Continuous Fermentation System Research Articles

Impact of cryoprotective agents on human gut microbes and invitro stabilized artificial gut microbiota communities.

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for invitro investigation of the molecular mechanisms behind microbe-microbe and/or microbe-host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at -80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through invitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species-specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota.

Continuous fermentation using high cell density cell recycle system for L-lactic acid production.

For complete utilization of high glucose at ∼100 g/L, a high cell density (HCD) continuous fermentation system was established using Lb. delbrueckii NCIM 2025 for the bioproduction of lactic acid (LA). An integrated membrane cell recycling system coupled with the continuous bioreactor, aided to achieve the highest 34.77 g/L h LA productivity and 0.94-0.98 g/g yield. ∼34 times higher productivity was observed (in comparison to batch fermentation conducted in this study), when the continuous operations were carried out at the maximum dilution rate and wet cell weight i.e. 0.36 h-1 and 230 g/L, respectively. These results show the potential of this method for large-scale lactic acid production because it not only produces high titers but also ensures that glucose is used effectively. The method's superior performance in comparison to earlier studies suggests it as an affordable and sustainable alternative for the production of LA.

The short-term and long-term effects of Dendrobium officinale leaves polysaccharides on the gut microbiota differ

The effect of active substances on the gut microbiota is not immediate, especially for some chronic diseases. This study used an in vitro continuous fermentation system to monitor the changes of gut microbiota in three different colon regions under long-term/short-term DOLP treatment. The results showed that the microbial diversity reached peak on 5th day and remained stable thereafter. Beta diversity analysis showed that the gut microbiota structures of the three regions gradually approached during fermentation. The contents of propionic acid, butyric acid, and valeric acid first increased and then decreased, while isobutyric acid and isovaleric acid first decreased and then increased. Alistipes and Oscillibacter were the key genera in regulating the gut microbiota structure. In summary, Long-term consumption of DOLP has varying effects on the gut microbiota over time, and for the purpose of adjusting the gut microbiota, the most effective duration appears to be continuous consumption for 5 days.

Multiscale kinetic modeling for biohydrogen production: A study on membrane bioreactors

Hydrogen can be a capable alternative to fossil fuels due to its carbon-free characteristics, in this content, biological hydrogen production is considered a practical approach because technology is green. Due to parameters affecting biohydrogen production, such as operating conditions, it is crucial to predict the process to see the proper yield. There are several conventional and unconventional models used in biohydrogen production prediction. This paper derived a triple first-order prediction model from a previously presented multi-scale kinetic model polynomial built upon the multi-stage growth hypothesis for bio-hydrogen production prediction. The original model was applied to batch and continuous stirred tank reactor studies for their model evaluation, this study evaluated the newly derived model for studies of membrane bioreactors. Due to their increased production yield, membrane bioreactors are an emerging field in biohydrogen production. Although the previous study was mainly applied for batch dark fermentations consisting of various microorganisms, the results presented in this study indicate that it is also applicable for continuous and photo fermentation systems. The original model results reported significant fitness accuracy among different datasheets compared to conventional models like the modified Gompertz model, considering essential factors impacting biohydrogen production suggested in the original model, this paper investigated eleven case studies of dynamic membrane bioreactors with modeling fitness of 99% for most cases. This study reports even higher fitness accuracy compared to the original model, even with different operating conditions.

Effects of Non-Protein Nitrogen Sources on In Vitro Rumen Fermentation Characteristics and Microbial Diversity

Here, the effects of non-protein nitrogen sources on fermentation parameters and microbial diversity were explored using three fistula goats as rumen fluid donors. The experiments involved six fermenters in a replicated 3 × 3 Latin square design with three dietary non-protein sources [ammonium chloride (A), biuret (B), and glutamine (G)] as treatment factors. A dual-flow continuous culture fermentation system was used. Microbial protein content in group B was significantly lower than that in the other two groups (P < 0.05). Ammonia nitrogen concentration significantly differed among the three groups (P < 0.01), following the order of G > A > B group. The acetate-to-propionate ratio in group G was significantly lower than that in the other two groups (P < 0.01). At the phylum level, the relative abundances of Cyanobacteria, Elusimicrobia, and Armatimonadetes were the highest in group G, being significantly higher than those in group B (P < 0.05). At the genus level, the relative abundance of Ruminococcus_1 was significantly higher in group A than in group B (P < 0.05). Overall, glutamine shifted the fermentation pathway from acetate to propionate, and the lower microbial crude protein content and relative abundances of the major fiber-degrading bacteria Ruminococcus_1 and protein-degrading bacteria Prevotellaceae_UCG-001 in group B indicate that biuret is not suitable as a dietary non-protein nitrogen source.

Impact of Food Origin Lactiplantibacillus plantarum Strains on the Human Intestinal Microbiota in an in vitro System.

We have previously isolated and characterized food-dwelling strains of Lactiplantibacillus (Lpb.) plantarum that are consumed naturally as part of the microbiota of table olives and raw milk cheeses. Despite being consumed at relatively high levels, the impact of such strains on the human gut microbiota is currently unclear. In the current study we evaluated the potential impact of food-dominant Lpb. plantarum strains on the human gut microbiota using a continuous fecal fermentation system. Daily inoculation of Lpb. plantarum strains led to significant, detectable levels in the fecal fermentation system. We examined the impact of the presence of Lpb. plantarum on the microbiota derived from two separate donors. For one donor, Lpb. plantarum increased alpha diversity and beta diversity. This was reflected in significant alterations in abundance of the unclassified genera, dominated by Enterobacteriaceae_unclass and Ruminococcaceae_unclass. The microbiota of the other donor was relatively unaffected following introduction of the Lpb. plantarum strains. Overall, the work describes the response of the human microbiota to the introduction of high levels of food-dominant Lpb. plantarum strains and indicates that the response may reflect interindividual differences between donor samples.

A novel integrated fermentation/recovery system for butanol production by Clostridium acetobutylicum

Intensive butanol production was carried out by immobilized cells of Clostridium acetobutylicum in a novel continuous fermentation system. The fermentation system consisted of four packed bed biofilm reactors (PBBR) connected in series. The novelty of the proposed system is: i) the use of a novel stagewise simulated moving bed (SMB) design/operational mode; ii) the integration of the reaction and the product recovery sections. The SMB operational mode was accomplished by equipping the PBBR with manifolds and time-actuated switching valves. PBBR integration with the product recovery section was also tested. An adsorption column was operated between the 3rd and 4th reactor of the series.The tuning of the PBBR system overall dilution rate (DOV) allowed to maximize: i) sugar conversion (DOV <0.150 h−1); ii) butanol concentration (0.650 h−1<DOV< 0.900 h−1); iii) butanol productivity (13.0 g/Lh at DOV = 0.900 h−1).The PBBR system was upgraded by integrating it with an in-line adsorption column and it guaranteed a twofold increase in butanol productivity (up to 22 g/Lh) and an average butanol concentration close to 24.0 g/L.

Model development and optimal control of a continuous packed bed bioreactor for the production of succinic acid

A dynamic model consisting of a system of partial differential equations, was created in order to describe and simulate the behaviour of a continuous immobilised-cell fermentation system for the production of succinic acid from glycerol. The model can confidently predict the steady state output of the process under a range of different initial and operating conditions. Moreover the model results, are verified by experimental results, further strengthening the credibility of the model. Then the constructed model was utilised to build a model reduction-based stochastic optimal control strategy for the complex bio-process. Proper Orthogonal Decomposition and artificial neural networks were employed to address the high dimensionality of the model, in a 2-step reduction scheme, and polynomial chaos expansion to address model uncertainty. Computational results show that the reduced model-based stochastic control policy can efficiently improve production satisfying process requirements for byproduct concentrations.

Establishment of an In Vitro System of the Human Intestinal Microbiota: Effect of Cultivation Conditions and Influence of Three Donor Stool Samples.

Fecal microbiota transplantation (FMT) is an alternative method for the treatment of gastrointestinal diseases with a high recovery rate. Disadvantages are ethical concerns, high donor requirements and the low storability of stool samples. The cultivation of an in vitro microbiota in a continuous bioreactor was established as an alternative to FMT to overcome these problems. In this study, the influence of the system parameters and donor stool characteristics was investigated. Each continuous colonic fermentation system was inoculated with feces from three different donors until a stable state was established. The influence of the fermentation conditions on the system’s behavior regarding cell count, metabolic activity, short-chain fatty acid profile and microbiota composition as well as richness and diversity was assessed. Cultivation conditions were found to affect the microbial system: the number of cells and the production of short-chain fatty acids increased. The abundance of Actinobacteria and Firmicutes decreased, Bacteroidetes increased, while Proteobacteria and Verrucomicrobia remained largely unaffected. Diversity in the in vitro system decreased, but richness was unaffected. The cultivation of stool from different donors revealed that the performance of the created in vitro system was similar and comparable, but unique characteristics of the composition of the original stool remained.

Changes in the volatile composition of apple and apple/pear ciders affected by the different dilution rates in the continuous fermentation system

The research concentrated on the analysis of biochemical parameters and volatile composition of the ciders prepared from two Polish apple varieties (Gala and Idared) with and without addition of the pear juice (Konferencja). Ciders have been prepared in a continuous fermentation system using FS-V-A bioreactor with different dilution rates (ranging from 0.0050 h−1 to 0.2222 h−1).Volatilome of ciders has been analysed by the means of headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry (HS-SPME-GC-MS). Performed analyses have shown that apple variety, dilution rate of fermenting cider, as well as pear juice addition has significant influence on the composition of the acquired cider volatiles. Ciders fermented at a lower dilution rate were characterised by a higher concentration of compounds such as ethyl decanoate, phenylethyl alcohol, while a higher dilution rate resulted in higher concentration of 2-hexanal, ethyl hexanoate, pentyl octanoate and (Z, E)-farnesene. Pear addition increased concentration of (2E, 4Z)-deca-2,4-dienoate and ethyl octanoate in the ciders.

Development of a Cell-recycled Continuous Fermentation Process for Enhanced Production of Succinic Acid by High-yielding Mutants of Actinobacillus succinogenes

Succinic acid (SA), a four-carbon dicarboxylic acid utilized as a platform chemical for valuable industrial products, is a major fermentation product of Actinobacillus succinogenes cells, synthetized during anaerobic metabolism. In this study, cell-recycled continuous fermentation (CRCF) was carried out in order to maximize the appreciably stable biocatalytic activity of SA-producing cells and the growth-associated mode of SA biosynthesis. Stable and long operations of CRCF could be carried out through an efficient decanter system developed in our laboratory, which could effectively separate highly dense microbial cells from the outlet stream. This allowed to overcome the wash-out phenomenon encountered at relatively low dilution rates in conventional continuous fermentation systems without cell recycling. Through careful assessment of the effects of dilution rate, composition of feeding medium, and cell recycling ratio on SA yield via the CRCF process, volumetric SA productivity could be enhanced to 3.86 g/(L·h), an approximately 5.1-fold increase compared to parallel continuous fermentation without cell recycling. A higher dilution rate and a 24% increment in SA production via increased density of active cells inside the bioreactor of the CRCF system were the probable factors inducing such a considerable enhancement in volumetric SA productivity during CRCF. Since volumetric productivity is the most important parameter determining the cost-effectiveness of a given fermentation bioprocess, it is quite evident that CRCF is a promising alternative to conventional batch or continuous fermentation without cell recycling for mass production of SA.

Comparison of bio-hydrogen and bio-methane production performance in continuous two-phase anaerobic fermentation system between co-digestion and digestate recirculation

The effect of co-digestion of food waste (FW) and cow dung (CD) with different ratios, and digestate recirculation with different recirculation ratios (RR) on the substrate degradation and energy production in continuous two-stage anaerobic fermentation system was investigated. Results from experiments indicated that co-digestion and digestate recirculation could promote the hydrogen production rate (HPR) and the methane production rate (MPR). Maximum HPR and MPR of 3.3 and 3.1 L/L/d were achieved for two-stage fermentation with recirculation system at RR of 0.4. Meanwhile, both co-digestion and digestate recirculation technology could reduce the amount of alkali addition to maintain pH in the hydrogen-reactor. Compared to digestate recirculation, co-digestion of FW and CD promote much more energy production, 654.9 and 4854.8 kJ/kgVSr were obtained from the co-digestion of FW and CD with the ratio of 2:1 in the hydrogen reactor and the methane reactor.

High L(+)-lactic acid productivity in continuous fermentations using bakery waste and lucerne green juice as renewable substrates

The biotechnological production of platform chemicals from renewable resources is in the scientific spotlight, as researchers seek to develop environmentally friendly and cost-efficient processes to compete with the petroleum-based ones. Lactic acid (LA) is an established platform chemical, registering an important market share, mainly owing to the increasing demand for polylactic acid. This study investigated the feasibility to produce LA from bakery waste hydrolysates and lucerne green juice (LGJ) as inexpensive substrates, using a Bacillus coagulans strain. A final LA concentration of 62.2 g/L, with a productivity of 2.59 g/(L.h) and a conversion yield of 0.57 g LA/ g bakery waste was achieved in batch fermentation mode. LA productivity reached 11.28 g/(L.h), using a continuous fermentation system coupled with cell retention membranes at a dilution rate of 0.2 h−1. The results indicate that bakery waste hydrolysates and LGJ can be utilized for the production of highly optical pure L(+)-LA.

3-phenyllactic acid productionby free-whole-cells of Lactobacillus crustorum in batch and continuous fermentation systems.

3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve3-PLA yields in batch and continuous fermentation systems using of free-whole-cellsofthe threeL.crustorum strains. The fermentation conditions of free-whole-cells of thethree L. crustorum strains for3-PLA productionwereoptimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P<0·05) greater 3-PLA production ability than the other strains after 10th recycle. The strain possesses three l-lactate dehydrogenase and three d-lactate dehydrogenase catalysing 3-PLA production from phenylpyruvic acid (PPA). Under the optimal conditions, thestrain produced 15·2mmoll-1 3-PLA (76% PPA conversion rate) in a batch fermentation system and 6·5mmoll-1 h-1 3-PLA (55% PPA conversion rate) in a continuous fermentation system using a 0·6 dilution rate. Free-whole-cells of L. crustorum NWAFU 1078 showed excellent reusability and higher 3-PLA yields under optimal biotransformation conditions in both batch and continuous fermentation systems. This study provides the possibility to use the free-whole-cells of L. crustorum NWAFU 1078 as a biocatalyst for effective production of 3-PLA.

81 Effects of engineered biocarbons on total gas and methane production, rumen fermentation and microbial protein synthesis in a semi continuous fermentation system (RUSITEC)

Abstract This study investigated the effects of engineered biocarbon on nutrient digestibility, rumen fermentation, total gas and methane (CH4) emissions, and microbial protein synthesis in a rumen simulation technique (RUSITEC) fed a barley silage-based TMR. The basal diet consisted of 60% barley silage, 27% barley grain, 10% canola meal and 3% minerals. Three pine-based biocarbon products CP016, CP024 and CP028. were added at 2% of substrate DM. Biocarbons differed in bulk density, surface area, pore volume, pH, but had similar chemical compositions. Treatments were assigned to sixteen vessels (n = 4/treatment) in two RUSITEC apparatuses in a randomized block design. The experiment period was 17 d, with a 10-d adaptation and 7-d sample collection period. Data were analyzed using the PROC MIXED in SAS, with treatment (T), day (D) and TxD interactions as fixed effects and RUSITEC apparatus and fermenters as random effects. Compared to the control, biocarbon did not affect total gas (P = 0.98), the amount of CH4 produced per unit of DM incubated (P = 0.48) or per unit of DM digested (P = 0.27). Biocarbon treatments averaged 6.5 g of CH4 /g DM incubated and 9.06 g CH4 /g DM digested as compared to 7.1 g of CH4 /g DM incubated and 10.46 g CH4 / g DM digested in the control, respectively. Biocarbon CP024 had the greatest numerical reduction, followed by CP028 then CP016 in all CH4 associated parameters. Biocarbon addition did not affect the disappearance of DM (P = 0.63), OM (P = 0.34), CP (P = 0.48), NDF (P = 0.12), or VFA (P = 0.65) and ammonia N levels (P = 0.99) and protozoal counts (P = 0.72). The amount of bacterial nitrogen (mg/d) associated with feed particles increased (P &amp;lt; 0.003), suggesting that biocarbon may have enhanced colonization. In conclusion, engineered biocarbon did not reduce CH4 emissions in the RUSITEC.

Optimization of the in situ recovery of butanol from ABE fermentation broth via membrane pervaporation

Butanol produced via the ABE fermentation is plagued with low final concentrations and low yield. The selective removal of butanol from the fermentation broth by integrating a separation process to the fermenter for the in situ recovery of butanol has been proposed by many researchers. In this investigation, the integration of a membrane pervaporation separation process with the continuous ABE fermentation system has been simulated and optimized using a multi-objective genetic algorithm. The ABE fermentation model proposed by Mulchandani and Volesky was used and the multi-objective optimization problem was defined to simultaneously maximize the butanol productivity, the overall butanol concentration and the sugar conversion. The three objective functions, if non-dominated, define the Pareto domain of the optimization problem along with the four decision variables, namely the dilution rate, the feed sugar concentration, the cell retention factor and the membrane surface area. The optimal solutions of the integrated process for two different pervaporation membrane models were compared to the stand-alone continuous fermentation. By adding an in situ separation system to the continuous ABE fermentation, the optimal butanol productivity and overall butanol concentration increased by approximately 250% compared to those of the non-integrated fermenter. Furthermore, the sugar conversion also increased.

Evaluation of a single-flow continuous culture fermenter system for determination of ruminal fermentation and enteric methane production.

A 4-unit, single-flow continuous culture fermenter system was developed to assess in vitro nutrient digestibility, volatile fatty acid (VFA) concentration and daily enteric methane (CH4 ) production of ruminant diets. The objective was to develop a closed-vessel system that maintained protozoal populations and provided accurate predictions of total CH4 production. A diet of 50% orchardgrass (Dactylis glomerata L.) and 50% alfalfa (Medicago sativa L.) was fed during 4, 10-day periods (7-day adaptation and 3-day collection). Fermenters were fed 82g of dry matter (DM)/day in four equal feedings. pH and temperature were taken every 2min, and CH4 concentration was measured every 10min. Samples for DM and protozoal counts were taken daily, and daily effluent samples were collected for determination of DM, VFA and NH3 -N concentrations. There was no effect (p>0.17) of adaptation versus collection days on vessel and effluent DM, temperature or pH. Initial protozoal counts decreased (p<0.01), but recovered to initial counts by the collection period. Total VFA, acetate, propionate and isobutyrate concentrations did not differ (p≥0.13) among periods or days of the collection period. There was no difference (p≥0.37) among days or periods in total daily CH4 production and CH4 production per g of OM, NDF, digestible OM or digestible NDF fed. Data collected throughout 4 experimental periods demonstrated that the system was able to reach a steady state in fermentation well within the 7-day adaptation period and even typically variable data (i.e., CH4 production) were stable within and across periods. While further research is needed to determine the relationship between this system and in vivo data, this continuous culture fermenter system provides a valid comparison of in vitro ruminal fermentation and enteric CH4 production of ruminant diets that can then be further validated with in vivo studies.

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of Thermotoga maritima

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H2 production limit. The study generated a kinetic model explaining H2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H2 production by Tma200 (0.217 mol-H2 g−1-biomass) and Tma100 (0.147 mol-H2 g−1-biomass) were higher than wild type (0.096 mol-H2 g−1 -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h−1) and Tma100 (0.495 h−1) are compared with the wild type (0.663 h−1). A higher maintenance energy along with growth and non-growth H2 coefficients corroborate the higher H2 productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H2 production.

Effects of live yeast (Saccharomyces cerevisiae) and type of cereal on rumen microbial fermentation in a dual flow continuous culture fermentation system.

Live yeast additives may help optimize ruminal fermentation of high-grain diets, especially when the starch of the ration is highly fermentable. Eight dual flow continuous culture fermenters were used in a 2×2 factorial design in two replicated periods of 9days (six for adaptation and three for sampling) to determine the effect of live yeast and type of cereal on rumen microbial fermentation and nutrient digestibility. Main factors were the addition of live yeast: no yeast (NY) vs. 2×107 CFU of yeast/g of diet (LY); and type of cereal in the diet: corn (CO) vs. barley (BA). All fermenters were fed 80g dry matter/day of a 10-90 forage to concentrate diet and pH was allowed to fluctuate with an upper (6.6) and lower (5.5) limit. Treatment BA increased OM digestion, valerate proportion, peptides and ammonia N fractions, ammonia N flow, crude protein degradation and target copies of Megasphaera elsdenii; and decreased NDF digestion, propionate proportion, branched-chain VFA (BCVFA) concentration, AA-N fraction and nonammonia N flow. Treatment LY increased BCVFA and decreased ammonia N fraction and flow, and the target copies of Streptococcus bovis. Treatment LY decreased the slope of pH drop, the area under pH 6.0 and the gas production. These results suggest potential benefits of LY in stabilizing the fermentation of BA-based diets.

Valorization of spent sulphite liquor for succinic acid production via continuous fermentation system

Spent sulphite liquor has been evaluated in continuous cultures for succinic acid production using Actinobacillus succinogenes and Basfia succiniciproducens. Continuous cultures were initially carried out at constant dilution rate (0.04 h−1) and varying commercial xylose concentrations (23–55 g/L) or constant xylose concentration (40 g/L) and varying dilution rates (0.02–0.25 h−1) showing that dilution rates of 0.02–0.15 h−1 led to satisfactory succinic acid production by both strains. In continuous cultures using nanofiltrated spent sulphite liquor, the highest yields were achieved at dilution rate of 0.02 h−1 (0.48 g/g for A. succinogenes and 0.55 g/g for B. succiniciproducens), while the highest productivities were obtained at dilution rates of 0.04 h−1 for A. succinogenes cultures (0.67 g/L/h) and 0.1 h−1 for B. succiniciproducens cultures (1.6 g/L/h). Metabolic flux analysis demonstrated higher biomass concentrations in A. succinogenes cultures carried out in both xylose and nanofiltrated spent sulphite liquor, while B. succiniproducens cultures were more robust regarding succinic acid production efficiency in either xylose or nanofiltrated spent sulphite liquor.