Automated liquid-handling operations for robust, resilient, and efficient bio-based laboratory practices

Increase in the adoption of liquid handling devices (LHD) can facilitate experimental activities. Initially adopted by businesses and industry-based laboratories, the practice has also moved to academic environments, where a wide range of non-standard/non-typical experiments can be performed. Current protocols or laboratory analyses require researchers to transfer liquids for the purpose of dilution, mixing, or inoculation, among other operations. LHD can render laboratories more efficient by performing more experiments per unit of time, by making operations robust and resilient against external factors and unforeseen events such as the COVID-19 pandemic, and by remote operation. The present work reviews literature that reported the adoption and utilisation of LHD available in the market and presents examples of their practical use. Applications demonstrate the critical role of automation in research development and its ability to reduce human intervention in the experimental workflow. Ultimately, this work will provide guidance to academic researchers to determine which LHD can fulfil their needs and how to exploit their use in both conventional and non-conventional applications. Furthermore, the breadth of applications and the scarcity of academic institutions involved in research and development that utilise these devices highlights an important area of opportunity for shift in technology to maximize research outcomes

From flask to large scale high cell density production of ω-transaminase using auto- induction media

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Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation

Background: The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. Results: Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. Conclusions: This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial ‘toolkits’ to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry

Combination of Genome-Scale Models and Bioreactor Dynamics to Optimize the Production of Commodity Chemicals

The current production of a number of commodity chemicals relies on the exploitation of fossil fuels and hence has an irreversible impact on the environment. Biotechnological processes offer an attractive alternative by enabling the manufacturing of chemicals by genetically modified microorganisms. However, this alternative approach poses some important technical challenges that must be tackled to make it competitive. On the one hand, the design of biotechnological processes is based on trial-and-error approaches, which are not only costly in terms of time and money, but also result in suboptimal designs. On the other hand, the manufacturing of chemicals by biological processes is almost exclusively carried out by batch or fed-batch cultures. Given that batch cultures are expensive and not easy to scale, technical means must be developed to make continuous cultures feasible and efficient. In order to address these challenges, we have developed a mathematical model able to integrate in a single model both the genome-scale metabolic model for the organism synthesizing the chemical of interest and the dynamics of the bioreactor in which the organism is cultured. Such a model is based on the use of Flexible Nets, a modeling formalism for dynamical systems. The integration of a microscopic (organism) and a macroscopic (bioreactor) model in a single net provides an overall view of the whole system and opens the door to global optimizations. As a case study, the production of citramalate with respect to the substrate consumed by E. coli is modeled, simulated and optimized in order to find the maximum productivity in a steady-state continuous culture. The predicted computational results were consistent with the wet lab experiments

Multi-product biorefinery from Arthrospira platensis biomass as feedstock for bioethanol and lactic acid production

With the aim to reach the maximum recovery of bulk and specialty bioproducts while minimizing waste generation, a multi-product biorefinery for ethanol and lactic acid production from the biomass of cyanobacterium Arthrospira platensis was investigated. Therefore, the residual biomass resulting from different pretreatments consisting of supercritical fluid extraction (SF) and microwave assisted extraction with non-polar (MN) and polar solvents (MP), previously applied on A. platensis to extract bioactive metabolites, was further valorized. In particular, it was used as a substrate for fermentation with Saccharomyces cerevisiae LPB-287 and Lactobacillus acidophilus ATCC 43121 to produce bioethanol (BE) and lactic acid (LA), respectively. The maximum concentrations achieved were 3.02 ± 0.07 g/L of BE by the MN process at 120 rpm 30 °C, and 9.67 ± 0.05 g/L of LA by the SF process at 120 rpm 37 °C. An economic analysis of BE and LA production was carried out to elucidate the impact of fermentation scale, fermenter costs, production titer, fermentation time and cyanobacterial biomass production cost. The results indicated that the critical variables are fermenter scale, equipment cost, and product titer; time process was analyzed but was not critical. As scale increased, costs tended to stabilize, but also more product was generated, which causes production costs per unit of product to sharply decrease. The median value of production cost was US$1.27 and US$ 0.39, for BE and LA, respectively, supporting the concept of cyanobacterium biomass being used for fermentation and subsequent extraction to obtain ethanol and lactic acid as end products from A. platensis

Multi-product biorefinery from Arthrospira platensis biomass as feedstock for bioethanol and lactic acid production

With the aim to reach the maximum recovery of bulk and specialty bioproducts while minimizing waste generation, a multi-product biorefinery for ethanol and lactic acid production from the biomass of cyanobacterium Arthrospira platensis was investigated. Therefore, the residual biomass resulting from different pretreatments consisting of supercritical fluid extraction (SF) and microwave assisted extraction with non-polar (MN) and polar solvents (MP), previously applied on A. platensis to extract bioactive metabolites, was further valorized. In particular, it was used as a substrate for fermentation with Saccharomyces cerevisiae LPB-287 and Lactobacillus acidophilus ATCC 43121 to produce bioethanol (BE) and lactic acid (LA), respectively. The maximum concentrations achieved were 3.02 ± 0.07 g/L of BE by the MN process at 120 rpm 30 °C, and 9.67 ± 0.05 g/L of LA by the SF process at 120 rpm 37 °C. An economic analysis of BE and LA production was carried out to elucidate the impact of fermentation scale, fermenter costs, production titer, fermentation time and cyanobacterial biomass production cost. The results indicated that the critical variables are fermenter scale, equipment cost, and product titer; time process was analyzed but was not critical. As scale increased, costs tended to stabilize, but also more product was generated, which causes production costs per unit of product to sharply decrease. The median value of production cost was US$1.27 and US$ 0.39, for BE and LA, respectively, supporting the concept of cyanobacterium biomass being used for fermentation and subsequent extraction to obtain ethanol and lactic acid as end products from A. platensis

Economic evaluation of the primary recovery of tetracycline with traditional and novel aqueous two-phase systems

Antibiotics are a key pharmaceutical to inhibit growth or kill microorganisms. They represent a profitable market and, in particular, tetracycline has been listed as an essential medicine by the WHO. Therefore it is important to improve their production processes. Recently novel and traditional aqueous two-phase systems for the extraction have been developed with positive results. The present work performs an economic analysis of the production and recovery of tetracycline through the use of several ATPS through bioprocess modeling using specialized software (BioSolve, Biopharm Services Ltd, UK) to determine production costs per gram (CoG/g). First, a virtual model was constructed using published data on the recovery of tetracycline and extended to incorporate uncertainties. To determine how the model behaved, a sensitivity analysis and Monte Carlo simulations were performed. Results showed that ATPS formed by cholinium chloride/K3PO4 was the best option to recover tetracycline, as it had the lowest CoG/g (US$ 672.83/g), offered the highest recovery yield (92.42%), second best sample input capacity (45% of the ATPS composition) and one of the lowest materials contribution to cost. The ionic liquid-based method of ATPS is a promising alternative for recovering tetracycline from fermentation broth.publishe

Extraction and Fractionation of Pigments from Saccharina latissima (Linnaeus, 2006) Using an Ionic Liquid plus Oil plus Water System

There is a strong industrial interest in the development of greener and more sustainable processes based on the use of renewable resources, and a biorefinery based on marine resources, such as macroalgae, stands as a major opportunity toward that end. In this work, Saccharina latissima (Linnaeus), a brown macroalga, was used as a source of pigments to develop an integrated platform that is able to promote the extraction and separation of chlorophyll and fucoxanthin in one single step. The process was studied, and its operational conditions were optimized with yields of extraction of chlorophyll and fucoxanthin of 4.93 ± 0.22 mgchl·gdry biomass–1 and 1956 ± 84 μgfuco·gdry biomass–1, respectively. These results were achieved with extraction systems composed of 84% of an aqueous solution of a tensioactive phosphonium-based ionic liquid (IL) at 350 mM + 16% of sunflower oil, during 40 min, using a solid–liquid ratio of 0.017 gdry biomass·mLsolvent–1. After the separation of both aqueous IL-rich and oil-rich phases, the IL content in both phases was investigated, the oil phase being free of IL. Envisioning the industrial potential of the process developed in this work, the recovery of the IL from the aqueous IL-rich phase of the initial system was attempted by a back-extraction using organic solvents immiscible in water, being shown that 82% of the IL can be recovered and reused in new cycles of extraction. The environmental and economic impacts of the final process proposed for the extraction and fractionation of chlorophyll and fucoxanthin were evaluated. Different scenarios were considered, but summing up the main results, the solvents’ recycling allowed better results, proving the economic and environmental viability of the overall process

Sustainable strategy based on induced precipitation for the purification of phycobiliproteins

Phycobiliproteins are fluorescent proteins mainly produced by red macroalgae and cyanobacteria. These proteins, essential to the survival of these organisms, find application in many fields of interest, from medical, pharmaceutical, and cosmetic to food and textile industries. The biggest obstacle to their use is the lack of simple environmental and economical sustainable methodologies to obtain these proteins with high purity. In this work, a new purification process is proposed based on the induced precipitation of the target proteins followed by ultrafiltration. Purities of 89.5% of both phycobiliproteins and 87.3% of R-phycoerythrin were achieved using ammonium sulfate and poly(acrylic acid) sodium salts as precipitation agents (followed by an ultrafiltration step), while maintaining high recovery yields and protein structure stability. Environmental analysis performed to evaluate the proposed process shows that the carbon footprint for the proposed process is much lower than that reported for alternative methodology, and the economic analysis reveals the cost-effective character associated to its high performance. This work is a step toward more sustainable and effective methodologies/processes with high industrial potential.publishe

Comparative Economic Analysis Between Endogenous and Recombinant Production of Hyaluronic Acid

Hyaluronic acid (HA) is a biopolymer with a wide range of applications, mainly in the cosmetic and pharmaceutical sectors. Typical industrial-scale production utilizes organisms that generate HA during their developmental cycle, such as Streptococcus equi sub. zooepidemicus. However, a significant disadvantage of using Streptococcus equi sub. zooepidemicus is that it is a zoonotic pathogen, which use at industrial scale can create several risks. This creates opportunities for heterologous, or recombinant, production of HA. At an industrial scale, the recovery and purification of HA follow a series of precipitation and filtration steps. Current recombinant approaches are developing promising alternatives, although their industrial implementation has yet to be adequately assessed. The present study aims to create a theoretical framework to forecast the advantages and disadvantages of endogenous and recombinant strains in production with the same downstream strategy. The analyses included a selection of the best cost-related recombinant and endogenous production strategies, followed by a sensitivity analysis of different production variables in order to identify the three most critical parameters. Then, all variables were analyzed by varying them simultaneously and employing multiple linear regression. Results indicate that, regardless of HA source, production titer, recovery yield and bioreactor scale are the parameters that affect production costs the most. Current results indicate that recombinant production needs to improve current titer at least 2-fold in order to compete with costs of endogenous production. This study serves as a platform to inform decision-making for future developments and improvements in the recombinant production of HA