Optimization of a Two-Species Microbial Consortium for Improved Mcl-PHA Production From Glucose–Xylose Mixtures

Polyhydroxyalkanoates (PHAs) have attracted much attention as a good substitute for petroleum-based plastics, especially mcl-PHA due to their superior physical and mechanical properties with broader applications. Artificial microbial consortia can solve the problems of low metabolic capacity of single engineered strains and low conversion efficiency of natural consortia while expanding the scope of substrate utilization. Therefore, the use of artificial microbial consortia is considered a promising method for the production of mcl-PHA. In this work, we designed and constructed a microbial consortium composed of engineered Escherichia coli MG1655 and Pseudomonas putida KT2440 based on the “nutrition supply–detoxification” concept, which improved mcl-PHA production from glucose-xylose mixtures. An engineered E. coli that preferentially uses xylose was engineered with an enhanced ability to secrete acetic acid and free fatty acids (FFAs), producing 6.44 g/L acetic acid and 2.51 g/L FFAs with 20 g/L xylose as substrate. The mcl-PHA producing strain of P. putida in the microbial consortium has been engineered to enhance its ability to convert acetic acid and FFAs into mcl-PHA, producing 0.75 g/L mcl-PHA with mixed substrates consisting of glucose, acetic acid, and octanoate, while also reducing the growth inhibition of E. coli by acetic acid. The further developed artificial microbial consortium finally produced 1.32 g/L of mcl-PHA from 20 g/L of a glucose–xylose mixture (1:1) after substrate competition control and process optimization. The substrate utilization and product synthesis functions were successfully divided into the two strains in the constructed artificial microbial consortium, and a mutually beneficial symbiosis of “nutrition supply–detoxification” with a relatively high mcl-PHA titer was achieved, enabling the efficient accumulation of mcl-PHA. The consortium developed in this study is a potential platform for mcl-PHA production from lignocellulosic biomass.

Production of a newly discovered PHA family member with an isobutyrate-fed enrichment culture

Using microbial enrichment cultures for the production of waste-derived polyhydroxyalkanoates (PHAs) is a promising technology to recover secondary resources. Volatile fatty acids (VFAs) form the preferred substrate for PHA production. Isobutyrate is a VFA appearing in multiple waste valorization routes, such as anaerobic fermentation, chain elongation, and microbial electrosynthesis, but has never been assessed individually on its PHA production potential. This research investigates isobutyrate as sole carbon source for a microbial enrichment culture in comparison to its structural isomer butyrate. The results reveal that the enrichment of isobutyrate has a very distinct character regarding microbial community development, PHA productivity, and even PHA composition. Although butyrate is a superior substrate in almost every aspect, this research shows that isobutyrate-rich waste streams have a noteworthy PHA-producing potential. The main finding is that the dominant microorganism, a Comamonas sp., is linked to the production of a unique PHA family member, poly(3-hydroxyisobutyrate) (PHiB), up to 37% of the cell dry weight. This is the first scientific report identifying microbial PHiB production, demonstrating that mixed microbial communities can be a powerful tool for discovery of new metabolic pathways and new types of polymers. Key points • PHiB production is a successful storage strategy in an isobutyrate-fed SBR • Isomers isobutyrate and butyrate reveal a very distinct PHA production behavior • Enrichments can be a tool for discovery of new metabolic pathways and polymers Graphical abstract

Sustainability Evaluation of Polyhydroxyalkanoate Production from Slaughterhouse Residues Utilising Emergy Accounting

High raw material prices and rivalry from the food industry have hampered the adoption of renewable resource-based goods. It has necessitated the investigation of cost-cutting strategies such as locating low-cost raw material supplies and adopting cleaner manufacturing processes. Exploiting waste streams as substitute resources for the operations is one low-cost option. The present study evaluates the environmental burden of biopolymer (polyhydroxyalkanoate) production from slaughtering residues. The sustainability of the PHA production process will be assessed utilising the Emergy Accounting methodology. The effect of changing energy resources from business as usual (i.e., electricity mix from the grid and heat provision utilising natural gas) to different renewable energy resources is also evaluated. The emergy intensity for PHA production (seJ/g) shows a minor improvement ranging from 1.5% to 2% by changing only the electricity provision resources. This impact reaches up to 17% when electricity and heat provision resources are replaced with biomass resources. Similarly, the emergy intensity for PHA production using electricity EU27 mix, coal, hydropower, wind power, and biomass is about 5% to 7% lower than the emergy intensity of polyethylene high density (PE-HD). In comparison, its value is up to 21% lower for electricity and heat provision from biomass.

Influence of the total concentration and the profile of volatile fatty acids on polyhydroxyalkanoates (PHA) production by mixed microbial cultures

Polyhydroxyalkanoates (PHAs) production from lignocellulosic biomass using mixed microbial cultures (MMC) is a potential cheap alternative for reducing the use of petroleum-based plastics. In this study, an MMC adapted to acidogenic effluent from dark fermentation (DF) of exhausted sugar beet cossettes (ESBC) has been tested in order to determine its capability to produce PHAs from nine different synthetic mixtures of volatile fatty acids (VFAs). The tests consisted of mixtures of acetic, propionic, butyric, and valeric acids in the range of 1.5–9.0 g/L of total acidity and with three different valeric:butyric ratios (10:1, 1:1, and 1:10). Experimental results have shown a consistent preference of the MMC for the butyric and valeric acids as carbon source instead other shorter acids (propionic or acetic) in terms of PHA production yield (estimated in dry cell weight basis), with a maximum value of 23% w/w. Additionally, valeric-rich mixtures have demonstrated to carry out a fast degradation process but with poor final PHA production compared with high butyric mixtures. Finally, high initial butyric and valeric concentrations (1.1 g/L and 4.1 g/L) have demonstrated to be counterproductive to PHA production.

Biosynthesis of Polyhydroxyalkanoate by Recombinant Bacteria: A Review

Polyhydroxyalkanoates (PHAs), a biodegradable polymer, has many industrial and medical applications such as heart valves, scaffold, suture and drug delivery. Because of their structural diversity and close analogy to plastics, PHAs have gained major interest over the world. Natural isolates, recombinant bacteria, and plants have all been found to improve the quality, quantity, and economics of PHA production. Their biodegradability makes them an especially appealing synthetic plastic substitute. PHB biosynthetic genes phbA, phbB, and phbC are grouped and systematised into a single phbCAB operon. In terms of alignment and clustering of the relevant genes, the PHB pathway varies greatly across bacterial genera. In this regard, the enzymes appear to have a high degree of sequence preservation. The structural studies further improve the mechanism of action of these enzymes and helped in improving and choosing the good candidates for increased production of PHB.

Modelling cell growth and polyhydroxyalkanoate (PHA) polymer synthesis by Pseudomonas putida LS46 under oxygen-limiting conditions

Background: Polyhydroxyalkanoates (PHAs) are biodegradable, biocompatible, and non-toxic polymers synthesized by bacteria that may be used to displace some petroleum-based plastic materials. One of the major barriers to the commercialization of PHA biosynthesis is the high cost of production. Objective: Oxygen-limitation is known to greatly influence bacterial cell growth and PHA production. In this study, the growth and synthesis of medium chain length PHAs (mcl-PHAs) by Pseudomonas putida LS46, cultured in batch-mode with octanoic acid, under oxygen-limited conditions, was modeled. Methods: Four models, including the Monod model, incorporated Leudeking–Piret (MLP), the Moser model incorporated Leudeking–Piret (Moser-LP), the Logistic model incorporated Leudeking–Piret (LLP), and the Modified Logistic model incorporated Leudeking–Piret (MLLP) were investigated. Kinetic parameters of each model were calibrated by using the multi-objective optimization algorithm, Pareto Archived Dynamically Dimensioned Search (PA-DDS), by minimizing the sum of absolute error (SAE) for PHA production and growth simultaneously. Results and Conclusions: Among the four models, MLP and Moser-LP models adequately represented the experimental data for oxygen-limited conditions. However, the MLP and Moser-LP models could not adequately simulate PHA production under oxygen-excess conditions. Modeling cell growth and PHA will assist in the development of a strategy for industrial-scale production.

Photoautotrophic and Mixotrophic Cultivation of Polyhydroxyalkanoate-Accumulating Microalgae Consortia Selected under Nitrogen and Phosphate Limitation

Microalgae consortia were photoautotrophically cultivated in sequencing batch photobioreactors (SBPRs) with an alteration of the normal growth and starvation (nutrient limitation) phases to select consortia capable of polyhydroxyalkanoate (PHA) accumulation. At the steady state of SBPR operation, the obtained microalgae consortia, selected under nitrogen and phosphate limitation, accumulated up to 11.38% and 10.24% of PHA in their biomass, which was identified as poly(3-hydroxybutyrate) (P3HB). Photoautotrophic and mixotrophic batch cultivation of the selected microalgae consortia was conducted to investigate the potential of biomass and PHA production. Sugar source supplementation enhanced the biomass and PHA production, with the highest PHA contents of 10.94 and 6.2%, and cumulative PHA productions of 100 and 130 mg/L, with this being achieved with sugarcane juice under nitrogen and phosphate limitation, respectively. The analysis of other macromolecules during batch cultivation indicated a high content of carbohydrates and lipids under nitrogen limitation, while higher protein contents were detected under phosphate limitation. These results recommended the selected microalgae consortia as potential tools for PHA and bioresource production. The mixed-culture non-sterile cultivation system developed in this study provides valuable information for large-scale microalgal PHA production process development following the biorefinery concept.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant

Background Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. Results Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. Conclusions Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Production of Polyhydroxyalkanoates in Unsterilized Hyper-Saline Medium by Halophiles Using Waste Silkworm Excrement as Carbon Source

The chlorophyll ethanol-extracted silkworm excrement was hardly biologically reused or fermented by most microorganisms. However, partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate polyhydroxyalkanoates. In this study, by using the nile red staining and gas chromatography assays, two endogenous haloarchaea strains: Haloarcula hispanica A85 and Natrinema altunense A112 of silkworm excrement were shown to accumulate poly(3-hydroxybutyrate) up to 0.23 g/L and 0.08 g/L, respectively, when using the silkworm excrement as the sole carbon source. The PHA production of two haloarchaea showed no significant decreases in the silkworm excrement medium without being sterilized compared to that of the sterilized medium. Meanwhile, the CFU experiments revealed that there were more than 60% target PHAs producing haloarchaea cells at the time of the highest PHAs production, and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells (to nearly 100%) and the production of PHAs. In conclusion, our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement, effectively. The introduce of halophiles could provide a potential way for open fermentation to further lower the cost of the production of PHAs.

PHA Production from Cheese Whey and “Scotta”: Comparison between a Consortium and a Pure Culture of Leuconostoc mesenteroides

It is urgent to expand the market of biodegradable alternatives to oil-derived plastics owing to (i) increasingly limited oil availability/accessibility, and (ii) the dramatic impact of traditional plastics on aquatic life, the food chain, all Earth ecosystems, and ultimately, human health. Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that can be obtained through microbial fermentation of agro-industrial byproducts, e.g., milk and cheese whey. Here, the PHA-accumulating efficiency of a mixed microbial culture (MMC, derived from activated sludges) grown on dairy byproducts (cheese and scotta whey) was measured. Bioreactor tests featuring temperature and pH control showed that both scotta and pre-treated Toma cheese whey could be used for PHA production by MMC, although scotta cheese whey supported higher PHA yield and productivity. The advantages of open MMCs include their plasticity and versatility to fast changing conditions; furthermore, no growth-medium sterilization is needed prior to fermentation. However, the use of pure cultures of efficient PHA producers may support better metabolic performances. Therefore, PHA-producing strains were isolated from an MMC, leading to the satisfactory identification of two bacterial strains, Citrobacter freundii and Leuconostoc spp., whose ability to accumulate PHAs in synthetic media was confirmed. A more detailed investigation by mass spectrometry revealed that the strain was L. mesenteroides. Although the validation of L. mesenteroides’ potential to produce PHA through fermentation of agro-industrial byproducts requires further investigations, this is the first study reporting PHA production with the Leuconostoc genus.