Nanobacterial Cellulose from Kombucha Fermentation as a Potential Protective Carrier of Lactobacillus plantarum under Simulated Gastrointestinal Tract Conditions.

Kombucha bacterial cellulose (KBC), a by-product of kombucha fermentation, can be used as a biomaterial for microbial immobilization. In this study, we investigated the properties of KBC produced from green tea kombucha fermentation on days 7, 14, and 30 and its potential as a protective carrier of Lactobacillus plantarum, a representative beneficial bacteria. The highest KBC yield (6.5%) was obtained on day 30. Scanning electron microscopy showed the development and changes in the fibrous structure of the KBC over time. They had crystallinity indices of 90-95%, crystallite sizes of 5.36-5.98 nm, and are identified as type I cellulose according to X-ray diffraction analysis. The 30-day KBC had the highest surface area of 19.91 m2/g, which was measured using the Brunauer-Emmett-Teller method. This was used to immobilize L. plantarum TISTR 541 cells using the adsorption-incubation method, by which 16.20 log CFU/g of immobilized cells was achieved. The amount of immobilized L. plantarum decreased to 7.98 log CFU/g after freeze-drying and to 2.94 log CFU/g after being exposed to simulated gastrointestinal tract conditions (HCl pH 2.0 and 0.3% bile salt), whereas the non-immobilized culture was not detected. This indicated its potential as a protective carrier to deliver beneficial bacteria to the gastrointestinal tract.

Intraspecific Diversity and Pathogenicity of Bacillus thuringiensis Isolates from an Emetic Illness.

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate.

Bacterial Communities Associated with Crude Oil Bioremediation through Composting Approaches with Indigenous Bacterial Isolate.

In this study, we aim to investigate the efficiency of crude oil bioremediation through composting and culture-assisted composting. First, forty-eight bacteria were isolated from a crude oil-contaminated soil, and the isolate with the highest crude oil degradation activity, identified as Pseudomonas aeruginosa, was selected. The bioremediation was then investigated and compared between crude oil-contaminated soil (S), the contaminated soil composted with fruit-based waste (SW), and the contaminated soil composted with the same waste with the addition of the selected bacterium (SWB). Both compost-based methods showed high efficiencies of crude oil bioremediation (78.1% and 83.84% for SW and SWB, respectively). However, only a slight difference between the treatments without and with the addition of P. aeruginosa was observed. To make a clear understanding of this point, bacterial communities throughout the 4-week bioremediation period were analyzed. It was found that the community dynamics between both composted treatments were similar, which corresponds with their similar bioremediation efficiencies. Interestingly, Pseudomonas disappeared from the system after one week, which suggests that this genus was not the key degrader or only involved in the early stage of the process. Altogether, our results elaborate that fruit-based composting is an effective approach for crude oil bioremediation.

Removal of Hydrogen Sulfide from Swine-Waste Biogas on a Pilot Scale Using Immobilized Paracoccus versutus CM1.

Hydrogen sulfide (H2S) is a toxic and corrosive component that commonly occurs in biogas. In this study, H2S removal from swine-waste biogas using sulfur-oxidizing Paracoccus versutus CM1 immobilized in porous glass (PG) and polyurethane foam (PUF) biofilters was investigated. Bacterial compositions in the biofilters were also determined using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The biofilters were first tested on a laboratory scale under three space velocities (SV): 20, 30, and 40 h−1. Within 24 h, at an SV of 20 h−1, PG and PUF biofilters immobilized with P. versutus CM1 removed 99.5% and 99.7% of H2S, respectively, corresponding to the elimination capacities (EC) of 83.5 and 86.2 gm−3 h−1. On a pilot scale, with the horizontal PG-P. versutus CM1 biofilter operated at an SV of 30 h−1, a removal efficiency of 99.7% and a maximum EC of 113.7 gm−3 h−1 were achieved. No reduction in methane content in the outlet biogas was observed under these conditions. The PCR-DGGE analysis revealed that Paracoccus, Acidithiobacillus, and Thiomonas were the predominant bacterial genera in the biofilters, which might play important roles in H2S removal. This PG−P. versutus CM1 biofiltration system is highly efficient for H2S removal from swine-waste biogas.

Genome Characterisation of an Isoprene-Degrading Alcaligenes sp. Isolated from a Tropical Restored Forest.

Isoprene is a climate-active biogenic volatile organic compound (BVOC), emitted into the atmosphere in abundance, mainly from terrestrial plants. Soil is an important sink for isoprene due to its consumption by microbes. In this study, we report the ability of a soil bacterium to degrade isoprene. Strain 13f was isolated from soil beneath wild Himalayan cherry trees in a tropical restored forest. Based on phylogenomic analysis and an Average Nucleotide Identity score of >95%, it most probably belongs to the species Alcaligenes faecalis. Isoprene degradation by Alcaligenes sp. strain 13f was measured by using gas chromatography. When isoprene was supplied as the sole carbon and energy source at the concentration of 7.2 × 105 ppbv and 7.2 × 106 ppbv, 32.6% and 19.6% of isoprene was consumed after 18 days, respectively. Genome analysis of Alcaligenes sp. strain 13f revealed that the genes that are typically found as part of the isoprene monooxygenase gene cluster in other isoprene-degrading bacteria were absent. This discovery suggests that there may be alternative pathways for isoprene metabolism.

Efficacy of Cordyceps militaris Extracts against Some Skin Pathogenic Bacteria and Antioxidant Activity.

Cordyceps militaris has been used for treating various diseases, as well as maintaining good overall health. The antibacterial properties of the C. militaris fruiting body and substrate, cultured in Chiang Mai (sample A and B) and Chiang Rai (sample C), Thailand, were investigated in this study. The aqueous and ethanolic extracts of C. militaris exhibited antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Cutibacterium acnes and methicillin-resistant S. aureus (MRSA) with the MIC/MBC ranging from 3.91 to 31.25 mg/mL. The ethanolic extracts of the fruiting body and substrate from sample B also inhibited all bacterial growth within 2-4 h of treatment. Furthermore, ethanolic extract from sample B showed the highest cordycepin content of 57.42 mg/g extract, whereas the highest adenosine content, 3.78 mg/g extract, was observed in the ethanolic extract from the fruiting body of sample A by HPLC. The ethanolic extracts from sample A also demonstrated the highest antioxidant activity and flavonoid content by 9.50 mg GAE/g extract and 10.59 mg QAE/g extract, respectively. However, the highest phenolic content of 49.04 mg GAE/g extract was found in the aqueous extract of sample A. In addition, the ethanolic extract of sample A at 2 and 4 mg/mL could significantly down-regulate the mecA gene expression in MRSA. Our findings reported the potential of C. militaris extract as a new substance for the treatment of skin pathogenic bacteria infections and an antioxidant agent.

Characterisation of Lactobacillus plantarum of Dairy-Product Origin for Probiotic Chèvre Cheese Production.

Probiotics are increasingly used as functional food ingredients. The objectives of this study were to isolate and characterise probiotic bacteria from dairy and fermented foods and to use a selected strain for the production of probiotic chèvre cheese. Tolerance to acid (pH 2.0) and bile salt (0.4% (w/v)) were first investigated, and then other probiotic properties were determined. Out of 241 isolates, 35 showed high tolerance to acid and bile salt, and 6 were chosen for further characterisation. They were Lactobacillus plantarum and L. fermentum, and possessed antibacterial activities against foodborne pathogens such as Bacillus cereus, Staphylococcus aureus, Salmonella enterica and Escherichia coli O157:H7. L. plantarum (isolate AD73) showed the highest percentage of adhesion (81.74 ± 0.16%) and was nontoxic to Caco-2 cells at a concentration of 108 CFU/mL. This isolate was therefore selected for the production of probiotic chèvre cheese from goat's milk and was prepared in a lyophilised form with a concentration of probiotic culture of 8.6 log CFU/g. The cheese had a shelf life of 8 days. On the expiry date, the probiotic, the starter and the yeast contents were 7.56 ± 0.05, 7.81 ± 0.03 and 5.64 log CFU/g, respectively. The level of the probiotics in this chèvre cheese was still sufficiently high to warrant its being a probiotic cheese.

Isoprene-Degrading Bacteria from Soils Associated with Tropical Economic Crops and Framework Forest Trees.

Isoprene, a volatile hydrocarbon emitted largely by plants, plays an important role in regulating the climate in diverse ways, such as reacting with free radicals in the atmosphere to produce greenhouse gases and pollutants. Isoprene is both deposited and formed in soil, where it can be consumed by some soil microbes, although much remains to be understood about isoprene consumption in tropical soils. In this study, isoprene-degrading bacteria from soils associated with tropical plants were investigated by cultivation and cultivation-independent approaches. Soil samples were taken from beneath selected framework forest trees and economic crops at different seasons, and isoprene degradation in soil microcosms was measured after 96 h of incubation. Isoprene losses were 4-31% and 15-52% in soils subjected to a lower (7.2 × 105 ppbv) and a higher (7.2 × 106 ppbv) concentration of isoprene, respectively. Sequencing of 16S rRNA genes revealed that bacterial communities in soil varied significantly across plant categories (framework trees versus economic crops) and the presence of isoprene, but not with isoprene concentration or season. Eight isoprene-degrading bacterial strains were isolated from the soils and, among these, four belong to the genera Ochrobactrum, Friedmanniella, Isoptericola and Cellulosimicrobium, which have not been previously shown to degrade isoprene.

Bacterial compositions of indigenous Lanna (Northern Thai) fermented foods and their potential functional properties.

Many indigenous fermented foods of Northern Thailand and neighbouring regions have traditionally been known for their health benefits. In this study, we explored the communities of bacteria in selected fermented foods which are commonly consumed among ethnic groups around Northern Thailand, for which information on their microbial compositions or their functional properties is still limited. The selected food groups included Thua Nao (alkaline fermented soybean product), Nham (fermented pork sausage/loaf), Nam phak (fermented Chinese cabbage) and Miang (fermented leaves from Miang Tea trees). Bacteria in these fermented foods were isolated and enumerated. Bacterial communities were determined using a culture-independent (pyrosequencing) approach. Lactic acid bacteria were recovered from all of these fermented food samples, with levels ranging from 3.1 to 7.5 log CFU/g throughout the fermentation processes. Analysis of the 16S rRNA gene from the fermented food samples using 454-pyrosequencing resulted in 113,844 sequences after quality evaluation. Lactic acid bacteria were found in high proportions in Nham, Nam phak and Miang. Bacillus was predominant in Thua nao, in which significant proportions of Lactic acid bacteria of the family Leuconostocaceae were also found. Groups of lactic acid bacteria found varied among different food samples, but three genera were predominant: Lactococcus, Lactobacillus and Leuconostoc, of which many members are recognised as probiotics. The results showed that these traditional Thai fermented food products are rich sources of beneficial bacteria and can potentially be functional/probiotic foods.

Antibiotic-Antiapoptotic Dual Function of Clinacanthus nutans (Burm. f.) Lindau Leaf Extracts against Bovine Mastitis.

Mastitis caused by bacterial infection has negative impacts on milk quality and animal health, and ultimately causes economic losses to the dairy industry worldwide. Gram-negative bacteria and their component lipopolysaccharide (LPS) can trigger the inflammatory response of endothelial cells (ECs) and subsequently promote EC dysfunction or injury, which is a critical pathogenesis of mastitis-causing sepsis shock. To control the bacterial infection and to minimise the LPS negative effects on ECs, we thus aimed to identify the potential herb extracts that comprised antibacterial activity and protective ability to inhibit LPS-induced cell death. Extracts from seven types of herbs derived from antibacterial screening were investigated for their protective effects on LPS-stimulated bovine endothelial cell line. Clinacanthus nutans (Burm. f.) Lindau (C. nutans) extract appeared to be the most effective antiapoptotic extract against LPS stimulation. Treatment of C. nutans extract in LPS-stimulated cells significantly lowered apoptotic cell death through modulating pro-survival Bcl-2 and pro-apoptotic Bax expression. The investigation of bioactive compounds using solvent fractionation, HPLC, and LC-MS/MS analysis revealed glyceryl 1,3-disterate (C39H76O5), kaempferol 3-O-feruloyl-sophoroside 7-O-glucoside (C43H48O24), and hydroxypthioceranic acid (C46H92O3) as the candidate components. Our findings indicated that C. nutans extract has great potential to be further developed as an alternative therapeutic agent for mastitis treatment.

Efficacy of Kombucha Obtained from Green, Oolong, and Black Teas on Inhibition of Pathogenic Bacteria, Antioxidation, and Toxicity on Colorectal Cancer Cell Line.

Kombucha tea is a refreshing beverage that is produced from the fermentation of tea leaves. In this study, kombucha tea was prepared using 1% green tea, oolong tea, and black tea, and 10% sucrose with acetic acid bacteria and yeast. The pH values of the kombucha tea were found to be in a range of 2.70-2.94 at 15 days of fermentation. The lowest pH value of 2.70 was recorded in the kombucha prepared from black tea. The total acidity of kombucha prepared from black tea was the highest by 16.75 g/L and it was still maintained after heat treatment by boiling and after autoclaved. Six organic acids: glucuronic, gluconic, D-saccharic acid 1,4-lactone, ascorbic, acetic, and succinic acid in kombucha tea were detected by HPLC with the optimization for organic acids detection using isocratic elution buffer with C18 conventional column. The highest level of organic acid was gluconic acid. Kombucha prepared from green tea revealed the highest phenolic content and antioxidation against DPPH radicals by 1.248 and 2.642 mg gallic acid/mL kombucha, respectively. Moreover, pathogenic enteric bacteria: Escherichia coli. E. coli O157:H7. Shigella dysenteriae, Salmonella Typhi, and Vibrio cholera were inhibited by kombucha and heat-denatured kombucha with diameter of the inhibition zones ranged from 15.0 ± 0.0-25.0 ± 0.0 mm. In addition, kombucha prepared from green tea and black tea demonstrated toxicity on Caco-2 colorectal cancer cells. Therefore, kombucha tea could be considered as a potential source of the antioxidation, inhibition of pathogenic enteric bacteria, and toxicity on colorectal cancer cells.

Biosurfactant-Producing Capability and Prediction of Functional Genes Potentially Beneficial to Microbial Enhanced Oil Recovery in Indigenous Bacterial Communities of an Onshore Oil Reservoir.

Microbial enhanced oil recovery (MEOR) is a bio-based technology with economic and environmental benefits. The success of MEOR depends greatly on the types and characteristics of indigenous microbes. The aim of this study was to evaluate the feasibility of applying MEOR at Mae Soon Reservoir, an onshore oil reservoir experiencing a decline in its production rate. We investigated the capability of the reservoir's bacteria to produce biosurfactants, and evaluated the potentials of uncultured indigenous bacteria to support MEOR by means of prediction of MEOR-related functional genes, based on a set of metagenomic 16s rRNA gene data. The biosurfactant-producing bacteria isolated from the oil-bearing sandstones from the reservoir belonged to one species: Bacillus licheniformis, with one having the ability to decrease surface tension from 72 to 32 mN/m. Gene sequences responsible for biosurfactant (licA3), lipase (lipP1) and catechol 2,3-dioxygenase (C23O) were detected in these isolates. The latter two, and other genes encoding MEOR-related functional proteins such as enoyl-CoA hydratase and alkane 1-monooxygenase, were predicted in the bacterial communities residing the reservoir's sandstones. Exposure of these sandstones to nutrients, consisting of KNO3 and NaH2PO4, resulted in an increase in the proportions of some predicted functional genes. These results indicated the potentials of MEOR application at Mae Soon site. Using the approaches demonstrated in this study would also assist evaluation of the feasibility of applying MEOR in oil reservoirs, which may be enhanced by an appropriate nutrient treatment.

Effect of inorganic nutrients on bacterial community composition in oil-bearing sandstones from the subsurface strata of an onshore oil reservoir and its potential use in Microbial Enhanced Oil Recovery.

Microbial Enhanced Oil Recovery (MEOR) is a promising strategy to improve recovery of residual oil in reservoirs, which can be performed by promoting specific indigenous microorganisms. In this study, we performed preliminary evaluation of the possibility of conducting MEOR at Mae Soon reservoir, an onshore reservoir in Northern Thailand. The reservoir's physicochemical characteristics, including the characteristics of the wells, the oil-bearing sandstone cores, and the reservoir's produced water, were determined. The microbiological characteristics of the oil wells in the reservoir were also investigated by submerging the reservoir's sandstone core samples, obtained from 6 oil wells, in the reservoir's produced water and in the produced water added with inorganic nutrients (KNO3 and NaH2PO4). The uncultured bacteria in both treatments were determined, using tagged 16S rRNA gene amplicon with Ion Torrent Sequencing Analysis. The effects of inorganic nutrients and the reservoir's parameters on the bacterial communities were analysed. A total number of 16,828 OTUs were taxonomically classified into 89 classes and 584 genera. In the controls (sandstone cores submerged in the produced water), the dominant bacterial populations were related to Deinococcus-Thermus, and Betaproteobacteria; while in the nutrient treated samples, there was a marked increase in the relative abundance of Gammaproteobacteria in three samples. Thermus, Acinetobacter, and Pseudomonas were the most abundant genera, and these are potential microorganisms for MEOR. Analysis of correlations between physiochemical properties of the reservoir and bacterial genera, using spearman's correlation analysis, suggested that some of the reservoir's properties, especially of the well and the rock, could influence some bacterial genera. To our knowledge, this is the first demonstration of the effect of inorganic nutrients on alteration of bacterial communities attached to reservoir's rock, and how the bacterial, physical, and chemical properties of a reservoir were co-analysed to serve as a basis for designing a MEOR process.