Food wastes for bioproduct production and potential strategies for high feedstock variability.

Unavoidable food wastes could be an important feedstock for industrial biotechnology, while their valorization could provide added value for the food processor. However, despite their abundance and low costs, the heterogeneous/mixed nature of these food wastes produced by food processors and consumers leads to a high degree of variability in carbon and nitrogen content, as well as specific substrates, in food waste hydrolysate. This has limited their use for bioproduct synthesis. These wastes are often instead used in anaerobic digestion and mixed microbial culture, creating a significant knowledge gap in their use for higher value biochemical production via pure and single microbial culture. To directly investigate this knowledge gap, various waste streams produced by a single food processor were enzymatically hydrolyzed and characterized, and the degree of variability with regard to substrates, carbon, and nitrogen was quantified. The impact of hydrolysate variability on the viability and performance of polyhydroxyalkanoates biopolymers production using bacteria (Cupriavidus necator) and archaea (Haloferax mediterranei) as well as sophorolipids biosurfactants production with the yeast (Starmerella bombicola) was then elucidated at laboratory-scale. After which, strategies implemented during this experimental proof-of-concept study, and beyond, for improved industrial-scale valorization which addresses the high variability of food waste hydrolysate were discussed in-depth, including media standardization and high non-selective microbial organisms growth-associated product synthesis. The insights provided would be beneficial for future endeavors aiming to utilize food wastes as feedstocks for industrial biotechnology.

Genetic and process engineering for polyhydroxyalkanoate production from pre- and post-consumer food waste.

Biopolymers produced as microbial carbon storage systems, such as polyhydroxyalkanoates (PHAs), offer potential to be used in place of petrochemically derived plastics. Low-value organic feedstocks, such as food waste, have been explored as a potential substrate for the microbial production of PHAs. In this review, we discuss the biosynthesis, composition and producers of PHAs, with a particular focus on the genetic and process engineering efforts to utilise non-native substrates, derived from food waste from across the entire supply chain, for microbial growth and PHA production. We highlight a series of studies that have achieved impressive advances and discuss the challenges of producing PHAs with consistent composition and properties from mixed and variable food waste and by-products.

Production of polyhydroxybutyrate by coupled saccharification-fermentation of inulin.

Inulin is a fructose-based polysaccharide that can be found in several plant species, from grass and onions to chicory roots; thus, it has the potential to be an excellent renewable source of fructose for several industrial applications. Among them, inulin hydrolysis can be coupled to a fermentation operation to produce polyhydroxybutyrate (PHB) using Cupriavidus necator H16. This work reports the PHB production process involving chicory root inulin hydrolysis using inulinase Novozym 960 followed by a C. necator fermentation. It was found that the maximum saccharification (95% wt.) was reached at 269 U/ginulin after 90 min. The hydrolysates obtained were then inoculated with C. necator, leading to a biomass concentration of 4 g/L with 30% (w/w) polymer accumulation. Although PHB production was low, during the first hours, the cell growth and polymer accumulation detected did not coincide with a fructose concentration decrease, suggesting a simultaneous saccharification and fermentation process, potentially alleviating the product inhibition inherent to the inulinase-fructose system. The characterization of the obtained PHB showed a polymer with more homogeneous values of Mw, and better thermal stability than PHB produced using pure fructose as a fermentation substrate. The results obtained demonstrate a viable alternative carbon substrate for PHB production, opening the possibility for inulin-rich renewable feedstock valorization.

Production of polyhydroxyalkanoates from hydrolysed rapeseed meal by Haloferax mediterranei.

Rapeseed meal (RSM) hydrolysate is a potential low-cost feedstock for the production of polyhydroxyalkanoates (PHAs) by the archaea, Haloferax mediterranei. Acidic and enzymatic hydrolysis were carried out to compare effectiveness. Enzymatic hydrolysis is more effective than acidic hydrolysis for fermentation substrate leading to increased PHA productivity. H. mediterranei didn't grow or produce PHA when acid hydrolysed RSM medium was present in proportions greater than 25% (vol.), potentially due to the effect of inhibitors such as furfural, hydroxymethylfurfural (HMF), etc. However, H. mediterranei was able to grow and produce PHA when using enzymatically hydrolysed RSM medium. The maximum PHA concentration of 0.512 g/L was found at 75% (vol.) in enzymatic RSM hydrolysate medium. The biopolymer obtained had improved thermal and mechanical properties compared to PHB homopolymer. RSM's potential as a low-cost alternative feedstock for improved PHA production under non-sterile conditions was successfully demonstrated, and its usage should be further explored.

Comprehensive optimization of tropical biomass hydrolysis for nitrogen-limited medium-chain polyhydroxyalkanoate synthesis.

Expanding the use of tropical biomass wastes for nitrogen-limited fermentation was investigated, specifically, the production of medium chain length polyhydroxyalkanoates. Comprehensive central composite design was conducted to assess pH, temperature, biomass solid loading, cellulase loading and amylase loading and their impact on the hydrolysis of palm, coconut and cassava wastes. Glucose yields of 33.3, 31.7 and 79.0% wt. with respect to total glucose were found for palm, coconut and cassava, respectively. Importantly, the impact on the total nitrogen derived during enzymatic hydrolysis of these tropical biomass was described for the first time. The level of nitrogen needs to be properly controlled as high nitrogen would result in low carbon to nitrogen ratio leading to low polyhydroxyalkanoates accumulation, but low nitrogen would hinder growth of the biopolymer producer. Maximum hydrolysate nitrogen, were 1.80, 1.55 and 0.871 g/l for palm, coconut and cassava, respectively. Using the surface responses, biomass media designed for high carbon-to-nitrogen were produced and validated using Pseudomonas putida. Low glucose-carbon to nitrogen were found for palm and coconut after scale-up, leading to the majority of their polyhydroxyalkanoates not being biomass-derived. However, cassava-derived biopolymers were successfully accumulated at 9.01 and 7.13% wt. for total medium chain length polyhydroxyalkanoates and 10-carbon polyhydroxyalkanoates, respectively. This study provides an important foundation for the expansion of tropical biomass wastes for biopolymer production and other nitrogen-limited applications in general.

Rapeseed meal valorization strategies via nitrogen- and oxygen-limited production of polyhydroxyalkanoates with Pseudomonas putida.

Rapeseed meal (RSM) is a candidate for biopolymer production due to its abundance, low cost and potential integration with other rapeseed-derived products. However, existing studies pursuing such schemes are limited. The feasibility of different strategies for RSM valorization via protein extraction and polyhydroxyalkanoate production were evaluated. Nitrogen-limited RSM media was produced from hydrolysis of residues which had undergone extensive protein extraction using sodium hydroxide. A study of oxygen-limited fermentation was also performed on hydrolysate of untreated RSM via batch feeding. The typical strategy of using a high carbon-to-nitrogen ratio may not be the most suitable route for polyhydroxyalkanoate (PHA) production using nitrogen-rich biomass as a feedstock. Central composite design-based experiments show that due to mass transfer limitations protein extraction at 1-L scale could only achieve yields around 50% and 69%, at room temperature and 60 °C, respectively. Protein extraction yields reduced with successive extractions, meaning that whilst the RSM hydrolysate is viable for growth, designing a valorization scheme which has the fermentation step dictated by the protein extraction may not be practical/economical. A better route which utilizes oxygen-limitation to initially induce stationary phase was identified, giving accumulation of polyhydroxyalkanoate once the oxygen levels began to recover; 8.93% and 1.75% PHA accumulation in fed-batch cultures of synthetic and RSM media, respectively. The findings demonstrate that decoupling of protein extraction performance from PHA synthesis is feasible. This study provides important insight into the degrees of freedom available in the design of a holistic valorization scheme of rapeseed meal, and high protein lignocellulosic biomass in general.