Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome

Potential of crassulacean acid metabolism (CAM) plants as biomass for bioenergy and biorefinery

Bioenergy crops have been exploited in the past decades as a mean to combat climate change and address energy security issues (IEA, 2014). However, the food vs fuel dilemma of employing energy crops has prompted scientists to search for alternative sustainable biomass options. Recently, Crassulacean Acid Metabolism (CAM) plants such as agave and prickly pear have gained research interest as alternative energy crops. Having evolved a specialised photosynthesis mode, CAM plants possess key features that enable them to thrive in arid and semi-arid regions where water is scarce and soil quality is poor. Stimulated by this interest, this study initially focused on evaluating the potential for methane production from CAM plants via anaerobic digestion and subsequently as a means of generating high-value products such as bioplastics. With over 16,000 CAM species from which to select, the first study screened a range of CAM plants, which were representative of 5 genera (Agave, Ananas, Euphorbia, Kalanchoe, and Opuntia) that encompass a variety of features typical of CAM plants. The biochemical methane potential (BMP) assay was employed to determine methane yields from the selected CAM plants. The BMP test was carried out using 250 ml glass bottles with 150 ml working volume and operating conditions of 39.0 ± 1.0 °C with constant agitation of 120 rpm. The results demonstrated that the BMPs of the CAM plants investigated ranged between 281 and 382 LCH4 kgVS-1, the highest of which was obtained for Agave angustifolia. The methane yields from the CAM plants tested were equivalent to that of maize, suggesting that they could have the potential as AD feedstocks and be highly attractive as a substrate in the biorefinery industry. Based on the results obtained from the screening experiment, Euphorbia tirucalli and Opuntia ficus-indica were selected as model CAM crops for further study. Euphorbia tirucalli was selected as it is also known as the petroleum plant with hydrocarbon components detected in their toxic latex resembling crude oil. Further, the effect of the inoculum to substrate (I/S) ratio, operating temperature, pH, and mixing (E:M) ratio via co-digestion on methane yields from E. tirucalli was investigated. The results demonstrated that the highest methane yields of E. tirucalli obtained in mesophilic and thermophilic conditions were 206 and 266 LCH4 kgVS-1, respectively, and an improvement of methane yields by 19–29% was observed under thermophilic conditions (55.0 ± 1.0 °C). The influence of the initial pH was observed but the results were not consistent. However, based on the energy balance assessment, mesophilic temperature (38.0 ± 1.0 °C) and pH 7.0 were found to provide optimal conditions for methane production. Methane yields obtained from the co-digestion of E. tirucalli and chicken manure were similar to those achieved in mono-digestion, regardless of the mixing (E:M) ratio. The second CAM crop selected, Opuntia ficus-indica, has previously been reported to have low lignin content but be rich in soluble sugars, which are desirable as an AD feedstock. A comprehensive study of O. ficus-indica was conducted using the BMP assay, to examine the influence of different factors such as substrate processing technique and plant age on methane production. The cladodes of O. ficus-indica were processed into 3 distinctive substrate types (ground cladode, liquid extract, and solid peel), which were demonstrated to produce the specific methane yields of 241, 222 and 271 L kgVS-1, respectively. Based on these findings, the processed O. ficus-indica cladode could be employed as an AD feedstock without the need for further processing. The liquid extract, which produced the highest methane yield, also had potential as a substrate for the production of higher-value biochemical products. A significant decrease in methane yields was detected in cladodes aged 3 years and older (p The concept of biorefinery was applied to this study. O. ficus-indica was selected as an example of biomass that could be systematically exploited firstly to a higher-added value product and subsequently to generate methane. The liquid extracted from O. ficus-indica (OLE) was found to be rich in sugars and organic acids. The liquid could potentially be directly exploited as a substrate for bio-fermentation, bypassing the conventional route of biomass conversion to volatile fatty acids (VFAs) via anaerobic digestion. Subsequently, proof-of-concept research was conducted using OLE as a substrate for polyhydroxyalkanoate (PHA) production employing the bacterium Cupriavidus necator H16. GC-MS metabolite analysis confirmed that the main compounds assimilated by this strain for their growth and PHA synthesis were fructose, citric/isocitric acid, and malic acid. C. necator H16 was able to produce polyhydroxybutyrate (PHB) of up to 2.12 g L-1, accounting for 45.5% of the cell dry weight. This study demonstrated for the first time that the synthesis of polyhydroxybutyrate (PHB) by C. necator using O. ficus-indica can be achieved.</p

Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

A new type of chromosome-free cells called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease, and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and were able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 days after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anti-cancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a novel and simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome

Investigating the design parameters for a permeable reactive barrier consisting of nanoscale zero-valent iron and bimetallic iron/copper for phosphate removal

There is a growing interest in deploying nanoscale zero valent iron (NZVI) in permeable reactive barriers (PRBs) for groundwater remediation. In the present study a series of packed-column experiments were conducted in order to investigate the effectiveness of phosphorus removal from groundwater using NZVI and bimetallic NZVI/Cu as reactive materials within PRBs. Seven sets of packed-column experiments were conducted in order to study the effect of different design parameters for PRB; including delivery approach of NZVI into porous media, PRB's configuration, coexisting groundwater ions and change in flowrate. Results implied that doping NZVI surface with copper had an anti-aggregation effect and enhanced its performance in terms of phosphorus removal 2.2 times higher than bare NZVI. Moreover, the lower flowrate (10 ml/min) demonstrated improved phosphorus removal by 22% compared with higher flowrate (60 ml/min). Additionally, groundwater ions barely interfered phosphorus removal process with only ±6%. Overall, geochemical properties and characteristics of the supporting materials were key parameters in the removal process of phosphorus by NZVI/Cu