assessing-viability-synthetic-bacterial-consortium-on-vitro-gut-host

Assessing the Viability of a Synthetic Bacterial Consortium on the <em>In Vitro</em> Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Ghent University

The methane removal capacity of mixed methane-oxidizing bacteria (MOB) culture in a biofilter setup using autoclaved aerated concrete (AAC) as a highly porous carrier material was tested. Batch experiment was performed to optimize MOB immobilization on AAC specimens where optimum methane removal was obtained when calcium chloride was not added during bacterial inoculation step and 10-mm-thick AAC specimens were used. The immobilized MOB could remove methane at low concentration (~1000 ppmv) in a biofilter setup for 127 days at average removal efficiency (RE) of 28.7 %. Unlike a plug flow reactor, increasing the total volume of the filter by adding a biofilter in series did not result in higher total RE. MOB also exhibited a higher abundance at the bottom of the filter, in proximity with the methane gas inlet where a high methane concentration was found. Overall, an efficient methane biofilter performance could be obtained using AAC as the carrier material.

Methane biofiltration using autoclaved aerated concrete as the carrier material.

Volatile fatty acids (VFA) are building blocks for the chemical industry. Sustainable, biological production is constrained by production and recovery costs, including the need for intensive pH correction. Membrane electrolysis has been developed as an in situ extraction technology tailored to the direct recovery of VFA from fermentation while stabilizing acidogenesis without caustic addition. A current applied across an anion exchange membrane reduces the fermentation broth (catholyte, water reduction: H2O + e(-) → ½ H2 + OH(-)) and drives carboxylate ions into a clean, concentrated VFA stream (anolyte, water oxidation: H2O → 2e(-) + 2 H(+) + O2).

Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation.

The choice of soilless growing medium for plant nutrition, growth and support is crucial for improving the eco-sustainability of the production in horticultural systems. As our current understanding of the functional microbial communities inhabiting this ecosystem is still limited, we examined the microbial community development of the two most important growing media (organic and mineral) used in open soilless horticultural systems. We aimed to identify factors that influence community composition over time, and to compare the distribution of individual taxa across growing media, and their potential functionality. High throughput sequencing analysis revealed a distinctive and stable microbial community in the organic growing medium. Humidity, pH, nitrate-N, ammonium-N and conductivity were uncovered as the main factors associated with the resident bacterial communities. Ammonium-N was correlated with Rhizobiaceae abundance, while potential competitive interactions among both Methylophilaceae and Actinobacteridae with Rhizobiaceae were suggested. Our results revealed that soilless growing media are unique niches for diverse bacterial communities with temporal functional stability, which may possibly impact the resistance to external forces. These differences in communities can be used to develop strategies to move towards a sustainable horticulture with increased productivity and quality. 

Mineral and organic growing media have distinct community structure, stability and functionality in soilless culture systems.

The objectives of this study are to identify oral commensal species which can inhibit the growth of the main periodontopathogens, to determine the antimicrobial substances involved in these inhibitory activities and to evaluate the influence of environmental factors on the magnitude of these inhibitions.

Antimicrobial effects of commensal oral species are regulated by environmental factors.

Vegetables and fruits are an important part of a healthy food diet, however, the eco-sustainability of the production of these can still be significantly improved. European farmers and consumers spend an estimated €15.5 billion per year on inorganic fertilizers and the production of N-fertilizers results in a high carbon footprint. We investigated if fertilizer type and medium constituents determine microbial nitrogen conversions in organic growing media and can be used as a next step towards a more sustainable horticulture. We demonstrated that growing media constituents showed differences in urea hydrolysis, ammonia and nitrite oxidation and in carbon dioxide respiration rate. Interestingly, mixing of the growing media constituents resulted in a stimulation of the function of the microorganisms. The use of organic fertilizer resulted in an increase in amoA gene copy number by factor 100 compared to inorganic fertilizers. Our results support our hypothesis that the activity of the functional microbial community with respect to nitrogen turnover in an organic growing medium can be improved by selecting and mixing the appropriate growing media components with each other. These findings contribute to the understanding of the functional microbial community in growing media and its potential role towards a more responsible horticulture. 

Growing media constituents determine the microbial nitrogen conversions in organic growing media for horticulture.

High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.

Absolute quantification of microbial taxon abundances.

Polyunsaturated fatty acids (PUFAs) may affect colon microbiome homeostasis by exerting (specific) antimicrobial effects and/or interfering with mucosal biofilm formation at the gut mucosal interface. We used standardized batch incubations and the Mucosal-Simulator of the Human Microbial Intestinal Ecosystem (M-SHIME) to show the in vitro luminal and mucosal effects of the main PUFA in the Western diet, linoleic acid (LA). High concentrations of LA were found to decrease butyrate production and Faecalibacterium prausnitzii numbers dependent on LA biohydrogenation to vaccenic acid (VA) and stearic acid (SA). In faecal batch incubations, LA biohydrogenation and butyrate production were positively correlated and SA did not inhibit butyrate production. In the M-SHIME, addition of a mucosal environment stimulated biohydrogenation to SA and protected F. prausnitzii from inhibition by LA. This was probably due to the preference of two biohydrogenating genera Roseburia and Pseudobutyrivibrio for the mucosal niche. Co-culture batch incubations using Roseburia hominis and F. prausnitzii validated these observations. Correlations networks further uncovered the central role of Roseburia and Pseudobutyrivibrio in protecting luminal and mucosal SHIME microbiota from LA-induced stress. Our results confirm how cross-shielding interactions provide resilience to the microbiome and demonstrate the importance of biohydrogenating, mucosal bacteria for recovery from LA stress.

Mucosa-associated biohydrogenating microbes protect the simulated colon microbiome from stress associated with high concentrations of poly-unsaturated fat.

Dysbiosis in the periodontal microbiota is associated with the development of periodontal diseases. Little is known about the initiation of dysbiosis. It was hypothesized that some commensal bacteria suppress the outgrowth of pathobionts by HO production. However, serum and blood components released due to inflammation can neutralize this suppressive effect, leading to the initiation of dysbiosis. Agar plate, dual-species and multi-species ecology experiments showed that HO production by commensal bacteria decreases pathobiont growth and colonization. Peroxidase and blood components neutralize this inhibitory effect primarily by an exogenous peroxidase activity without stimulating growth and biofilm formation of pathobionts directly. In multi-species environments, neutralization of HO resulted in 2 to 3 log increases in pathobionts, a hallmark for dysbiosis. Our data show that in oral biofilms, commensal species suppress the amounts of pathobionts by HO production. Inflammation can neutralize this effect and thereby initiates dysbiosis by allowing the outgrowth of pathobionts.

Dysbiosis by neutralizing commensal mediated inhibition of pathobionts.

To identify potential oral prebiotics that selectively stimulate commensal, albeit beneficial bacteria of the resident oral microbial community while suppressing the growth of pathogenic bacteria.

Nutritional stimulation of commensal oral bacteria suppresses pathogens: the prebiotic concept.

The link between gut microbiota and human health is well-recognized and described. This ultimate impact on the host has contributed to explain the mutual dependence between humans and their gut bacteria. Gut microbiota can be manipulated through passive or active strategies. The former includes diet, lifestyle, and environment, while the latter comprise antibiotics, pre- and probiotics. Historically, conventional probiotic strategies included a phylogenetically limited diversity of bacteria and some yeast strains. However, biotherapeutic strategies evolved in the last years with the advent of fecal microbiota transplant (FMT), successfully applied for treating CDI, IBD, and other diseases. Despite the positive outcomes, long-term effects resulting from the uncharacterized nature of FMT are not sufficiently studied. Thus, developing strategies to simulate the FMT, using characterized gut colonizers with identified phylogenetic diversity, may be a promising alternative. As the definition of probiotics states that the microorganism should have beneficial effects on the host, several bacterial species with proven efficacy have been considered next generation probiotics. Non-conventional candidate strains include , and members of the Clostridia clusters IV, XIVa, and XVIII. However, viable intestinal delivery is one of the current challenges, due to their stringent survival conditions. In this review, we will cover current perspectives on the development and assessment of next generation probiotics and the approaches that industry and stakeholders must consider for a successful outcome.

Emerging Trends in "Smart Probiotics": Functional Consideration for the Development of Novel Health and Industrial Applications.

Dietary supplementation of docosahexaenoic acid (DHA)-enriched products inhibits the final step of biohydrogenation in the adult rumen, resulting in the accumulation of 18:1 isomers, particularly of ()-11 18:1. Occasionally, a shift toward the formation of 10 intermediates at the expense of 11 intermediates can be triggered. However, whether similar impact would occur when supplementing DHA-enriched products during pregnancy or early life remains unknown. Therefore, the current  study aimed to investigate the effect of a nutritional intervention with DHA in the early life of goat kids on rumen biohydrogenation and microbial community. Delivery of DHA was achieved by supplementing DHA-enriched microalgae (DHA Gold) either to the maternal diet during pregnancy (prenatal) or to the diet of the young offspring (postnatal). At the age of 12 weeks, rumen fluid was sampled for analysis of long-chain fatty acids and microbial community based on bacterial 16S rRNA amplicon sequencing. Postnatal supplementation with DHA-enriched microalgae inhibited the final biohydrogenation step, as observed in adult animals. This resulted particularly in increased ruminal proportions of 11 18:1 rather than a shift to 10 intermediates, suggesting that both young and adult goats might be less prone to dietary induced shifts toward the formation of 10 intermediates, in comparison with cows. Although  species have been identified as the most important biohydrogenating bacteria, this genus was more abundant when complete biohydrogenation, i.e. 18:0 formation, was inhibited.  abundance was positively correlated with 18:0 accumulation, whereas  spp.  spp. and  spp. were more abundant in situations with greater 10 accumulation. Extensive comparisons made between current results and literature data indicate that current associations between biohydrogenation intermediates and rumen bacteria in young goats align with former observations in adult ruminants.

Article	Year
Methane biofiltration using autoclaved aerated concrete as the carrier material. Applied microbiology and biotechnology\| PubMed ID: 25967655	2015
Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation. Biotechnology for biofuels\| PubMed ID: 26697110	2015
Mineral and organic growing media have distinct community structure, stability and functionality in soilless culture systems. Scientific reports\| PubMed ID: 26728128	2016
Antimicrobial effects of commensal oral species are regulated by environmental factors. Journal of dentistry\| PubMed ID: 26875613	2016
Growing media constituents determine the microbial nitrogen conversions in organic growing media for horticulture. Microbial biotechnology\| PubMed ID: 27005434	2016
Absolute quantification of microbial taxon abundances. The ISME journal\| PubMed ID: 27612291	2017
Mucosa-associated biohydrogenating microbes protect the simulated colon microbiome from stress associated with high concentrations of poly-unsaturated fat. Environmental microbiology\| PubMed ID: 27883264	2017
Dysbiosis by neutralizing commensal mediated inhibition of pathobionts. Scientific reports\| PubMed ID: 27897256	2016
Nutritional stimulation of commensal oral bacteria suppresses pathogens: the prebiotic concept. Journal of clinical periodontology\| PubMed ID: 28128467	2017
Emerging Trends in "Smart Probiotics": Functional Consideration for the Development of Novel Health and Industrial Applications. Frontiers in microbiology\| PubMed ID: 29033923	2017
Rumen Biohydrogenation and Microbial Community Changes Upon Early Life Supplementation of 22:6-3 Enriched Microalgae to Goats. Frontiers in microbiology\| PubMed ID: 29636742	2018

Emma Hernandez-Sanabria

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