Responses of potential ammonia oxidation and ammonia oxidizers community to arsenic stress in seven types of soil.

Soil arsenic contamination is of great concern because of its toxicity to human, crops, and soil microorganisms. However, the impacts of arsenic on soil ammonia oxidizers communities remain unclear. Seven types of soil spiked with 0 or 100 mg arsenic per kg soil were incubated for 180 days and sampled at days 1, 15, 30, 90 and 180. The changes in the community composition and abundance of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) were analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis, clone library sequencing, and quantitative PCR (qPCR) targeting amoA gene. Results revealed considerable variations in the potential ammonia oxidation (PAO) rates in different soils, but soil PAO was not consistently significantly inhibited by arsenic, probably due to the low bioavailable arsenic contents or the existence of functional redundancy between AOB and AOA. The variations in AOB and AOA communities were closely associated with the changes in arsenic fractionations. The amoA gene abundances of AOA increased after arsenic addition, whereas AOB decreased, which corroborated the notion that AOA and AOB might occupy different niches in arsenic-contaminated soils. Phylogenetic analysis of amoA gene-encoded proteins revealed that all AOB clone sequences belonged to the genus Nitrosospira, among which those belonging to Nitrosospira cluster 3a were dominant. The main AOA sequence detected belonged to Thaumarchaeal Group 1.1b, which was considered to have a high ability to adapt to environmental changes. Our results provide new insights into the impacts of arsenic on the soil nitrogen cycling.

Microbial Community Profiling Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE).

In their natural environments, microorganisms usually live in organized communities. Profiling analysis of microbial communities has recently assumed special relevance as it allows a thorough understanding of the diversity of the microbiota, its behavior over time, and the establishment of patterns associated with health and disease. The application of molecular biology approaches holds the advantage of including culture-difficult and as-yet-uncultivated phylotypes in the profiles, providing a more comprehensive picture of the microbial community. This chapter focuses on two particular techniques, namely terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE), both of which have been widely used in environmental studies and have been recently successfully used by the authors in the study of the oral microbial communities associated with conditions of health and disease.

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.

Effects on Microbiota Composition after Consumption of Quinoa Beverage Fermented by a Novel Xylose-Metabolizing L. plantarum Strain.

Demands for novel lactic acid bacteria with potential to be used as probiotics along with healthy fermented plant-based products increase worldwide. In this study, a novel Lactiplantibacillus plantarum P31891 strain with enzymatic capacity to degrade tannins and ferment xylose was used as starter culture for fermentation of a quinoa-based beverage. The probiotic potential of the selected strain was evaluated in healthy volunteers. Twenty participants consumed the beverage for 14 days; microbiota changes in saliva and faecal samples were analyzed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), Next Generation Sequencing (NGS) and qPCR; and gastrointestinal well-being and digestive symptoms were recorded. The results indicated that the consumption of the beverage with Lactiplantibacillus plantarum P31891 in a probiotic dose (1012 CFU/mL) increased the number of Lactobacillus in the feces but not in saliva. Overall, the bacterial community did not seem to be influenced by the bacterium or by the beverage, as expressed by the diversity indexes, but specific genera were affected, as reflected in changes in amplicon sequence variants. Consequently, Lactiplantibacillus plantarum P31891 showed potential to be categorized as a probiotic strain in the fermented quinoa-based beverage.

Microbiome Diversity and Community-Level Change Points within Manure-based small Biogas Plants.

Efforts to integrate biogas plants into bioeconomy concepts will lead to an expansion of manure-based (small) biogas plants, while their operation is challenging due to critical characteristics of some types of livestock manure. For a better process understanding, in this study, three manure-based small biogas plants were investigated with emphasis on microbiome diversity. Due to varying digester types, feedstocks, and process conditions, 16S rRNA gene amplicon sequencing showed differences in the taxonomic composition. Dynamic variations of each investigated biogas plant microbiome over time were analyzed by terminal restriction fragment length polymorphism (TRFLP), whereby nonmetric multidimensional scaling (NMDS) revealed two well-running systems, one of them with a high share of chicken manure, and one unstable system. By using Threshold Indicator Taxa Analysis (TITAN), community-level change points at ammonium and ammonia concentrations of 2.25 g L-1 and 193 mg L-1 or volatile fatty acid concentrations of 0.75 g L-1were reliably identified which are lower than the commonly reported thresholds for critical process stages based on chemical parameters. Although a change in the microbiome structure does not necessarily indicate an upcoming critical process stage, the recorded community-level change points might be a first indication to carefully observe the process.

Seasonal dynamics of the bacterial communities associated with cyanobacterial blooms in the Han River.

DNA-based analyses of bacterial communities were performed to identify the bacteria co-occurring with cyanobacterial blooms in samples collected at a single site over 2 years. Microcystis aeruginosa was the most predominant species (81% in 2018, and 94% in 2019) within the phylum Cyanobacteria, and microcystins were detected during all cyanobacterial blooms. The stereo microscope and scanning electron microscope observations showed bacterial associations on and around the aggregated M. aeruginosa cells. Culture-independent analyses of filtered bacterial communities showed that the Flavobacterium species in phylum Bacteroidetes (19%) was dominant in the cyanobacterial phycosphere, followed by the Limnohabitans species in Betaproteobacteria (11%). Using principal component analysis, major bacterial genus, including Microcystis and Flavobacterium species, were clustered during cyanobacterial blooms in both years. To identify key bacterial species that develop long-term symbiosis with M. aeruginosa, another culture-independent analysis was performed after the environmental sample had been serially subcultured for 1 year. Interestingly, Brevundimonas (14%) was the most dominant species, followed by Porphyrobacter (7%) and Rhodobacter (3.5%) within the Alphaproteobacteria. Screening of 100 colonies from cyanobacterial bloom samples revealed that the majority of culturable bacteria belonged to Gammaproteobacteria (28%) and Betaproteobacteria (57%), including Pseudomonas, Curvibacter, and Paucibacter species. Several isolates of Brevundimonas, Curvibacter, and Pseudomonas species could promote the growth of axenic M. aeruginosa PCC7806. The sensitivity of M. aeruginosa PCC7806 cells to different environmental conditions was monitored in bacteria-free pristine freshwater, indicating that nitrogen addition promotes the growth of M. aeruginosa.

Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study plant-associated communities of bacteria and fungi.

High-throughput sequencing technologies have revolutionized the study of plant-associated microbial populations, but they are relatively expensive. Molecular fingerprinting techniques are more affordable, yet yield considerably less information about the microbial community. Does this mean they are no longer useful for plant microbiome research? In this paper, we review the past 10 years of studies on plant-associated microbiomes using molecular fingerprinting methodologies, including single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), amplicon length heterogeneity PCR (LH-PCR), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA), and terminal restriction fragment length polymorphism (TRFLP). We also present data juxtaposing results from TRFLP methods with those generated using Illumina sequencing in the comparison of rhizobacterial populations of Brazilian maize and fungal surveys in Canadian tomato roots. In both cases, the TRFLP approach yielded the desired results at a level of resolution comparable to that of the MiSeq method, but at a fraction of the cost. Community fingerprinting methods (especially TRFLP) remain relevant for the identification of dominant microbes in a population, the observation of shifts in plant microbiome community diversity, and for screening samples before their use in more sensitive and expensive approaches.

Season of Birth Impacts the Neonatal Nasopharyngeal Microbiota.

Objective: Pathogenic airway bacteria colonizing the neonatal airway increase the risk of childhood asthma, but little is known about the determinants of the establishment and dynamics of the airway microbiota in early life. We studied associations between perinatal risk factors and bacterial richness of the commensal milieu in the neonatal respiratory tract. Methods: Three hundred and twenty-eight children from the Copenhagen Prospective Studies on Asthma in the Childhood2000 (COPSAC2000) at-risk birth cohort were included in this study. The bacterial richness in each of the nasopharynxes of the 1-month old, asymptomatic neonates was analyzed by use of a culture-independent technique (T-RFLP). Information on perinatal risk factors included predisposition to asthma, allergy and eczema; social status of family; maternal exposures during pregnancy; mode of delivery; and postnatal exposures. The risk factor analysis was done by conventional statistics and partial least square discriminant analysis (PLSDA). Results: The nasopharyngeal bacterial community at 1-month displayed an average of 35 (IQR: 14-55, range 1-161) phylogenetically different bacteria groups. Season of birth was associated with nasopharyngeal bacterial richness at 1-month of age with a higher bacterial richness (p = 0.003) and more abundant specific bacterial profiles representing Gram-negative alpha-proteobacteria and Gram-positive Bacilli in the nasopharynx of summer-born children. Conclusion: Early postnatal bacterial colonization of the upper airways is significantly affected by birth season, emphasizing a future focus on the seasonality aspect in modelling the impact of early dynamic changes in airway bacterial communities in relation to later disease development.

Effect of a Profound Feedstock Change on the Structure and Performance of Biogas Microbiomes.

In this study the response of biogas-producing microbiomes to a profound feedstock change was investigated. The microbiomes were adapted to the digestion of either 100% sugar beet, maize silage, or of the silages with elevated amounts of total ammonium nitrogen (TAN) by adding ammonium carbonate or animal manure. The feedstock exchange resulted in a short-range decrease or increase in the biogas yields according to the level of chemical feedstock complexity. Fifteen taxa were found in all reactors and can be considered as generalists. Thirteen taxa were detected in the reactors operated with low TAN and six in the reactors with high TAN concentration. Taxa assigned to the phylum Bacteroidetes and to the order Spirochaetales increased with the exchange to sugar beet silage, indicating an affinity to easily degradable compounds. The recorded TAN-sensitive taxa (phylum Cloacimonetes) showed no specific affinity to maize or sugar beet silage. The archaeal community remained unchanged. The reported findings showed a smooth adaptation of the microbial communities, without a profound negative impact on the overall biogas production indicating that the two feedstocks, sugar beet and maize silage, potentially do not contain chemical compounds that are difficult to handle during anaerobic digestion.

Effect of soil organic matter on petroleum hydrocarbon degradation in diesel/fuel oil-contaminated soil.

The purpose of this study is to investigate the effect of soil organic matter (SOM) content levels on the biodegradation of total petroleum hydrocarbons (TPH). Batch experiments were conducted with soils with 2% or 10% organic matter that had been contaminated by diesel or fuel oil. In addition to the TPH (diesel or fuel oil) degradation efficiency, a comprehensive investigation was conducted on the TPH-degrading microbial community using molecular tools including oligonucleotide microarray technique and terminal restriction fragment length polymorphism analysis (T-RFLP). TPH was reduced from 10,000 mg/kg to 1849-4352 mg/kg dry weight soil. Higher biodegradation efficiencies and kinetic rate constants were observed in higher SOM contents. Hydrocarbon fractional analyses were conducted to explain the optimal operation with relatively low resin and aromatic fractions detected at the end of the remediation. The bacterial and fungal counts in the 10% SOM were approximately 10 CFU/g to 102 CFU/g above those in the 2% SOM, and the lowest fungal level was found when the least TPH degradability was measured. The internal transcribed spacer microarray identified the microorganisms that were introduced and proved their survival. The associated growth pattern confirmed that different kinds of contamination oils affected the microbial community diversity over time. Both the microarray and T-RFLP profiles indicated that Gordonia alkanivorans, G. desulfuricans, and Rhodococcus erythoropolis were the dominant bacteria, while Fusarium oxysporum and Aspergillus versicolor were the dominant fungi. The T-RFLP-derived nonmetric multidimensional scaling concluded that the dynamics of the microbial communities were impacted by the TPH degradation stages.

[Water Quality Characteristics and Distribution of Bacterial Communities During Thermal Stratification in the Miyun Reservoir].

As an important urban drinking water source, reservoirs are a special type of water body formed by artificial dams. Water quality of reservoirs directly affects the residents' drinking water safety. In order to reveal the characteristics of stratification and vertical changes of bacterial communities in the Miyun Reservoir, a drinking water source of Beijing, vertical stratified samples were collected during the stable stratified period of the reservoir (autumn). The vertical distribution characteristics of bacterial communities in the Miyun Reservoir were studied by using 16S rDNA terminal restriction fragment length polymorphism (T-RFLP) and quantitative PCR. Cluster analysis and multivariate statistical analysis were used to reveal the response relationships between bacterial communities and environmental factors. The results were as follows. ①The thermocline of the Miyun Reservoir was located at a water depth of 20-30 m, and the water temperature range was 15-19℃. The cluster analysis data of the seven sampled water layers were divided into an aerobic area (upper layer) and anoxic area (lower layer). The temperature, dissolved oxygen, and pH gradually decreased below 15 m. The electrical conductivity, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and total nitrogen changed significantly after 15 m. The water quality showed obvious features in the vertical direction. ② The redundancy analysis (RDA) results showed that there were obvious vertical changes in the dissolved oxygen, pH, electrical conductivity, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen between the aerobic and anoxic water layer. Those factors were the main environmental factors affecting the vertical distribution of the bacterial communities in the Miyun Reservoir. ③ The total bacterial number fluctuated with changes in the water depth. The Shannon-Wiener index and the number of T-RFs of bacteria in the aerobic zone were significantly higher than those in the anoxic zone, which indicates that there was significant stratification in the distribution of bacterial communities in the water of the Miyun Reservoir in autumn. This study explored the effects of water stratification on reservoir water quality and bacterial communities, and the findings provide a scientific basis for predicting water quality changes and reservoir management.

Foliar Application of Vegetal-Derived Bioactive Compounds Stimulates the Growth of Beneficial Bacteria and Enhances Microbiome Biodiversity in Lettuce.

Many studies on plant biostimulants and organic fertilizers have been focused on the ability of these products to increase crop productivity and ameliorate crop tolerance to abiotic stresses. However, little information is available on their effect on plant microbiota, whereas it is well known that microorganisms associated with plant play crucial roles on the health and productivity of their host. The aim of this study was to evaluate the effect of a vegetal-derived protein hydrolysate (PH), a vegetal-derived PH enriched with copper (Cu-PH), and a tropical plant extract enriched with micronutrients (PE) on shoot growth and the epiphytic bacterial population of lettuce plants and the ability of these products to enhance the growth of beneficial or harmful bacteria. The three plant-derived products enhanced shoot biomass of lettuce plants indicating a biostimulant effect of the products. Data obtained using culture-independent (Terminal Restriction Fragment Length Polymorphism and Next Generation Sequencing) and culture-dependent approaches indicated that foliar application of commercial products altered the composition of the microbial population and stimulated the growth of specific bacteria belonging to Pantoea, Pseudomonas, Acinetobacter, and Bacillus genus. Data presented in this work demonstrated that some of these strains exhibited potential plant growth-promoting properties and/or biocontrol activity against fungi and bacteria phytopathogens including Fusarium, Trichoderma, and Erwinia species. No indication of potential health risks associated to the enrichment of human or plant bacterial pathogens emerged by the analysis of the microbiota of treated and no-treated plants. Overall, the findings presented in this study indicate that the commercial organic-based products can enhance the growth of beneficial bacteria occurring in the plant microbiota and signals produced by these bacteria can act synergistically with the organic compounds to enhance plant growth and productivity.

Long-term dynamics of the bacterial community in a Swedish full-scale wastewater treatment plant.

The operational efficiency of activated sludge wastewater treatment plants depends to a large extent on the microbial community structure of the activated sludge. The aims of this paper are to describe the composition of the bacterial community in a Swedish full-scale activated sludge wastewater treatment plant, to describe the dynamics of the community and to elucidate possible causes for bacterial community composition changes. The bacterial community composition in the activated sludge was described using 16S rRNA gene libraries and monitored for 15 months by a terminal restriction fragment (T-RF) length polymorphism (T-RFLP) analysis of the 16S rRNA gene. Despite variable environmental conditions, a large fraction of the observed T-RFs were present at all times, making up at least 50% in all samples, possibly representing a relatively stable core fraction of the bacterial community. However, the proportions of the different T-RFs in this fraction as well as the T-RFs in the more variable fraction showed a significant variation over time and temperature. The difference in community composition between summer and winter coincided with observed differences in floc structure. These observations suggest a relationship between floc properties and bacterial community composition, although additional experiments are required to determine causality.

Comparison of gut microbiota composition between laboratory-bred marmosets (Callithrix jacchus) with chronic diarrhea and healthy animals using terminal restriction fragment length polymorphism analysis.

Chronic diarrhea in laboratory-bred marmosets poses a serious health problem during experiments. Despite a growing demand for laboratory-bred experimental marmosets, the mechanisms underlying the development of diarrhea and measures for its treatment and prevention remain unclear. To explore the factors affecting development of chronic diarrhea in laboratory-bred marmosets, the gut microbiota composition (GMC) of 58 laboratory-bred marmosets, including 19 animals with chronic diarrhea, was analyzed using terminal restriction fragment length polymorphism. We found that the GMCs in these animals cluster into two groups that differ significantly in rate of chronic diarrhea (56.5% in one group, Cluster 1, and 17.1% in Cluster 2). Additionally, a higher α-diversity and a lower proportion of Bifidobacterium spp. according to quantitative PCR was found the animals in the Cluster 1 than in those in Cluster 2. Taken together, our findings indicate that there is a relationship between GMC and development of chronic diarrhea in laboratory-bred marmosets. This is the first study to highlight the potential of assessing GMC in relation to development of chronic diarrhea in laboratory-bred marmosets.

Assessing the Bacterial Community Structure in the Rhizoplane of Wetland Plants.

Plant-microorganism interaction in the rhizosphere is important for nutrient cycling, carbon sequestration in natural ecosystems, contaminant elimination and ecosystem functioning. Abundance of microbial communities and variation in species composition can be an imperative determinant of phytoremediation capability. In the present study we have assessed the bacterial community structure in the rhizoplane of wetland plants, Acorus calamus, Typha latifolia, and Phragmites karka using Terminal restriction fragment length polymorphism technique. The most dominant phylum, in the plants under study, was phylum Firmicutes, followed by Proteobacteria and Actinobacteria. Bacterial groups belonging to phylum Chloroflexi, Acidobacteria, Deferribacteres and Thermotogae also showed their presence in P. karka and T. latifolia but were absent in A. calamus. Diversity indices of bacterial community were assessed. The results of this study show the presence of bacterial phyla which play an important role in bioremediation of contaminants. Thus these plants can be used as potential candidates of phytoremediation.

Substrate-Induced Response in Biogas Process Performance and Microbial Community Relates Back to Inoculum Source.

This study investigated whether biogas reactor performance, including microbial community development, in response to a change in substrate composition is influenced by initial inoculum source. For the study, reactors previously operated with the same grass⁻manure mixture for more than 120 days and started with two different inocula were used. These reactors initially showed great differences depending on inoculum source, but eventually showed similar performance and overall microbial community structure. At the start of the present experiment, the substrate was complemented with milled feed wheat, added all at once or divided into two portions. The starting hypothesis was that process performance depends on initial inoculum source and microbial diversity, and thus that reactor performance is influenced by the feeding regime. In response to the substrate change, all reactors showed increases and decreases in volumetric and specific methane production, respectively. However, specific methane yield and development of the microbial community showed differences related to the initial inoculum source, confirming the hypothesis. However, the different feeding regimes had only minor effects on process performance and overall community structure, but still induced differences in the cellulose-degrading community and in cellulose degradation.

Effect of fermented vegetable beverage containing Pediococcus pentosaceus in patients with mild to moderate ulcerative colitis.

In patients with ulcerative colitis (UC), probiotics are often employed as an adjuvant therapeutic option. In the present study, a fermented vegetable beverage containing Pediococcus pentosaceus strain IDS885 was administered to patients with active UC for 8 weeks. A total of 11 patients with mildly to moderately active UC were randomly assigned into two groups: Group A (n=6), in which the subjects consumed the fermented beverage for 8 weeks immediately following enrollment, and Group B (n=5), in which the subjects were followed up for 8 weeks following enrollment and then consumed the beverage over the ensuring 8 weeks. The subjects whose Rachmilewitz clinical activity index (CAI) had decreased by ≥1 point were defined as responders, whereas the subjects whose score had either been unchanged or increased were defined as non-responders. A total of 7 subjects (5 in Group A and 2 in Group B) completed the 8-week consumption regimen. No significant changes were observed in the Rachmilewitz CAI and ulcerative colitis endoscopic index of severity prior to and following the consumption period. Regarding the gastrointestinal symptom rating scale (GSRS), the total GSRS score and Question 12, 'Loose stools' were significantly improved following consumption (P=0.042 and 0.048, respectively). Organic acid analysis revealed that the levels of acetic acid, propionic acid and n-butyric acid tended to be continuously higher in the responders than in the non-responders. In conclusion, the fermented vegetable beverage ameliorated loose stool symptoms, although the activity of UC did not improve.

Numerical analyses of intestinal microbiota by data mining.

The human intestinal microbiota has a close relationship with health control and causes of diseases, and a vast number of scientific papers on this topic have been published recently. Some progress has been made in identifying the causes or species of related microbiota, and successful results of data mining are reviewed here. Humans who are targets of a disease have their own individual characteristics, including various types of noise because of their individual life style and history. The quantitatively dominant bacterial species are not always deeply connected with a target disease. Instead of conventional simple comparisons of the statistical record, here the Gini-coefficient (i.e., evaluation of the uniformity of a group) was applied to minimize the effects of various types of noise in the data. A series of results were reviewed comparatively for normal daily life, disease and technical aspects of data mining. Some representative cases (i.e., heavy smokers, Crohn's disease, coronary artery disease and prediction accuracy of diagnosis) are discussed in detail. In conclusion, data mining is useful for general diagnostic applications with reasonable cost and reproducibility.