Dynamic immobilization of bacterial cells on biofilm in a polyester nonwoven chemostat.

Cell immobilization plays an important role in biocatalysis for high-value products. It is necessary to maintain the viability of immobilized cells for bioconversion using viable cells as biocatalysts. In this study, a novel polyester nonwoven chemostat was designed for cell immobilization to investigate biofilm formation and the dynamic balance between adsorption and desorption of cells on polyester nonwoven. The polyester nonwoven was suitable for cell immobilization, and the cell numbers on the polyester nonwoven can reach 6.5 ± 0.38 log CFU/mL. After adding the polyester nonwoven to the chemostat, the fluctuation phenomenon of free bacterial cells occurred. The reason for this phenomenon was the balance between adsorption and desorption of bacterial cells on the polyester nonwoven. Bacterial cells could adhere to the surface of polyester nonwoven via secreting extracellular polymeric substances (EPS) to form biofilms. As the maturation of biofilms, some dead cells inside the biofilms can cause the detachment of biofilms. This process of continuous adsorption and desorption of cells can ensure that the polyester nonwoven chemostat has lasting biological activity.

Kinetics-based development of two-stage continuous fermentation of 1,3-propanediol from crude glycerol by Clostridium butyricum.

BACKGROUND: Glycerol, as a by-product, mainly derives from the conversion of many crops to biodiesel, ethanol, and fatty ester. Its bioconversion to 1,3-propanediol (1,3-PDO) is an environmentally friendly method. Continuous fermentation has many striking merits over fed-batch and batch fermentation, such as high product concentration with easy feeding operation, long-term high productivity without frequent seed culture, and energy-intensive sterilization. However, it is usually difficult to harvest high product concentrations. RESULTS: In this study, a three-stage continuous fermentation was firstly designed to produce 1,3-PDO from crude glycerol by Clostridium butyricum, in which the first stage fermentation was responsible for providing the excellent cells in a robust growth state, the second stage focused on promoting 1,3-PDO production, and the third stage aimed to further boost the 1,3-PDO concentration and reduce the residual glycerol concentration as much as possible. Through the three-stage continuous fermentation, 80.05 g/L 1,3-PDO as the maximum concentration was produced while maintaining residual glycerol of 5.87 g/L, achieving a yield of 0.48 g/g and a productivity of 3.67 g/(L·h). Based on the 14 sets of experimental data from the first stage, a kinetic model was developed to describe the intricate relationships among the concentrations of 1,3-PDO, substrate, biomass, and butyrate. Subsequently, this kinetic model was used to optimize and predict the highest 1,3-PDO productivity of 11.26 g/(L·h) in the first stage fermentation, while the glycerol feeding concentration and dilution rate were determined to be 92 g/L and 0.341 h-1, separately. Additionally, to achieve a target 1,3-PDO production of 80 g/L without the third stage fermentation, the predicted minimum volume ratio of the second fermenter to the first one was 11.9. The kinetics-based two-stage continuous fermentation was experimentally verified well with the predicted results. CONCLUSION: A novel three-stage continuous fermentation and a kinetic model were reported. Then a simpler two-stage continuous fermentation was developed based on the optimization of the kinetic model. This kinetics-based development of two-stage continuous fermentation could achieve high-level production of 1,3-PDO. Meanwhile, it provides a reference for other bio-chemicals production by applying kinetics to optimize multi-stage continuous fermentation.

Dynamic flux balance analysis of 1,3-propanediol production by clostridium butyricum fermentation.

To study the relationship between the yield of 1,3-propanediol (1,3-PDO) and the flux change of the Clostridium butyricum metabolic pathway, an optimized calculation method based on dynamic flux balance analysis was used by combining genome-scale flux balance analysis with a kinetic model. A more comprehensive and extensive metabolic pathway was obtained by optimization calculations. The primary extended branches include: the dihydroxyacetone node, which enters the pentose phosphate pathway; the α-oxoglutarate node, which has synthetic metabolic pathways for glutamic acid and amino acids; and the serine and homocysteine nodes, which produce cystathionine before homocysteine enters the methionine cycle pathway. According to the expanded metabolic network, the flux distribution of key nodes in the metabolic pathway and the relationship between the flux distribution ratio of nodes and the yield of 1,3-PDO were analyzed. At the dihydroxyacetone node, the flux of dihydroxyacetone converted to dihydroxyacetone phosphate was positively correlated with the yield of 1,3-PDO. As an important intermediate product, the flux change in the metabolic pathway of α-oxoglutarate reacting with amino acids to produce glutamic acid is positively correlated with the yield. When pyruvate was used as the central node to convert into lactic acid and α-oxoglutarate, the proportion of branch flux was negatively correlated with the yield of 1,3-PDO. These studies provide a theoretical basis for the optimization and further study of the metabolic pathway of C. butyricum.

The pretreatment of the sustainable biomass feedstock of Pennisetum giganteum for biorefinery using deep eutectic solvents.

In this study, Pennisetum giganteum (PG) was investigated as lignocellulosic feedstock to be pretreated by the acidic and basic deep eutectic solvents (DESs) to generate monomeric sugars. The basic DESs showed excellent efficiency of delignification and saccharification. ChCl/MEA can remove 79.8 % lignin and reserve 89.5 % cellulose. As a result, 95.6 % glucose and 88.0 % xylose yield were obtained, significantly enhanced 9.4 and 15.5 times in contrast with those of the unpretreated PG. The 3D microstructures of raw and pretreated PG were constructed for the first time to better investigate the pretreatment effect on its structure. The increasing porosity (20.5 %) and the reducing CrI (42.2 %) contributed in enhancing enzymatic digestion. Moreover, the recyclability of DES indicated that at least 90 % DES was recovered and 59.5 % lignin still can removed with 79.8 % glucose were obtained after five recycling cycles. Meanwhile, 51.6 % lignin was recovered throughout the recycling process.

Extractive adsorption of 1,3-propanediol on a novel polystyrene macroporous resin enclosing medium and long-chain alcohols as extractant.

Extractive adsorption is an integrated separation method employing a novel resin with both particle and liquid characteristics in terms of adsorption and extraction. In this study, the novel extractive adsorption polystyrene-divinylbenzene (PS-DVB) macroporous resin was synthesized by suspension polymerization, in which n-octanol (OL-PS-DVB) or mixed alcohols of n-octanol, undecyl alcohol, and tetradecyl alcohol (MA-PS-DVB) were added as porogen and enclosed in the resin skeleton after the reaction. The characterization of the two novel resins of OL-PS-DVB and MA-PS-DVB showed that they have large specific surface areas of 48.7 and 17.4 m2/g, respectively. Additionally, the two synthesized resins have much higher static adsorption capacities of 1,3-propanediol (511 and 473 mg/g) and dynamic adsorption capacities (312 and 267 mg/g) than traditional resins, because extractants enclosed in the resin can increase the adsorption capacity. Through Langmuir equation, the theoretical static maximum adsorption capacity of the mixed alcohols resin is 515 mg/g at 298 K and Gibbs free energy change of adsorption was -3781 J/mol, indicating that the adsorption process was spontaneous. In addition, the sorbent concentration effect in the resin was generated at high 1,3-propanediol (1,3-PDO) concentrations. The fitting of the Flocculation model can reveal that there is a possible relation between adsorption and flocculation. Compared to OL-PS-DVB, MA-PS-DVB showed better performance in the recovery yield of 1,3-PDO and other byproducts, the removal rates of the inorganic salt and protein, and the efficiency of recycled resin. For MA-PS-DVB, the recovery of 1,3-PDO, butyrate acid, acetic acid, and residual glycerol was 97.1%, 94.7%, 93.3%, and 90.3%, respectively. Simultaneously, the resin of MA-PS-DVB could remove 93.8% of inorganic salts and 90.9% of proteins in the concentrated fermentation broth. The two synthesized resins of OL-PS-DVB and MA-PS-DVB still had 90% or 92% of capacity for extractive adsorption of 1,3-propanediol after 10 times of recycling, which exhibited potential application in the separation of 1,3-propanediol from fermentation broth.

In situ carbon dioxide capture to co-produce 1,3-propanediol, biohydrogen and micro-nano calcium carbonate from crude glycerol by Clostridium butyricum.

BACKGROUND: Climate change caused by greenhouse gas emission has become a global hot topic. Although biotechnology is considered as an environmentally friendly method to produce chemicals, almost all biochemicals face carbon dioxide emission from inevitable respiration and energy metabolism of most microorganisms. To cater for the broad prospect of biochemicals, bioprocess optimization of diverse valuable products is becoming increasingly important for environmental sustainability and cleaner production. Based on Ca(OH)2 as a CO2 capture agent and pH regulator, a bioprocess was proposed for co-production of 1,3-propanediol (1,3-PDO), biohydrogen and micro-nano CaCO3 by Clostridium butyricum DL07. RESULTS: In fed-batch fermentation, the maximum concentration of 1,3-PDO reached up to 88.6 g/L with an overall productivity of 5.54 g/L/h. This productivity is 31.9% higher than the highest value previously reports (4.20 g/L/h). In addition, the ratio of H2 to CO2 in exhaust gas showed a remarkable 152-fold increase in the 5 M Ca(OH)2 group compared to 5 M NaOH as the CO2 capture agent. Green hydrogen in exhaust gas ranged between 17.2% and 20.2%, with the remainder being N2 with negligible CO2 emissions. During CO2 capture in situ, micro-nano calcite particles of CaCO3 with sizes in the range of 300 nm to 20 µm were formed simultaneously. Moreover, when compared with 5M NaOH group, the concentrations of soluble salts and proteins in the fermentation broth of 5 M Ca(OH)2 group were notably reduced by 53.6% and 44.1%, respectively. The remarkable reduction of soluble salts and proteins would contribute to the separation of 1,3-PDO. CONCLUSIONS: Ca(OH)2 was used as a CO2 capture agent and pH regulator in this study to promote the production of 1,3-PDO. Meanwhile, micro-nano CaCO3 and green H2 were co-produced. In addition, the soluble salts and proteins in the fermentation broth were significantly reduced.

Bu Shen Yi Sui Capsules Promote Remyelination by Regulating MicroRNA-219 and MicroRNA-338 in Exosomes to Promote Oligodendrocyte Precursor Cell Differentiation.

Remyelination is a refractory feature of demyelinating diseases such as multiple sclerosis (MS). Studies have shown that promoting oligodendrocyte precursor cell (OPC) differentiation, which cannot be achieved by currently available therapeutic agents, is the key to enhancing remyelination. Bu Shen Yi Sui capsule (BSYSC) is a traditional Chinese herbal medicine over many years of clinical practice. We have found that BSYSC can effectively treat MS. In this study, the effects of BSYSC in promoting OPCs differentiation and remyelination were assessed using an experimental autoimmune encephalomyelitis (EAE) model in vivo and cultured OPCs in vitro. The results showed that BSYSC reduced clinical function scores and increased neuroprotection. The expression of platelet-derived growth factor receptor α (PDGFR-α) was decreased and the level of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was increased in the brains and spinal cords of mice as well as in OPCs after treatment with BSYSC. We further found that BSYSC elevated the expression of miR-219 or miR-338 in the serum exosomes of mice with EAE, thereby suppressing the expression of Sox6, Lingo1, and Hes5, which negatively regulate OPCs differentiation. Therefore, serum exosomes of BSYSC-treated mice (exos-BSYSC) were extracted and administered to OPCs in which miR-219 or miR-338 expression was knocked down by adenovirus, and the results showed that Sox6, Lingo1, and Hes5 expression was downregulated, MBP expression was upregulated, OPCs differentiation was increased, and the ability of OPCs to wrap around neuronal axons was improved. In conclusion, BSYSC may exert clinically relevant effects by regulating microRNA (miR) levels in exosomes and thus promoting the differentiation and maturation of OPCs.

Chinese Medicine Formula Siwu-Yin Inhibits Esophageal Precancerous Lesions by Improving Intestinal Flora and Macrophage Polarization.

Siwu-Yin (SWY), a traditional Chinese medicinal formula, can replenish blood and nourish Yin. It was recorded in ancient Chinese medicine books in treating esophageal dysphagia, which has similar symptoms and prognosis with esophageal precancerous lesions and esophageal cancer. However, its effect has not been established in vivo. This study explores the antiesophageal cancer effect of SWY on rats with esophageal precancerous lesions. By performing 16S rRNA gene sequencing and metabolomics, it was suggested that SWY may improve the composition of intestinal flora of rats by regulating the synthesis and secretion of bile acids. In addition, flow cytometry results showed that SWY treatment modified tumor microenvironment by improving macrophage polarization and therefore inhibiting the occurrence of esophageal precancerous lesions.

Bu Shen Yi Sui Capsule Alleviates Neuroinflammation and Demyelination by Promoting Microglia toward M2 Polarization, Which Correlates with Changes in miR-124 and miR-155 in Experimental Autoimmune Encephalomyelitis.

BACKGROUND: Bu Shen Yi Sui capsule (BSYS) is a traditional Chinese medicine prescription that has shown antineuroinflammatory and neuroprotective effects in treating multiple sclerosis (MS) and its animal model of experimental autoimmune encephalomyelitis (EAE). Microglia play an important role in neuroinflammation. The M1 phenotype of microglia is involved in the proinflammatory process of the disease, while the M2 phenotype plays an anti-inflammatory role. Promoting the polarization of microglia to M2 in MS/EAE is a promising therapeutic strategy. This study is aimed at exploring the effects of BSYS on microglial polarization in mice with EAE. METHODS: The EAE model was established by the intraperitoneal injection of pertussis toxin and subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG)35-55 in C57BL/6J mice. The mice were treated with BSYS (3.02 g/kg), FTY720 (0.3 mg/kg), or distilled water by intragastric administration. H&E and LFB staining, transmission electron microscopy, qRT-PCR, immunofluorescence, ELISA, fluorescence in situ hybridization, and western blotting were used to detect the histological changes in myelin, microglial M1/M2 polarization markers, and the expression of key genes involved in EAE. Results and Conclusions. BSYS treatment of EAE mice increased the body weight, decreased the clinical score, and reduced demyelination induced by inflammatory infiltration. BSYS also inhibited the mRNA expression of M1 microglial markers while increasing the mRNA level of M2 markers. Additionally, BSYS led to a marked decrease in the ratio of M1 microglia (iNOS+/Iba1+) and an obvious increase in the number of M2 microglia (Arg1+/Iba1+). In the EAE mouse model, miR-124 expression was decreased, and miR-155 expression was increased, while BSYS treatment significantly reversed this effect and modulated the levels of C/EBP α, PU.1, and SOCS1 (target genes of miR-124 and miR-155). Therefore, the neuroprotective effect of BSYS against MS/EAE was related to promoting microglia toward M2 polarization, which may be correlated with changes in miR-124 and miR-155 in vivo.

Metabolism, morphology and transcriptome analysis of oscillatory behavior of Clostridium butyricum during long-term continuous fermentation for 1,3-propanediol production.

BACKGROUND: Oscillation is a special cell behavior in microorganisms during continuous fermentation, which poses threats to the output stability for industrial productions of biofuels and biochemicals. In previous study, a spontaneous oscillatory behavior was observed in Clostridium butyricum-intensive microbial consortium in continuous fermentation for 1,3-propanediol (1,3-PDO) production from glycerol, which led to the discovery of oscillation in species C. butyricum. RESULTS: Spontaneous oscillations by C. butyricum tended to occur under glycerol-limited conditions at low dilution rates. At a glycerol feed concentration of 88 g/L and a dilution rate of 0.048 h-1, the oscillatory behavior of C. butyricum was observed after continuous operation for 146 h and was sustained for over 450 h with an average oscillation period of 51 h. During oscillations, microbial glycerol metabolism exhibited dramatic periodic changes, in which productions of lactate, formate and hydrogen significantly lagged behind that of other products including biomass, 1,3-PDO and butyrate. Analysis of extracellular oxidation-reduction potential and intracellular ratio of NAD+/NADH indicated that microbial cells experienced distinct redox changes during oscillations, from oxidized to reduced state with decreasing of growth rate. Meanwhile, C. butyricum S3 exhibited periodic morphological changes during oscillations, with aggregates, elongated shape, spores or cell debris at the trough of biomass production. Transcriptome analysis indicated that expression levels of multiple genes were up-regulated when microbial cells were undergoing stress, including that for pyruvate metabolism, conversion of acetyl-CoA to acetaldehyde as well as stress response. CONCLUSION: This study for the first time systematically investigated the oscillatory behavior of C. butyricum in aspect of occurrence condition, metabolism, morphology and transcriptome. Based on the experimental results, two hypotheses were put forward to explain the oscillatory behavior: disorder of pyruvate metabolism, and excessive accumulation of acetaldehyde.

Sequential fed-batch fermentation of 1,3-propanediol from glycerol by Clostridium butyricum DL07.

The demand for 1,3-propanediol (1,3-PDO) has increased sharply due to its role as a monomer for the synthesis of polytrimethylene terephthalate (PTT). Although Clostridium butyricum is considered to be one of the most promising bioproducers for 1,3-PDO, its low productivity hinders its application on industrial scale because of the longer time needed for anaerobic cultivation. In this study, an excellent C. butyricum (DL07) strain was obtained with high-level titer and productivity of 1,3-PDO, i.e., 104.8 g/L and 3.38 g/(L•h) vs. 94.2 g/L and 3.04 g/(L•h) using pure or crude glycerol as substrate in fed-batch fermentation, respectively. Furthermore, a novel sequential fed-batch fermentation was investigated, in which the next bioreactor was inoculated by C. butyricum DL07 cells growing at exponential phase in the prior bioreactor. It could run steadily for at least eight cycles. The average concentration of 1,3-PDO in eight cycles was 85 g/L with the average productivity of 3.1 g/(L•h). The sequential fed-batch fermentation could achieve semi-continuous production of 1,3-PDO with higher productivity than repeated fed-batch fermentation and would greatly contribute to the industrial production of 1,3-PDO by C. butyricum. KEY POINTS: • A novel C. butyricum strain was screened to produce 104.8 g/L 1,3-PDO from glycerol. • Corn steep liquor powder was used as a cheap nitrogen source for 1,3-PDO production. • A sequential fed-batch fermentation process was established for 1,3-PDO production. • An automatic glycerol feeding strategy was applied in the production of 1,3-PDO.

Bioconversion of Raw Glycerol From Waste Cooking-Oil-Based Biodiesel Production to 1,3-Propanediol and Lactate by a Microbial Consortium.

Waste cooking oil (WCO) is a sustainable alternative to raw vegetable oils and fats for biodiesel production considering both environmental and economic benefits. Raw glycerol from WCO-based biodiesel production (GWCO) is difficult to utilize via biological method, as multiple toxic impurities have inhibitory effects on microbial growth especially for pure cultures. In this work, four microbial consortia were selected from activated sludge by 30 serial transfers under different conditions. The obtained consortia exhibited lower diversity and species difference with the transfers. The consortium LS30 exhibited unique advantages for bioconversion of GWCO to 1,3-propanediol (1,3-PDO) and lactate (LA). Moreover, the fermentation could be performed economically under microaerobic and non-sterile conditions. The consortium consisted of 57.97% Enterobacter and 39.25% Escherichia could effectively convert 60 g/L GWCO to 1,3-PDO and LA in batch fermentation. In addition, this consortium exhibited better tolerance to fatty acid-derived crude glycerol (100 g/L), which demonstrated that specific toxic impurities in GWCO did pose a great challenge to microbial growth and metabolism. In fed batch fermentation, 27.77 g/L 1,3-PDO and 14.68 g/L LA were achieved. Compared with the consortium, a long lag phase in cell growth associated with a decreased glycerol consumption was observed in four single-strain fermentations. Furthermore, neither the consortium DL38 with excellent glycerol tolerance nor consortium C2-2M with high yield of 1,3-PDO could effectively transform GWCO into valuable products. The results demonstrated that the selected microbial consortium has the advanced adaptability to the toxic impurities in GWCO compared with other reported consortia and isolated single strain. This process can contribute to added-value use of GWCO.

Stability and oscillatory behavior of microbial consortium in continuous conversion of crude glycerol to 1,3-propanediol.

Microbial consortium is an alternative for bioconversion of crude glycerol to value-added products whereas concerns about the process stability in long-term operation existed. The aim of this study is to evaluate the feasibility of using an anaerobic microbial consortium as inoculum for continuous conversion of crude glycerol to 1,3-propanediol (1,3-PDO). Performances of continuous fermentations with the consortium inoculum were evaluated under different dilution rates and glycerol feed concentrations. The highest 1,3-PDO production of 57.86 g/L was achieved with a productivity of 5.55 g/(L·h). Analyses of kinetic data showed that the consortium maintained a consistent pattern for 1,3-PDO production under different operating conditions despite changes in community composition. The continuous fermentation by the consortium was able to operate for a longer period of time (31 volume changes) than that using pure culture (24 volume changes) with the average 1,3-PDO concentration of 53.52 g/L and productivity of 6.69 g/(L·h) under glycerol-excess condition, which could be contributed to the intraspecies diversity among Clostridium butyricum in the consortium. Under glycerol-limited conditions, however, a spontaneous oscillation of the consortium was observed after continuous operation for about 120 h, along with severe fluctuations of the microbial community. The oscillatory behavior could be reduced by increasing the dilution rates and was likely the metabolic feature of C. butyricum.

Selection and characterization of an anaerobic microbial consortium with high adaptation to crude glycerol for 1,3-propanediol production.

Crude glycerol is an ideal feedstock for bioproduction of 1,3-propanediol (1,3-PDO) while pure culture always shows low substrate tolerance and limited productivity. In this study, an anaerobic microbial consortium for conversion of crude glycerol was selected and its 1,3-PDO production capacity was evaluated. The consortium was obtained from anaerobic activated sludge by 19 serial transfers and mainly consisted of 94.64% Clostridiaceae and 4.47% Peptostreptococcaceae. The consortium adapted well with high glycerol concentration of 120 g/L as well as wide substrate concentration fluctuation from 15 to 80 g/L, producing 60.61 and 82.66 g/L 1,3-PDO in the batch and fed-batch fermentation, with the productivity of 3.79 and 3.06 g/(L∙h), respectively, which are among the best results published so far. Furthermore, mini consortia isolated by serial dilution exhibited similar microbial composition but gradually decreasing tolerance to crude glycerol. Four randomly selected Clostridium butyricum displayed different substrate tolerance and insufficient 1,3-PDO production capacity. This work demonstrated that the high adaptation to crude glycerol of the consortium was the collaborative effort of different individuals.

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale.

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m(3). The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l(-1), 57% and 2.1 g l(-1 )h(-1), respectively, are close to those of 75 g l(-1), 61%, and 2.2 g l(-1 )h(-1) under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.