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Carotenoids are a group of isoprenoid pigments naturally synthesized by plants and microorganisms, which are applied industrially in food, cosmetic, and pharmaceutical product formulations. In addition to their use as coloring agents, carotenoids have been proposed as health additives, being able to prevent cancer, macular degradation, and cataracts. Moreover, carotenoids may also protect cells against oxidative damage, acting as an antioxidant agent. Considering the interest in greener and sustainable industrial processing, the search for natural carotenoids has increased over the last few decades. In particular, it has been suggested that the use of bioprocessing technologies can improve carotenoid production yields or, as a minimum, increase the efficiency of currently used production processes. Thus, this review provides a short but comprehensive overview of the recent biotechnological developments in carotenoid production using microorganisms. The hot topics in the field are properly addressed, from carotenoid biosynthesis to the current technologies involved in their extraction, and even highlighting the recent advances in the marketing and application of “microbial” carotenoids. It is expected that this review will improve the knowledge and understanding of the most appropriate and economic strategies for a biotechnological production of carotenoids.

Summary A key barrier to market penetration for sophorolipid biosurfactants is the ability to improve productivity and utilize alternative feedstocks to reduce the cost of production. To do this, a suitable screening tool is required that is able to model the interactions between media components and alter conditions to maximize productivity. In the following work, a central composite design is applied to analyse the effects of altering glucose, rapeseed oil, corn steep liquor and ammonium sulphate concentrations on sophorolipid production with Starmerella bombicola ATCC 222144 after 168 h. Sophorolipid production was analysed using standard least squares regression and the findings related to the growth (OD600) and broth conditions (glucose, glycerol and oil concentration). An optimum media composition was found that was capable of producing 39.5 g l–1 sophorolipid. Nitrogen and rapeseed oil sources were found to be significant, linked to their role in growth and substrate supply respectively. Glucose did not demonstrate a significant effect on production despite its importance to biosynthesis and its depletion in the broth within 96 h, instead being replaced by glycerol (via triglyceride breakdown) as the hydrophilic carbon source at the point of glucose depletion. A large dataset was obtained, and a regression model with applications towards substrate screening and process optimisation developed. By applying a central composite circumscribed design of experiment, it has been possible to model the contribution of media components towards sophorolipid production. Through this, an optimised control has been developed alongside a predictive tool with applications in feedstock screening.

In this work, the viability of continuous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production with controlled composition in Haloferax mediterranei when fed volatile fatty acids is demonstrated. Continuous fermentations showed to greatly outperform batch fermentations with continuous feeding. Operating the bioreactor continuously allowed for PHBV productivity normalised by cell density to increase from 0.29 to 0.38 mg L −1 h −1 , in previous continuously fed-fed batch fermentations, to 0.87 and 1.43 mg L −1 h −1 in a continuous mode of operation for 0.1 and 0.25 M carbon concentrations in the media respectively. Continuous bioreactor experiments were carried out for 100 h, maintaining control over the copolymer composition at around 30 mol% 3-hydroxyvalerate 3HV. This work presents the first continuous production of PHBV in Haloferax mediterranei which continuously delivers polymer at a higher productivity, compared to fed-batch modes of operation. Operating bioreactors continuously whilst maintaining control over copolymer composition brings new processing opportunities for increasing biopolymer production capacity, a crucial step towards the wider industrialisation of polyhydroxyalkanoates (PHAs).

The incidence of anterior cruciate ligament (ACL) ruptures is approximately 50 per 100,000 people. ACL rupture repair methods that offer better biomechanics have the potential to reduce long term osteoarthritis. To improve ACL regeneration biomechanically similar, biocompatible and biodegradable tissue scaffolds are required. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with high 3-hydroxyvalerate (3HV) content, based scaffold materials have been developed, with the advantages of traditional tissue engineering scaffolds combined with attractive mechanical properties, e.g., elasticity and biodegradability. PHBV with 3HV fractions of 0 to 100 mol% were produced in a controlled manner allowing specific compositions to be targeted, giving control over material properties. In conjunction electrospinning conditions were altered, to manipulate the degree of fibre alignment, with increasing collector rotating speed used to obtain random and aligned PHBV fibres. The PHBV based materials produced were characterised, with mechanical properties, thermal properties and surface morphology being studied. An electrospun PHBV fibre mat with 50 mol% 3HV content shows a significant increase in elasticity compared to those with lower 3HV content and could be fabricated into aligned fibres. Biocompatibility testing with L929 fibroblasts demonstrates good cell viability, with the aligned fibre network promoting fibroblast alignment in the axial fibre direction, desirable for ACL repair applications. Dynamic load testing shows that the 50 mol% 3HV PHBV material produced can withstand cyclic loading with reasonable resilience. Electrospun PHBV can be produced with low batch variability and tailored, application specific properties, giving these biomaterials promise in tissue scaffold applications where aligned fibre networks are desired, such as ACL regeneration.

Two different methods of production of bio-hydrogenated diesel (BHD), simply called green diesel from rice bran oil (RBO), were performed. In the first route, a direct hydrotreating reaction of RBO to BHD catalysed by Pd/Al2O3 was performed in a high-pressure batch reactor. Operating conditions were investigated as follows: catalyst loading (0.5 to 1.5% wt.), temperature (325 to 400 °C), initial hydrogen (H2) pressure (40 to 60 bar) and reaction time (30 to 90 min). The optimal condition was obtained at 1% wt catalyst loading, 350 °C, 40 bar H2 pressure and 60 min. Yields of crude/refined biofuels and BHD achieved were approximately 98%, 81.71% and 73.71%, respectively. In another route, transesterification together with hydrotreating reactions of rice bran methyl ester (RBME) to BHD was performed using the optimal conditions obtained from the first route. The amount of 98% crude biofuel was obtained and was equivalent to production yields of refined biofuel (85.71%) and BHD (68.51%). Furthermore, physical and chemical properties of both RBO/RBME green diesel were also considered following ASTM standard methods. In summary, both catalytic reactions were achieved in the range of a low-speed industrial diesel and were further recommended for BHD or green diesel production from RBO.

Rhamnolipids (RL) are microbial amphiphilic molecules containing a hydrophilic rhamnose head and a hydrophobic fatty acid tail. RL are of interest to academia and industry due to their potential as a biosurfactant, being used to substitute petroleum-based surfactants in traditional applications or in novel bioremediation and biomedical applications. Currently, commercialization of RL is still in a nascent state, and improved RL production in terms of titers, yields, and productivities could benefit their techno-economic viability and market competitiveness. This review provides a detailed assessment of recent studies that have achieved higher RL production through improvements in microbial producers, media formulation, fermentation design, and operations. Key successes and areas for future work are identified and discussed in detail, as well as put into context with pilot-scale and techno-economic analysis of RL production from the wider literature. This review provides an updated perspective on the current status of RL production. The discussions and insights provided could potentially be used to improve future RL and biosurfactant commercialization efforts.

The extreme haloarchaea Haloferax mediterranei accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) without the need for specific precursors. In this study, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and, for the first time, nitrate. With excess nitrogen, ammonium and nitrate cultures generated 10.7 g/L biomass containing 4.6 wt% PHBV and 5.6 g/L biomass with 9.3 wt% PHBV, respectively. Copolymer composition varied with the nitrogen source used: PHBV from ammonium cultures had 16.9 mol% 3-hydroxyvalerate (HV), while PHBV from nitrate cultures contained 12.5 mol% HV. Nitrogen limitation was achieved with carbon-to-nitrogen (C/N) molar ratios of 25 or higher. Nitrogen limitation reduced biomass generation and polymer concentration, but polymer accumulation increased to 6.6 and 9.4% for ammonium and nitrate, respectively, with C/N 42. PHBV composition was also affected and cultures with lower C/N ratios produced richer HV polymers. Copolymer formation was not a uniform process: HV was only detected after a minimum accumulation of 0.45 g/L PHB and lasted for a maximum of 48 h. The understanding of copolymer synthesis and the influence of culture conditions such as the nitrogen source will help in designing novel strategies for the production of PHBV with more regular structure and material properties.

The effects of foaming on the production of the hydrophobin protein HFBII by fermentation have been investigated at two different scales. The foaming behaviour was characterised in standard terms of the product enrichment and recovery achieved. Additional specific attention was given to the rate at which foam, product and biomass overflowed from the fermentation system in order to assess the utility of foam fractionation for HFBII recovery. HFBII was expressed as an extracellular product during fed-batch fermentations with a genetically modified strain of Saccharomyces cerevisiae, which were carried out with and without the antifoam Struktol J647. In the presence of antifoam, HFBII production is shown to be largely unaffected by process scale, with similar yields of HFBII on dry matter obtained. More variation in HFBII yield was observed between fermentations without antifoam. In fermentations without antifoam, a maximum HFBII enrichment in the foam phase of 94.7 was measured with an overall enrichment, averaged over all overflowed material throughout the whole fermentation, of 54.6 at a recovery of 98.1%, leaving a residual HFBII concentration of 5.3 mg L−1 in the fermenter. It is also shown that uncontrolled foaming resulted in reduced concentration of biomass in the fermenter vessel, affecting total production. This study illustrates the potential of foam fractionation for efficient recovery of HFBII through simultaneous high enrichment and recovery which are greater than those reported for similar systems.

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator . The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration ( C 0 ) and the specific growth rate ( µ ) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations ( C 0  = 2.9–23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L ( C 0  = 19.1 g/L) saponified SCG oil. Peak µ -values of 0.139 h −1 and 0.145 h −1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates ( µ  = 0.35 h −1 ) than on the crude SCG oil ( µ  = 0.23 h −1 ). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA. Key points • The bioavailability of plant oil substrates can be improved via saponification. • Cell growth and inhibition were accurately described by the Han-Levenpsiel model. • Mixing crude and saponified oils enable variation of free fatty acid content.

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration (C.sub.0) and the specific growth rate ([micro]) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations (C.sub.0 = 2.9-23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L (C.sub.0 = 19.1 g/L) saponified SCG oil. Peak [micro]-values of 0.139 h.sup.-1 and 0.145 h.sup.-1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates ([micro] = 0.35 h.sup.-1) than on the crude SCG oil ([micro] = 0.23 h.sup.-1). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA.