Supplementing Glycerol to Inoculum Induces Changes in pH, SCFA Profiles and Microbiota Composition in In-Vitro Batch Fermentation

Glycerol was generally added to the inoculum as a cryoprotectant. However, it was also a suitable substrate for microbial fermentation, which may produce more SCFAs, thereby decreased pH of the fermentation broth. This study investigated the effect of supplementing glycerol to inoculum on in vitro fermentation and whether an enhanced buffer capacity of medium could maintain the pH stability during in vitro batch fermentation, subsequently improving the accuracy of short chain fatty acids (SCFAs) determination, especially propionate. Two ileal digesta were fermented by pig fecal inoculum with or without glycerol (served as anti-frozen inoculum or frozen inoculum) in standard buffer or enhanced buffer solution (served as normal or modified medium). Along with the fermentation, adding glycerol decreased the pH of fermentation broth (p < 0.05). However, modified medium could alleviate the pH decrement compared with normal medium (p < 0.05). The concentration of total propionic acid production was much higher than that of other SCFAs in anti-frozen inoculum fermentation at 24 and 36 h, thereby increasing the variation (SD) of net production of propionate. The α-diversity analysis showed that adding glycerol decreased Chao1 and Shannon index under normal medium fermentation (p < 0.05) compared to modified medium (p < 0.05) along with fermentation. PCoA showed that all groups were clustered differently (p < 0.01). Adding glycerol improved the relative abundances of Firmicutes, Anaerovibrio, unclassified_f_Selenomonadaceae, and decreased the relative abundance of Proteobacteria (p < 0.05). The relative abundances of Firmicutes, such as Lactobacillus, Blautia and Eubacterium_Ruminantium_group in modified medium with frozen inoculum fermentation were higher than (p < 0.05) those in normal medium at 36 h of incubation. These results showed that adding glycerol in inoculum changed the fermentation patterns, regardless of substrate and medium, and suggested fermentation using frozen inoculum with modified medium could maintain stability of pH, improve the accuracy of SCFA determination, as well as maintain a balanced microbial community.

Evaluation of the Antibacterial and Prebiotic Potential of Ascophyllum nodosum and Its Extracts Using Selected Bacterial Members of the Pig Gastrointestinal Microbiota

Ascophyllum nodosum and its extracts are promising antibacterial and prebiotic dietary supplements for pigs. The objectives of this study were to evaluate the effects of the increasing concentrations of: (1) two whole biomass samples of A. nodosum with different harvest seasons, February (ANWB-F) and November (ANWB-N), in a weaned pig faecal batch fermentation assay, and (2) A. nodosum extracts produced using four different extraction conditions of a hydrothermal-assisted extraction methodology (ANE1–4) and conventional extraction methods with water (ANWE) and ethanol (ANEE) as solvent in individual pure culture growth assays using a panel of beneficial and pathogenic bacterial strains. In the batch fermentation assay, ANWB-F reduced Bifidobacterium spp. counts (p < 0.05) while ANWB-N increased total bacterial counts and reduced Bifidobacterium spp. and Enterobacteriaceae counts (p < 0.05). Of the ANE1–4, produced from ANWB-F, ANWE and ANEE that were evaluated in the pure culture growth assays, the most interesting extracts were the ANE1 that reduced Salmonella Typhimurium, enterotoxigenic Escherichia coli and B. thermophilum counts and the ANE4 that stimulated B. thermophilum growth (p < 0.05). In conclusion, the extraction method and conditions influenced the bioactivities of the A. nodosum extracts with ANE1 and ANE4 exhibiting distinct antibacterial and prebiotic properties in vitro, respectively, that merit further exploration.

Correction: Garcia et al. Primary Model for Biomass Growth Prediction in Batch Fermentation. Symmetry 2021, 13, 1468

The authors wish to make the following corrections to this paper [...]

Submerged Fermentation of Animal Fat By-Products by Oleaginous Filamentous Fungi for the Production of Unsaturated Single Cell Oil

Animal waste fats were explored as a fermentation substrate for the production of high-value unsaturated single cell oil (SCO) using oleaginous fungi, Mucor circinelloides and Mortierella alpina. Both strains showed good growth and lipid accumulation when using animal fat as a single carbon source. The biomass concentration of 16.7 ± 2.2 gDCW/L and lipid content of 54.1%wt (of dry cell weight) were obtained for Mucor circinelloides in shake flask experiments, surpassing the biomass yield achieved in batch and fed-batch fermentation. In contrast, Mortierella alpina gave the highest biomass concentration (8.3 ± 0.3 gDCW/L) and lipid content (55.8%wt) in fed-batch fermentation. Fat grown Mortierella alpina was able to produce arachidonic acid (ARA), and the highest ARA content of 23.8%wt (of total lipid weight) was in fed-batch fermentation. Gamma-linolenic acid (GLA) was produced by both fungal strains. At the end of fed-batch fermentation, the GLA yields obtained for Mucor circinelloides and Mortierella alpina were 4.51%wt and 2.77%wt (of total lipid weight), respectively. This study demonstrates the production of unsaturated SCO-rich fungal biomass from animal fat by fermentation.

Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12

In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

Improvement of 1,3-Propanediol Production From Crude Glycerol by Co-cultivation of Anaerobic and Facultative Microbes Under Non-strictly Anaerobic Conditions

Background: Natural microbial consortia could efficiently produce 1,3-propanediol, the most promising bulk biochemical derived from glycerol that can be used as a monomer in the synthesis of polytrimethylene terephthalate (PTT). While natural microbial communities are made up of a diverse range of microbes with frequently unknown functions, the construction of synthetic microbial consortia allows for creating more defined systems with lower complexity.Results: In this study, the synthetic microbial consortia were constructed by combing facultative microbes of Klebsiella pneumoniae DUT2 (KP) and/or Escherichia coli DUT3 (EC) cultures with the strict anaerobic microbe of Clostridium butyricum DUT1 (CB) cultures under micro-aerobic conditions. The function of EC and KP during the fermentation process was to deplete oxygen and provide an anaerobic environment for CB. Furthermore, KP competes with CB to consume crude glycerol and produce 1,3-PDO. The interaction of commensalism and competition resulted in synthetic microbial consortia that could efficiently convert crude glycerol to 1,3-PDO even under micro-aerobic conditions. In a batch fermentation, the synthetic CB:KP co-culture at an initial abundance ratio of 92.5:7.5 yielded a maximum 1,3-PDO concentration of 52.08 g/L, with a yield of 0.49 g/g and a productivity of 1.80 g/(L.h), which increased by 10%, 9%, and 12%, respectively, when compared to the CB mono-culture under strictly anaerobic conditions. Compared to the KP mono-culture, the final 1,3-PDO concentration, yield, and productivity by the synthetic CB:KP consortia increased by 16%, 19%, and 84%, respectively. The synthetic CB:KP:EC co-culture achieved the highest 1,3-PDO flux of 49.17% at an initial abundance ratio of 85:7.5:7.5, while 7.43%, 5.77%, 3.15% 4.24%, and 2.13% of flux was distributed to butyric acid, acetic acid, lactic acid, ethanol, and succinic acid pathways. In a fed-batch fermentation, synthetic CB:KP:EC co-culture demonstrated a maximum 1,3-PDO concentration of 77.68 g/L with a yield of 0.51 g/g which is 30% and 13% higher than the production by the CB mono-culture at 0.02 vvm N2 supply. The initial abundance of CB guaranteed to be at least 85% facilitates 1,3-PDO production from crude glycerol efficiently by the development of synthetic microbial consortia. Conclusion: Under micro-aerobic conditions, the synthetic microbial consortia demonstrated excellent performance on 1,3-propanediol production via the interaction of commensalism and competition. The experimental results demonstrated the potential benefit of using the synthetic microbial consortia to produce 1,3-propanediol from crude glycerol.