Co-substrate Strategy Research Articles

Polyhydroxybutyrate (PHB) Biosynthesis by an Engineered Yarrowia lipolytica Strain Using Co-Substrate Strategy

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h−1. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica.

Irreversible biosynthesis of D-Allulose from D-glucose in Escherichia coli through fine-tuning of carbon flux and cofactor regeneration engineering.

As a rare hexose with low calories and various physiological functions, D-allulose has drawn increasing attention. The current industrial production of D-allulose from D-fructose or D-glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of D-fructose to D-allulose could be enhanced via phosphorylation-dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. After modifying the glycolytic pathway of Escherichia coli and optimizing the whole-cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L-1 D-allulose from 30 g L-1 D-glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and D-allulose production increased by 55.3% to 11.76 ± 0.58 g L-1 (0.53 g g-1 ). Fine-tuning of carbon flux caused a 48% reduction in D-fructose accumulation to 1.47 ± 0.15 g L-1 . After implementing fed-batch co-substrate strategy, the D-allulose titer reached 15.80 ± 0.31 g L-1 (0.62 g g-1 ) with a D-glucose conversion rate of 84.8%. This study presented a novel strategy for high-yield D-allulose production from low-cost substrate. This article is protected by copyright. All rights reserved.

Substrates of Opposite Polarities and Downstream Processing for Efficient Production of the Biosurfactant Mannosylerythritol Lipids from Moesziomyces spp.

Biosurfactants can replace fossil-driven surfactants with positive environmental impacts, owing to their low eco-toxicity and high biodegradability. However, their large-scale production and application are restricted by high production costs. Such costs can be reduced using renewable raw materials and facilitated downstream processing. Here, a novel strategy for mannosylerythritol lipid (MEL) production explores the combination of hydrophilic and hydrophobic carbon sources sideways with a novel downstream processing strategy, based on nanofiltration technology. Co-substrate MEL production by Moesziomyces antarcticus was threefold higher than using D-glucose with low levels of residual lipids. The use of waste frying oil instead of soybean oil (SBO) in co-substrate strategy resulted in similar MEL production. Moesziomyces antarcticus cultivations, using 3.9 M of total carbon in substrates, yields 7.3, 18.1, and 20.1 g/L of MEL, and 2.1, 10.0, and 5.1 g/L of residual lipids, for D-glucose, SBO, and a combination of D-Glucose and SBO, respectively. Such approach makes it possible to reduce the amount of oil used, offset by the equivalent molar increase in D-glucose, improving sustainability and decreasing residual unconsumed oil substrates, facilitating downstream processing. Moesziomyces spp. also produces lipases that broken down the oil and, thus, residual unconsumed oils are in the form of free fatty-acids or monoacylglycerol, which are smaller molecules than MEL. Therefore, nanofiltration of ethyl acetate extracts from co-substrate-based culture broths allows to improve MEL purity (ratio of MEL per total MEL and residual lipids) from 66 to 93% using 3-diavolumes.

Production of Mannosylerythritol Lipids Using Oils from Oleaginous Microalgae: Two Sequential Microorganism Culture Approach.

Mannosylerythritol lipids (MELs) are biosurfactants with excellent biochemical properties and a wide range of potential applications. However, most of the studies focusing on MELs high titre production have been relying in the use of vegetable oils with impact on the sustainability and process economy. Herein, we report for the first time MELs production using oils produced from microalgae. The bio-oil was extracted from Neochloris oleoabundans and evaluated for their use as sole carbon source or in a co-substrate strategy, using as an additional carbon source D-glucose, on Moesziomyces spp. cultures to support cell growth and induce the production of MELs. Both Moesziomyces antarcticus and M. aphidis were able to grow and produce MELs using algae-derived bio-oils as a carbon source. Using a medium containing as carbon sources 40 g/L of D-glucose and 20 g/L of bio-oils, Moesziomyces antarcticus and M. aphidis produced 12.47 ± 0.28 and 5.72 ± 2.32 g/L of MELs, respectively. Interestingly, there are no significant differences in productivity when using oils from microalgae or vegetable oils as carbon sources. The MELs productivities achieved were 1.78 ± 0.04 and 1.99 ± 0.12 g/L/h, respectively, for M. antarcticus fed with algae-derived or vegetable oils. These results open new perspectives for the production of MELs in systems combining different microorganisms.

Ethanol as an efficient cosubstrate for the biodegradation of azo dyes by Providencia rettgeri: Mechanistic analysis based on kinetics, pathways and genomics

Azo dyes pose hazards to ecosystems and human health and the cosubstrate strategy has become the focus for the bioremediation of azo dyes. Herein, Brilliant Crocein (BC), a model pollutant, was biodegraded by Providencia rettgeri domesticated from activated sludge. Additional ethanol, as a cosubstrate, could accelerate P. rettgeri growth and BC biodegradation, as reflected by the Gompertz models. This phenomenon was attributed to the smaller metabolites and greater number of potential pathways observed under the synergistic effect of ethanol. Genomic analysis of P. rettgeri showed that functional genes related to azo bond cleavage, redox reactions, ring opening and hydrolysis played crucial roles in azo dye biodegradation. Furthermore, the mechanism proposed was that ethanol might stimulate the production of additional reducing power via the expression of related genes, leading to the cleavage of azo bonds and aromatic rings. However, biodegradation without ethanol could only partly cleave the azo bonds.

Using easy-to-biodegrade co-substrate to eliminate microcystin toxic on electrochemically active bacteria and enhance bioelectricity generation from cyanobacteria biomass

Cyanobacterial biomass is a promising natural resource for power generation, through the reactions bio-catalyzed by electrochemically active bacteria (EAB). However, the major limitation is the involvement of Microcystin-LR (MC-LR) in inhibiting EAB activation. In this work, toxic M. aeruginosa biomass was employed as analyte of a microbial fuel cell (MFC), and sodium acetate was applied as easy-to-biodegrade co-substrate to alleviate the MC-LR stress on EAB survival. The running stability was continuously enhanced with the increment of co-substrate concentration. The sufficient co-substrate supply (6.0 mM) eliminated the negative effects of MC-LR on the cyanobacteria biomass fed-MFC performance; it contributed 12.7% extension on the electric cyclic terms and caused the productions of the power density which was comparable and even 3.8% higher than its corresponding control (MFC treated with acetate alone). The co-substrate addition also increased coulombic efficiency by 60.1%, microcystin-LR removal efficiency increased by 64.7%, and diversified the microbial community with more species able to biodegrade the MC-LR, bio-transforming the metabolites and EAB. Microcystin-degrading bacteria, such as Sphingopyxis sp., Burkholderia-Paraburkholderia, and Bacillus sp., were remarkably increased, and EAB, including Shewanella sp., Desulfovibrio desulfuricans, Aeromonas hydrophila, were also much more enriched in co-substrate use protocol. Therefore, this study verified a co-substrate strategy for simultaneously eliminating MC-LR toxin and enhancing bioelectricity generation from cyanobacterial biomass via an MFC.

Increased Selectivity for Butanol in Clostridium Pasteurianum Fermentations via Butyric Acid Addition or Dual Feedstock Strategy

Volatility of the petroleum market has renewed research into butanol as an alternate fuel. In order to increase the selectivity for butanol during glycerol fermentation with Clostridium pasteurianum, butyric acid can be added to the medium. In this manuscript, different methods of extracellular butyric acid addition are explored, as well as self-generation of butyric acid fermented from sugars in a co-substrate strategy. Molasses was used as an inexpensive sugar substrate, and the optimal molasses to glycerol ratio was found to allow the butyric acid to be taken back up into the cells and increase the productivity of butanol from all carbon sources. When butyric acid is added directly into the media, there was no significant difference between chemically pure butyric acid, or butyric acid rich supernatant from a separate fermentation. When low concentrations of butyric acid (1 or 2 g/L) are added to the initial media, an inhibitory effect is observed, with no influence on the butanol selectivity. However, when added later to the fermentation, over 1 g/L butyric acid is taken into the cells and increased the relative carbon yield from 0.449 to 0.519 mols carbon in product/mols carbon in substrate. An optimized dual substrate fermentation strategy in a pH-controlled reactor resulted in the relative carbon yield rising from 0.439 when grown on solely glycerol, to 0.480 mols C product/mols C substrate with the dual substrate strategy. An additional benefit is the utilization of a novel source of sugars to produce butanol from C. pasteurianum. The addition of butyric acid, regardless of how it is generated, under the proper conditions can allow for increased selectivity for butanol from all substrates.

Co-substrate addition accelerated amoxicillin degradation and detoxification by up-regulating degradation related enzymes and promoting cell resistance

β-Lactam antibiotics are the most commonly used antibiotics, and are difficult to remove by conventional biological treatments because of their persistent and toxic nature. The addition of co-substrates has been successfully employed to improve the removal of refractory pollutants. So, we hypothesized that the co-substrate strategy would increase antibiotic degradation and benefit microbial survival. In this work, we reported that co-substrate (acetate) addition up-regulated key degrading enzymes and resistance related genes in a model bacteria strain (L. aquatilis) when being treated with 0.055 mM amoxicillin (AMO). β-Lactamase, amidases, transaminase, and amide C-N hydrolase showed increased activation. As a result, AMO removal reached ∼95 %, a ∼60 % increase over the control. Furthermore, the addition of acetate drove the down-stream TCA cycle, which accelerated the detoxification of the intermediates and reduced the microbial inhibition by the antibiotic products to as low as ∼15 %. Besides, the expression levels of genes encoding the efflux pump, penicillin binding proteins, and β-Lactamase were up-regulated, and the inhibition of peptidoglycan biosynthesis was down-regulated. The cell density was enhanced by ∼170 % and showed improved DNA replication. In conclusion, the addition of the co-substrate accelerated AMO degradation and detoxification by up-regulating degrading enzymes and promoting cell resistance.

Cosubstrate strategy for enhancing lignocellulose degradation during rumen fermentation in vitro: Characteristics and microorganism composition

To enhance the degradation of wheat straw (WS) and corn straw (CS) in rumen fermentation, characterization of degradation and ruminal microorganisms of monosubstrate (WS/CS) groups and a cosubstrate strategy with food waste (FW) group was performed. The cellulose, hemicellulose, and lignin degradation efficiency of WS and CS; soluble chemical oxygen demand; volatile fatty acid yields; and activity of ligninolytic, cellulolytic, and hemicellulolytic enzymes for the cosubstrate group were improved compared with those for the corresponding monosubstrate groups. An accurate and a good of fit of the Weibull kinetic model, decreased crystallinity index values, and characteristic absorbance bands in the Fourier transform-infrared spectra further confirmed that cosubstrate addition with FW decreased the resistance of cellulose and hemicellulose to biodegradation. High-throughput sequencing results suggested that the bacterial diversity in CS rumen fermentation and fungal diversity and richness in WS rumen fermentation were promoted with FW as a cosubstrate. The cosubstrate addition with FW significantly affected the composition of the ruminal bacteria and fungi in rumen fermentation. The relative abundances (RAs) of rumen bacteria were increased in the cosubstrate CS/WS and FW fermentation conditions, and the enhancement of CS degradation with FW supplementation was stronger than that of WS rumen fermentation with FW supplementation. The RAs of the ruminal fungal genera Ustilago and Fusarium were promoted in CS and WS fermentation with FW, respectively. Moreover, the fermentation properties and rumen flora in the FW rumen fermentation also provided some evidence to suggest an enhancement of the cosubstrate strategy compared with the monosubstrate strategy.

Co-substrate strategy for improved power production and chlorophenol degradation in a microbial fuel cell

A co-substrate strategy has the potential to contribute toward minimizing the poisoning of refractories on the anode respiring bacteria (ARB), which is a necessity for microbial fuel cell (MFC) to recover energy from toxic wastewater. However, little is known about the underlying mechanisms. This study employed 4-chlorophenol (4-CP) as target refractory pollutant and acetate as co-substrate. The co-substrate could stimulate 4-CP sensitive extracellular electron transfer (EET) enzymes’ activity under 4-CP stress, such as succinate dehydrogenase (complex II) and cytochromes c OmcA and OmcB. For the microbial community, ARB were still abundant during 4-CP toxification; Azospirillum and Dechloromonas were enriched to conduct aromatic ring breakage and dechlorination. Thus, a 4.3-fold increased power generation was achieved, and the 4-CP and chemical oxygen demand (COD) removals increased by ∼42% and ∼53%, respectively. An easy-to-degrade substrate could improve power re-coverage from refractory wastewater by enhancing EET enzyme activity and optimizing the microbial community.

Enhancing chlorophenol biodegradation: Using a co-substrate strategy to resist photo-H2O2 stress in a photocatalytic-biological reactor

Intimately coupled photocatalysis and biodegradation (ICPB) is an emerging technology for treating refractory wastewater. The prerequisite of successful ICPB is that photocatalytic reactive oxygen species (ROSs) attack refractory pollutants and yield biodegradable intermediates, but “instantly” extinct before they transfer to the biofilms. However, photocatalytic induced H2O2 (a typical ROS) is different from others due to its long half-life. Photo-H2O2 in ICPB could potentially damage the biofilm; however, this has not been verified to date. In this study, we show that photo-H2O2 damages biofilms in ICPB, and we successfully addressed this issue using a strategy with the co-substrate (NaAC). Under H2O2 stress, the living cell percentage in ICPB dropped significantly, and the cell morphology presented the disinfection of bacteria cells, with enlarged size and disappeared organelles. By adding NaAC, the living cell percentage increased by 19.6% and the bacterial intracellular structure became integrated. The Shannon index increased from 3.45 to 4.05, indicating enhanced microbial diversity. Thauera and Dechloromonas were dominant genera, which played important roles in the breaking of the aromatic ring and in dechlorinating. As a result, 4-CP removal and mineralization increased by ∼27% and ∼23%, respectively. Here, we identified the negative effects of photo-H2O2 on the microbial survival of ICPB and for the first time propose an efficient co-substrate strategy to remedy this issue.