The magnetotactic bacteria (MTB) model Magnetospirillum gryphiswaldense MSR-1 (Mgryph) is typically known for its capacity to produce magnetic nanoparticles with unique properties, namely, magnetosomes. However, the magnetosome fraction represents only around 4% of the total cell mass. Therefore, the downstream processing of Mgryph generates a substantial amount of under-utilized microbial biomass waste (MBW) rich in proteins and polyhydroxyalkanoates (PHAs), which can be used, for example, as animal feed and biodegradable bioplastics, respectively. In this work, we have designed an integrated Mgryph-based biorefinery through the utilization of the MBW for the recovery of PHA and soluble proteins using NaClO extraction, revealing that poly­(3-hydroxybutyrate-co-3-hydroxyvalerate) is produced with a relative abundance of 99:1 mol % (3HB/3HV). We have further upgraded PHA into crotonic acid using pyrolysis, which can be used in adhesive and biofuel manufacturing. The effect of the MBW concentration used in the NaClO extraction step (10, 30, and 50 g MBW L–1) was evaluated to determine PHA recovery yields, purity, and purification factor, as well as the thermal stability and fraction of volatile components. The condition using 10 g MBW L–1 was the best among those tested with 51.3 ± 0.14% PHA content in the extract, 97.8% extraction yield, and 2.93 purification factor. The thermogravimetric analysis of PHA extracts from Mgryph showed a degradation range between 232 and 292 °C and a purity of up to 73 ± 4%. Under optimal extraction conditions (10 g MBW L–1), 53.3% of the total cellular protein was recovered. Analysis of products from isothermal pyrolysis of PHA extracts at 300 °C yielded up to 86.0 ± 1.5% of crotonic acid. To the best of our knowledge, our study is the first to extract PHA from Mgryph, thus representing a benchmark for future optimization studies for PHA recovery in this microorganism. Moreover, this work explores the development of an integrated Mgryph-based biorefinery for valorizing MBW into added value biochemicals, which can be used in a wide range of applications, thus representing an opportunity to improve the efficiency of magnetosome production toward the development of sustainable bioprocesses.

Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform.

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