia. Genome sequences were analyzed employing BLAST Ring Image Generator (BRIG) software. The results showed that all of the samples, including isolates IIEMP-3 and IWJB-6 did not harbor rmpA, i.e. DNA sequence for K. pneumoniae virulence factor. Conclusion, significance and impact of study: Klebsiella could be found in almost all tempeh samples from Indonesia and could be harmless for human due to the absence of rmpA and other virulence-associated genes. The significance of this study showed that IIEMP-3 and IWJB-6 isolates were more closely related to K. variicola. However, K. variicola At22 harbored sdsA gene which is lacking in those two tempeh isolates. Combined with PCR analysis for specific gene/s; our study suggested that isolates from Indonesian tempeh were closely related to K. variicola, and proposed to be designated as K. variicola subsp. tempehensis.

Effect of biofertilizer on the diversity of nitrogen - fixing bacteria and total bacterial community in lowland paddy fields in Sukabumi West Java, Indonesia

Aims: Some of methanotrophic bacteria and nitrous oxide (N2O) reducing bacteria have been proven able to support the plant growth and increase productivity of paddy. However, the effect of application of the methanotrophics and N2O reducing bacteria as a biofertilizer to indigenous nitrogen-fixing bacteria and total bacterial community are still not well known yet. The aim of the study was to analyze the diversity of nitrogen-fixing bacteria and total bacterial communty in lowland paddy soils. Methodology and results: Soil samples were taken from lowland paddy fields in Pelabuhan Ratu, Sukabumi, West Java, Indonesia. There were two treatments applied to the paddy field i.e biofertilizer-treated field (biofertilizer with 50 kg/ha NPK) and control (250 kg/ha NPK fertilizer). There were nine different nifH bands which were successfully sequenced and most of them were identified as unculturable bacteria and three of them were closely related to Sphingomonas sp., Magnetospirillum sp. and Ideonella dechloratans respectively. In addition, there were 20 different 16S rDNA bands which were successfully sequenced. Phylogenetic analysis of the sequence showed that there were 5 phyla of bacteria, i.e. Proteobacteria (Alphaproteobacteria and Gammaproteobacteria), Chlorofexi, Gemmatimonadetes, Clostridia, and Bacteroidetes respectively. Alphaproteobacteria was the most dominant group in lowland paddy field. Microbial diversities in the biofertilizer-treated field were lower than that of 100% fertilizer-treated field either based on nifH and 16S rDNA genes. Conclusion, significance and impact study: Biofertilizer treatment has lower microbial diversity than control, either based on nifH and 16S rDNA genes.

AHL-lactonase characteristics of Bacillus thuringiensis SGT3g and its effectiveness in inhibiting pathogenicity of Dickeya dadantii

ABSTRACT Aims: Dickeya dadantii is a pathogenic bacterium causing bacterial soft rot disease in plants. The bacterium uses a homoserine lactone signal in its quorum sensing process to express the virulence factor genes. Anti-quorum sensing is a new approach to control plant pathogenic bacteria. The aims of this study are to characterize AHL-lactonase enzyme produced by Bacillus thuringiensis SGT3g and to determine its effectiveness in inhibiting virulence of D. dadantii. Methodology and results: Activity of AHL-lactonase was determined using Chromobacterium violaceum as a bacterial biosensor. The crude extract enzymes of AHL-lactonase on both as extracellular and intracellular enzymes were analyzed their enzyme activity of protein precipitation and dialysis products. The optimum activity of AHL-lactonase was found at 30 °C and pH 5-8. Bacillus thuringiensis SGT3g was capable to reduce soft rot symptom disease caused by D. dadantii on Phalaenopsis orchid leaves after 24 h of incubation. Conclusion, significance and impact study: Bacillus thuringiensis SGT3g was capable to degrade AHL signal of C. violaceum and D. dadantii. The activity AHL-lactonase of B. thuringiensis SGT3g had a wide range of pH and temperature. The lactonase could reduce soft rot symptom disease caused by D. dadantii without any growth inhibition of D. dadantii on orchid leaves. Bacillus thuringiensis SGT3g can be used as an alternative biopesticide to control phytopathogenic bacteria due to its capability to suppress bacterial pathogenic virulence.

Molecular diversity pattern of intestinal lactic acid bacteria in Cemani chicken, Indonesian native chicken, as revealed by terminal restriction fragment length polymorphisms

Aims: An ecological study was conducted to investigate the diversity pattern of the lactic acid bacteria in the gastrointestinal tract of Cemani chicken, a native Indonesian chicken, using a molecular approach based on 16S rRNA genes. Methodology and results: Digesta samples of seven chickens were collected for terminal restriction fragment length polymorphism (T-RFLP) analysis. The molecular diversity of lactic acid bacteria in crop, ventriculus, ileum and cecum were determined. The results showed that microbial composition of lactic acid bacteria in cecum was relatively different with other upper gastrointestinal tract. Lactic acid bacteria phylotypes and diversity in ileum were higher than those in the crop, ventriculus, and cecum. Conclusion, significance and impact of study: We confirmed that cecum of native chicken has a different environment as compared to other gastrointestinal regions showing the lowest value of the Sorensen’s index. This first report of LAB diversity pattern in Cemani chicken contributes a more comprehensive understanding of the microbial ecology in the chicken.

Cupredoxin domain of particulate methane monooxygenase (pMMO) gene expression in recombinant Escherichia coli

Aims: Particulate methane monooxygenase (pMMO) is an integral membrane protein that converts methane to methanol as the first step in the metabolic pathway of methanotroph bacteria. Methanotroph have a slow growth rate that make researcher have to develop an alternative approach by expressing the pMMO genes in Escherichia coli. However, it was very difficult to express all the pMMO encoded genes in E. coli and it is suspected that the protein might be toxic to E. coli. Therefore, this research tried another approach by expressing the active site of pMMO enzyme; cupredoxin domain of pmoB subunit encoded by spmoB gene. Methodology and results: The spmoB gene from Methylococcus capsulatus (Bath) was expressed in E. coli BL21 (DE3) under T7 promoter and pET15b as the expression vector. Several modifications were made so this gene would be expressed in the cytoplasm. Expression analysis with SDS-PAGE showed that overexpression of this gene could be done at several concentrations of IPTG and incubation temperature. The spmoB gene expression produced a recombinant protein with a size approximately 38.9 kDa. Assay of spmoB protein activity showed that the amount of methanol accumulated during methane oxidation by the recombinant strain was 0.114 mmol/mL culture.h. Conclusion, significance and impact study: We successfully expressed spmoB gene in E. coli BL21 (DE3) without high production of toxic compounds and it has methane oxidation activity. This result allowed further characterization of its potential applications.

The effectiveness of methanotrophic bacteria and Ochrobactrum anthropi to reduce CH4 and N2O emissions and to promote paddy growth in lowland paddy fields

Aims: Paddy field is one of the sources of greenhouse gasses such as methane (CH4) and nitrous oxide (N2O), which causes global warming and other negative effects in agricultural sector. An alternative to optimize paddy productivity and reduce emissions of CH4 and N2O is by using methanotrophic bacteria and Ochrobactrum anthropi BL2. Methodology and results: This study consisted of two parts, i.e. positive control and experimental treatments. Positive control consisted of 250 kg/ha NPK inorganic fertilizer NPK (15:15:15) (100% of the recommended normal dose) without any methanotrophic bacteria. Meanwhile the experimental treatment consisted of 50 kg/ha inorganic fertilizers NPK (20% of the recommended normal dose) with methanotrophic bacteria (Methylocystis rosea BGM 1, M. parvus BGM 3, Methylococcus capculatus BGM 9, Methylobacter sp. SKM 14) and N2O reducing bacteria (Ochrobactrum anthropi BL2). Using weight indicator of 1000 grams, all the bacteria are capable of increasing paddy productivity by 42.07%, compared to conventional method which can only increase the productivity by 2.51% (Cepy and Wangiyana, 2011). The increasing productivity and growth of paddy plants were due to the nitrogen fixation activity of M. rosea BGM 1, M. capculatus BGM 9, and Methylobacter sp. SKM 14. In the experimental treatment using bacteria, the emission of CH4 and N2O was reduced with the highest CH4 and N2O sinks of 24018.8 mol CH4/day/ha and 68.48 mol N2O/day/ha, respectively. However, the positive control treatment with 100% of the recommended fertilizer dose showed the highest CH4 and N2O emissions which were up to 74346.45 mol CH4/day/ha and 26.21 mol N2O/day/ha, respectively. Conclusion, significance and impact study: All the methanotropic bacteria and O. anthropi BL2 are significantly increase paddy production, compared to positive control treatment. The addition of bacteria in paddy fields results in CH4 and N2O sinks.