PREreview of Complex waste stream valorisation through combined enzymatic hydrolysis and catabolic assimilation byPseudomonas putida

This article introduces an approach to transform municipal waste into valuable products through enzymatic pre-treatment bioconversion using an engineered strain of Pseudomonas putida. The authors focused on enabling engineer this strain to assimilate various compounds, including terephthalic acid (TPA), ethylene glycol (EG), lactic acid, glucose, xylose, ferulic acid, p-coumaric acid, glycerol, and fatty acids. This choice is significant as TPA and EG are key monomers of polyethylene terephthalate (PET) plastics prevalent in municipal waste, and other compounds are typical in wood, agricultural, and food wastes.  To achieve this, the authors modified a previously reported strain, known to co-utilize certain sugars and aromatic compounds to introduce pathways for TPA, EG, and xylose metabolism. The methodology is thorough, and the choice of microorganisms and products is aptly justified. As a result, regardless of the amount and concentration of each available substrate, the engineered P. putida strain could convert the available carbon into biomass. Also, hydrolysates obtained from the enzymatic hydrolysis of mimicked municipal waste were fed as carbon sources for the production of a human therapeutic protein (IFNα2a) and bioplastic monomers (PHA) providing a proof-of-concept for the biological upcycling of mimicked municipal solid waste. Although the work is very detailed, complete and scientifically exciting, caution against overstating these findings is advisable, as the demonstrated processes may not directly translate to an economically feasible municipal waste treatment scenario, a limitation they acknowledge. 

Major issues

• Mimiched waste: The study’s use of a simulated medium is logical for establishing a proof-of-concept, but extending these conclusions to actual municipal waste is premature. Real waste comprises diverse, unknown materials that could inhibit the process. Therefore, claims about the biological upcycling of municipal waste should be tempered until the strain's effectiveness in real-world conditions is validated. 

  Mannose metabolism: The article overlooks the presence of mannan, a key polysaccharide in various leguminous food wastes and softwoods, likely found in paper waste. It would be good to understand how mannose is metabolized by the  P. putida strain, as this sugar metabolism is usually underexplored and would be very relevant as a product from municipal waste samples rich in food and agricultural wastes.

  Enzymatic pretreatment: The rationale behind the chosen enzymatic pre-treatment lacks clarity, particularly considering its cost implications and potential inefficiencies in processing real municipal solid waste (MSW). Possible, chemical, or hydrothermal pretreatments would be a better fit to mimic a real process, but of course, they would generate small amounts of degradation products that might inhibit or reduce the efficiency of the P. putida strain. Nevertheless, this should be evaluated, as an enzymatic pretreatment such as the one proposed will probably have a very high cost and will hamper the process's economic feasibility. Also, the efficiency in converting the substrates in products seems low, as 25% solids are used, but products are in the range of a few g/L. So, it would be good to show the conversion yields of the mimic municipal wastes by this process. 

  Discussion: The discussion would benefit from greater depth, particularly in addressing potential inhibitors or competing substances for the selected microorganism and examining the conversion yield. Also, one of the target products selected for this proof-of-concept is pharmaceutical-grade, which requires a much more stringent production process. It would be good to discuss this aspect further. This can have a significant impact on the financial viability of production.

Minor issues

• Figures: Certain graphics, like those depicting metabolic pathways, require revision for clarity. A simplified schematic for each pathway could be more effective instead of duplicating the same pathway and highlighting it in bold. Also, some images present huge amounts of data, being somewhat polluted. It is advisable that the authors choose to present the most significant data within the main paper text and include other evidence or explanations as supplementary material to simplify the readability and overall comprehension of the central idea in the Figures. 

  Abstract: The abstract is overly general. The abstract currently lacks specific quantitative data from the study. Including such data would give a clearer overview of the research findings and implications.

Competing interests

The author declares that they have no competing interests.