Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding.

Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.

Priority changes between biofilm exopolysaccharides synthesis and rhamnolipids production are mediated by a c-di-GMP-specific phosphodiesterase NbdA in Pseudomonas aeruginosa.

The synthesis of biofilm exopolysaccharides and rhamnolipids (RLs) are two interrelated processes in Pseudomonas aeruginosa, but how bacteria coordinate these two processes remains unclear. We collected a P. aeruginosa KT1115 with rugose small colony variant (RSCV) phenotype from soil, and used it to study the dynamic regulation mechanism of biofilm polysaccharide and RLs synthesis. The results showed that the overproduction of biofilm exopolysaccharides at biofilm stage ultimately contributed the surge of RLs production at RLs stage. This phenomenon was further verified by comparing PAO1 with its engineered RSCV mutant, PAO1ΔwspF. Further genomic, transcriptomic analyses and gene deletion revealed that downregulation of c-di-GMP level was the key to switch biofilm exopolysaccharides accumulation to RLs surge, by transcriptionally upregulating a c-di-GMP phosphodiesterase NbdA. Overall, this study demonstrates the importance of c-di-GMP in coordinating biofilm exopolysaccharides and RLs synthesis, and provides an inspiration for enhancing RLs production through regulating c-di-GMP level.

Construction and comparison of synthetic microbial consortium system (SMCs) by non-living or living materials immobilization and application in acetochlor degradation.

The microbial degradation of pesticides by pure or mixed microbial cultures has been thoroughly explored, however, they are still difficult to apply in real environmental remediation. Here, we constructed a synthetic microbial consortium system (SMCs) through the immobilization technology by non-living or living materials to improve the acetochlor degradation efficiency. Rhodococcus sp. T3-1, Delftia sp. T3-6 and Sphingobium sp. MEA3-1 were isolated for the SMCs construction. The free-floating consortium with the composition ratio of 1:2:2 (Rhodococcus sp. T3-1, Delftia sp. T3-6 and Sphingobium sp. MEA3-1) demonstrated 94.8% degradation of acetochlor, and the accumulation of intermediate metabolite 2-methyl-6-ethylaniline was decreased by 3 times. The immobilized consortium using composite materials showed synergistic effects on the acetochlor degradation with maximum degradation efficiency of 97.81%. In addition, a novel immobilization method with the biofilm of Myxococcus xanthus DK1622 as living materials was proposed. The maximum 96.62% degradation was obtained in non-trophic media. Furthermore, the immobilized SMCs showed significantly enhanced environmental robustness, reusability and stability. The results indicate the promising application of the immobilization methods using composite and living materials in pollutant-contaminated environments.

Genome Characterization of Pseudomonas aeruginosa KT1115, a High Di-rhamnolipid-Producing Strain with Strong Oils Metabolizing Ability.

In this study, a wild-type Pseudomonas aeruginosa strain KT1115 with the capability of converting rapeseed oils into di-rhamnolipids, a class of biosurfactants with extensive application potential, was successfully isolated and characterized. Di-rhamnolipids production by microorganism culture provided a mild, eco-friendly, and secure approach for surfactants production instead of conventional chemical synthesis. However, few studies have been attempted to explore the metabolic mechanism behind the high di-rhamnolipids production by P. aeruginosa. Here, we presented the graft genome of a wild-type P. aeruginosa strain KT1115, with emphasis on the analysis of oils metabolism and rhamnolipid synthesis. The availability of the genome sequence provides additional insight into the genetic mechanism enhancing di-rhamnolipids biosynthesis.

Enhanced rhamnolipids production using a novel bioreactor system based on integrated foam-control and repeated fed-batch fermentation strategy.

BACKGROUND: Rhamnolipids are the best known microbial-derived biosurfactants, which has attracted great interest as potential ''green" alternative for synthetic surfactants. However, rhamnolipids are the major contributors to severe foam problems, which greatly inhibit the economics of industrial-scale production. In this study, a novel foam-control system was established for ex situ dealing with the massive overflowing foam. Based on the designed facility, foam reduction efficiency, rhamnolipids production by batch and repeated fed-batch fermentation were comprehensively investigated. RESULTS: An ex situ foam-control system was developed to control the massive overflowing foam and improve rhamnolipids production. It was found that the size of individual bubble in the early stage was much larger than that of late fermentation stage. The foam liquefaction efficiency decreased from 54.37% at the beginning to only 9.23% at the end of the fermentation. This difference of bubble stability directly resulted in higher foam reduction efficiency of 67.46% in the early stage, whereas the small uniform bubbles can only be reduced by 57.53% at the later fermentation stage. Moreover, reduction of secondary foam is very important for foam controlling. Two improved designs of the device in this study obtained about 20% improvement of foam reduction efficiency, respectively. The batch fermentation result showed that the average volume of the overflowing foam was reduced from 58-640 to 19-216 mL/min during the fermentation process, presenting a notable reduction efficiency ranging from 51.92 to 73.47%. Meanwhile, rhamnolipids production of batch fermentation reached 45.63 g/L, and the yield 0.76 g/g was significantly better than ever reported. Further, a repeated fed-batch fermentation based on the overall optimization was carried out. Total rhamnolipids concentration reached 48.67 g/L with the yield around of 0.67-0.83 g/g, which presented an improvement of 62% and 49% compared with conventional batch fermentation by using various kinds of defoamers, respectively. CONCLUSIONS: The ex situ foam-control system presented a notable reduction efficiency, which helped greatly to easily solve the severe foaming problem without any defoamer addition. Moreover, rhamnolipids production and yield by repeated fed-batch fermentation obtained prominent improvement compared to conventional batch cultivation, which can further facilitate economical rhamnolipids production at large scales.

High Di-rhamnolipid Production Using Pseudomonas aeruginosa KT1115, Separation of Mono/Di-rhamnolipids, and Evaluation of Their Properties.

Rhamnolipids (RLs) are important bioproducts that are regarded as promising biosurfactant for applications in oil exploitation, cosmetics, and food industry. In this study, the newly isolated Pseudomonas aeruginosa KT1115 showed high production of di-RLs. The highest yield of RLs by P. aeruginosa KT1115, reaching 44.39 g/L after 8 days of fermentation in a 5 L bioreactor, was obtained from rapeseed oil-nitrate medium after process optimization. Furthermore, we established a new separation process that achieved up to 91.82% RLs recovery with a purity of 89% and further obtained mono/di-rhamnolipids. Finally, ESI-MS analysis showed that the RLs produced by strain KT1115 have a high proportion of di-RLs (mono-RLs: di-RLs = 11.47: 88.53), which have a lower critical micelle-forming concentration (8 mN/m) and better emulsification ability with kerosene (52.1% EI24) than mono-RLs (167 mN/m and 41.4% EI24, respectively). These results demonstrated that P. aeruginosa KT1115 is a potential industrial producer of di-RLs, which have improved applicability and offer significant commercial benefits.

Biomethane Production From Lignocellulose: Biomass Recalcitrance and Its Impacts on Anaerobic Digestion.

Anaerobic digestion using lignocellulosic material as the substrate is a cost-effective strategy for biomethane production, which provides great potential to convert biomass into renewable energy. However, the recalcitrance of native lignocellulosic biomass makes it resistant to microbial hydrolysis, which reduces the bioconversion efficiency of organic matter into biogas. Therefore, it is necessary to critically investigate the correlation between lignocellulose characteristics and bioconversion efficiency. Accordingly, this review comprehensively summarizes the anaerobic digestion process and rate-limiting step, structural and compositional properties of lignocellulosic biomass, recalcitrance and inhibitors of lignocellulose and their major effects on anaerobic digestion for biomethane production. Moreover, various type of pretreatment strategies applied to lignocellulosic biomass was discussed in detail, which would contribution to cell wall degradation and improvement of biomethane yields. In the view of current knowledge, high energy input and cost requirements are the main limitations of these pretreatment methods. In addition to optimization of fermentation process, further studies should focus much more on key structural influence factors of biomass recalcitrance and anaerobic digestion efficiency, which will contribute to improvement of biomethane production from lignocellulose.

Performance evaluation of a lab-scale moving bed biofilm reactor (MBBR) using polyethylene as support material in the treatment of wastewater contaminated with terephthalic acid.

Untreated terephthalic acid (TPA) wastewaters with high organic loads will cause severe environmental pollution problems. In this study, a lab-scale moving bed biofilm reactor, where biomass of Delftia sp. WL-3 is attached to polypropylene carrier elements, has been tested for TPA bioremediation at 25-27 °C. The system achieved stable operation after a short 15-day start-up period. During the operation period of 65 days, stable chemical oxygen demand (COD) and TPA removal efficiencies of 68% and 76% were maintained with an organic load rate (OLR) and hydraulic retention time of 2.5 kg COD·(m3·d)-1 and 24 h, respectively. In addition, the Scanning Electron Microscope (SEM) showed that high-densities of WL-3 biomass accumulated on the surface of the carrier and formed a rich biofilm, indicating polypropylene carrier can improve the degradation efficiency. On the contrary, the biodegradation ability of stain WL-3 without the polypropylene carrier declined significantly with removal efficiencies of 10% and 15% for COD and TPA. Furthermore, the system exhibited excellent robustness to different OLR and influent matrix ratios, indicating its potential for applications in the treatment of TPA-containment wastewater in the field.