Enhancing Bioremediation Potential of Pseudomonas putida by Developing Its Acid Stress Tolerance With Glutamate Decarboxylase Dependent System and Global Regulator of Extreme Radiation Resistance

The extensive use of acids in a variety of manufacturing industries results in the increase of discharged acidic waste stream into the environment. Such co-pollution of acids and other organic pollutants limits the biodegradation capability of neutrophilic degraders. With high-throughput genetic techniques, we aim to improve the acid tolerance of a pollutant-degrading bacterium, Pseudomonas putida S16 by genetically engineering it with the glutamate decarboxylase (GAD)-dependent system and the global regulator (IrrE) of extreme radiation resistance. The engineered strains holding either GAD system or irrE regulator could grow under pH 4.5, compared to the wild type. They could also degrade over 90% of a selected pollutant (benzoate or nicotine) under pH 5.0 in 48 h, while no biodegradation was detected with the wild type under the same conditions. We conclude that acid stress tolerance by the possession of the GAD system or IrrE regulator in pollutant-degrading bacteria would be a promising approach to enhance their viability and biodegrading activities in bioremediation of acidic wastes

Triggering of Polymer-Degrading Enzymes from Layered Double Hydroxides for Recycling Strategies

The use of degrading enzymes in polymer formulation is a very attractive strategy to manage the end-of-life of plastics. However, high temperatures cause the denaturation of enzymes and the loss of their catalytic activity; therefore, protection strategies are necessary. Once protected, the enzyme needs to be released in appropriate media to exert its catalytic activity. A successful protection strategy involves the use of layered double hydroxides: cutinase, selected as a highly degrading polyester hydrolytic enzyme, is thermally protected by immobilization in Mg/Al layered double hydroxide structures. Different triggering media are here evaluated in order to find the best releasing conditions of cutinase from LDH. In detail, phosphate and citrate-phosphate buffers, potassium carbonate, sodium chloride, and sodium sulfate solutions are studied. After the comparison of all media in terms of protein release and activity retained, phosphate buffer is selected as the best candidate for the release of cutinase from LDH, and the effect of pH and concentration is also evaluated. The amount of the enzyme released is determined with the Lowry method. Activity tests are performed via spectrophotometry

Advances in combined enzymatic extraction of ferulic acid from wheat bran

Wheat bran could be utilised as feedstock for innovative and sustainable biorefinery processes. Here, an enzymatic hydrolysis process for ferulic acid (FA) extraction was optimised step by step for total wheat bran (Tritello) and then also applied to the outer bran layer (Bran 1). Proteins, reducing sugars, total phenols and FA were quantified. The highest FA yields (0.82-1.05\u202fg/kg bran) were obtained either by rehydrating the bran by autoclaving (Tritello) or by steam explosion (Bran 1) using a bran/water ratio of 1:20, followed by enzymatic pre-treatment with Alcalase and Termamyl, to remove protein and sugars, and a final enzymatic hydrolysis with Pentopan and feruloyl esterase to solubilise phenol. FA was recovered from the final digestate via solid phase extraction. A 40-fold scale-up was also performed and the release of compounds along all the process steps and at increasing incubation times was monitored. Results showed that FA was initially present at a minimum level while it was specifically released during the enzymatic treatment. In the final optimized process, the FA extraction yield was higher than that obtained with NaOH control hydrolysis while, in comparison with other FA enzymatic extraction methods, fewer process steps were required and no buffers, strong acid/alkali nor toxic compounds were used. Furthermore, the proposed process may be easily scaled-up, confirming the feasibility of wheat bran valorisation by biorefinery processes to obtain valuable compounds having several areas of potential industrial exploitation

Comparative preliminary evaluation of two in-stream water treatment technologies for the agricultural reuse of drainage water in the Nile delta

In the Nile Delta, a complex network of canals collects drainage water from surface-irrigated fields, but also municipal wastewater. The goal of this work was to assess the technical, environmental and financial feasibility of the upgrade of a drainage canal (DC) into either an in-stream constructed wetland (ICW) or a canalized facultative lagoon (CFL), in order to produce a water re-usable in agriculture according to the Egyptian law. The model-based design of the proposed technologies was derived from field experimental data for the ICW and laboratory data for the CFL. Both technologies, integrated by a sedimentation pond and a disinfection canal, led to the attainment of the water quality standards imposed by Egyptian Law 92/2013 for the reuse of drainage water. The life cycle assessment indicated that the upgrade of an existing DC to either an ICW or a CFL results in an extremely small environmental burden, 64 0.3% of that of a traditional activated sludge process. The cost/benefit analysis (CBA) was based on the assumptions that (i) farmers currently irrigate a non-food crop (cotton) with the low-quality drainage water present in the DC, and (ii) thanks to the upgrade to a ICW or CFL, farmers will irrigate a food crop characterized by a higher market price (rice). The CBA indicated that the DC upgrade to an ICW represents an attractive investment, as it leads to a financial rate of return > 10% over a wide range of cotton market prices. Conversely, the upgrade to a CFL is less attractive due to high investment costs. In conclusion, the upgrade of DCs to ICWs appears a promising option for the treatment of drainage canal water in the Nile Delta, thanks to the high pollutant removal performances, low cost and negligible environmental burden. This article is protected by copyright. All rights reserved

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Recycling by-design of plastic through formulation with thermally protected enzymes in layered double hydroxide structures

The use of polymer-degrading enzymes in polymer formulation is a very attractive strategy for the life-cycle of plastics. The design of a material able to degrade on-demand could help reach the ambitious targets pursued by recent European policies. Only 32% of the resulting waste from the 50 million tonnes of plastics consumed each year in Europe is actually recycled. Since packaging accounts for more than 40% of all plastics produced every year, the improvement of its circularity from origin to subsequent life cycles is now a priority. The present research suggests a solution to improve the recyclability of plastics via a life cycle approach employing thermally stable enzymes as innovative materials providing a new potential for plastic and its end life. More specifically, cutinase, selected as a highly degrading polyester hydrolytic enzyme, was thermally protected by immobilization in Mg/Al layered double hydroxide structures. The cutinase immobilization efficiency was found to be high, as well as its release ability in an appropriate medium. The thermal stability of cutinase was strongly improved after immobilization, as highlighted by a 6-times increase of its half-life at 90 ◦C, compared to the free enzyme, and by a high activity retention (>60%) after short exposure to temperatures up to 200 ◦C. Moreover, it was demonstrated that a film of poly (butylene succinate-co-adipate) formulated with 5 wt% of immobilized cutinase, completely degraded within 24 h