The aim of this study was to investigate the effect of iron (Fe) availability on butyrate production in the complex bacterial ecosystem of the human gut. Hence, different Fe availabilities were mimicked in an in vitro colonic fermentation model (the polyfermenter intestinal model called PolyFermS) inoculated with immobilized gut microbiota from a child and in batch cultures of the butyrate producer Roseburia intestinalis. Shifts in the microbial community (16S rRNA sequencing and quantitative PCR), metabolic activity (high-performance liquid chromatography), and expression of genes involved in butyrate production were assessed. In the PolyFermS, moderate Fe deficiency resulted in a 1.4-fold increase in butyrate production and a 5-fold increase in butyryl-coenzyme A (CoA):acetate CoA-transferase gene expression, while very strong Fe deficiency significantly decreased butyrate concentrations and butyrate-producing bacteria compared with the results under normal Fe conditions. Batch cultures of R. intestinalis grown in a low-Fe environment preferentially produced lactate and had reduced butyrate and hydrogen production, in parallel with upregulation of the lactate dehydrogenase gene and downregulation of the pyruvate:ferredoxin-oxidoreductase gene. In contrast, under high-Fe conditions, R. intestinalis cultures showed enhanced butyrate and hydrogen production, along with increased expression of the corresponding genes, compared with the results under normal-Fe conditions. Our data reveal the strong regulatory effect of Fe on gut microbiota butyrate producers and on the concentrations of butyrate, which contributes to the maintenance of host gut health.

SummaryStrict anaerobic gut microbes have been suggested as ‘next‐generation probiotics’ for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (−80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant‐free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03–2% in protectant‐free cultures to 11–37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process‐ and species‐specific.

SummaryInterest in faecal microbiota transplantation (FMT) has increased as therapy for intestinal diseases, but safety issues limit its widespread use. Intestinal fermentation technology (IFT) can produce controlled, diverse and metabolically active ‘artificial’ colonic microbiota as potential alternative to common FMT. However, suitable processing technology to store this artificial microbiota is lacking. In this study, we evaluated the impact of the two cryoprotectives, glycerol (15% v/v) and inulin (5% w/v) alone and in combination, in preserving short‐chain fatty acid formation and recovery of major butyrate‐producing bacteria in three artificial microbiota during cryopreservation for 3 months at −80°C. After 24 h anaerobic fermentation of the preserved microbiota, butyrate and propionate production were maintained when glycerol was used as cryoprotectant, while acetate and butyrate were formed more rapidly with glycerol in combination with inulin. Glycerol supported cryopreservation of the Roseburia spp./Eubacterium rectale group, while inulin improved the recovery of Faecalibacterium prausnitzii. Eubacterium hallii growth was affected minimally by cryopreservation. Our data indicate that butyrate producers, which are key organisms for gut health, can be well preserved with glycerol and inulin during frozen storage. This is of high importance if artificially produced colonic microbiota is considered for therapeutic purposes.

The human gut microbiota (HGM) plays a pivotal role in health and disease. Consequently, nutritional and medical research focusing on HGM modulation strategies as a means of improving host health is steadily increasing. In vitro HGM fermentation models offer a valid complement to human and animal studies when it comes to the mechanistic exploration of novel modulation approaches and their direct effects on HGM composition and activity, while excluding interfering host effects. However, in vitro cultivation of HGM can be challenging due to its high oxygen sensitivity and the difficulties of accurately modeling the physio-chemical complexity of the gut environment. Despite the increased use of in vitro HGM models, there is no consensus about appropriate model selection and operation, sometimes leading to major deficiencies in study design and result interpretation. In this review paper, we aim to analyze crucial aspects of the application, setup and operation, data validation and result interpretation of in vitro HGM models. When carefully designed and implemented, in vitro HGM modeling is a powerful strategy for isolating and investigating biotic and abiotic factors in the HGM, as well as evaluating their effects in a controlled environment akin to the gut. Furthermore, complementary approaches combining different in vitro and in vivo models can strengthen the design and interpretation of human studies.

Biofilm-associated, sessile communities represent the major bacterial lifestyle, whereas planktonic cells mainly appear during initial colonization of new surfaces. Previous research, mainly performed with pathogens, demonstrated increased environmental stress tolerance of biofilm-growing compared to planktonic bacteria. The lifestyle-specific stress response of colonic microbiota, both natural and fermentation produced, has not been addressed before. Planktonic and sessile “artificial” colonic microbiota delivered by PolyFermS continuous fermentation models can provide a controllable and reproducible alternative to fecal transplantation in treating gastrointestinal disorders. We therefore characterized planktonic and sessile microbiota produced in two PolyFermS models inoculated with immobilized fecal microbiota and comparatively tested their levels of tolerance of frozen storage (–80°C) and freeze-dried storage (4°C) for 9 months to mimic preservation strategies for therapeutic applications. Sessile microbiota harbored next to shared taxa a unique community distinguishable from planktonic microbiota. Synergistetes and Proteobacteria were highly represented in sessile microbiota, while Firmicutes were more abundant in planktonic microbiota. The community structure and metabolic activity of both microbiota, monitored during standardized reactivation batch fermentations, were better preserved after frozen storage than dried storage, indicated by higher Bray-Curtis similarity and enhanced recovery of metabolite production. For both lifestyles, reestablishment of Bacteroidaceae was impaired after frozen and dried storage along with reduced propionate formation. In contrast, butyrate production was maintained after reactivation despite compositional rearrangements within the butyrate-producing community. Unexpectedly, the rate of recovery of metabolite production was lower after preservation of sessile than planktonic microbiota. We speculate that higher functional dependencies between microbes might have led to the lower stress tolerance of sessile than planktonic microbiota. IMPORTANCE Fecal microbiota transplantation has been successfully applied in the treatment of recurrent Clostridium difficile infection and has been suggested as an alternative therapy for other intestinal disorders such as inflammatory bowel disease or metabolic syndrome. “Artificial” colonic microbiota delivered by PolyFermS continuous fermentation models can provide a controllable and reproducible alternative to fecal transplantation, but effective preservation strategies must be developed. In this study, we systematically investigated the response of sessile and planktonic artificial colonic microbiota to cryopreservation and lyophilization. We suggest that functional redundancy is an important factor in providing functional stability with respect to exposure to stress during processing and storage. Functional redundancy in compositionally reduced microbial systems may be considered when designing microbial products for therapy.

Dietary fibers such as arabinoxylan (AX) are promising food constituents to prevent particular diet-related chronic diseases because of their prebiotic properties. Arabinoxylan fermentation by the gut microbiota depends on the structural architecture of AX, which can be modified during food processing and consequently affect its prebiotic potential, but it is little investigated. Therefore, the aim of this study was to evaluate the effects of naturally occurring and processing-induced structural alterations of the soluble AX of wheat bran and rye flour on the in vitro human colon fermentation. It was found that fermentation behavior is strongly linked to the AX fine structure and their processing-induced modifications. The short-chain fatty acid (SCFA) metabolism, acidification kinetics, bacterial growth, and bacterial composition revealed that wheat bran AX (WBAX) was fermented faster than rye flour AX. Increased levels of bound phenolic acids resulting from processing were identified as the inhibiting factor for AX fermentation kinetics. Bacterial genera promoted by AX varied between AX source and processing type, but also between microbiota. Extruded WBAX promoted butyrate production and growth of butyrate-producing Faecalibacterium in the butyrogenic microbiota while it did not enhance fermentation and inhibited the growth of Prevotella in the propiogenic microbiota. We anticipate that the findings of this study are a starting point for further investigation on the impact of processing-induced changes on the prebiotic potential of dietary fibers prior to human studies.

No abstract

No abstract