Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array¹. Spacer insertion is carried out by the Cas1–Cas2 integrase complex^2–4. A substantial fraction of CRISPR–Cas systems use a Fe–S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM)^5,6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3′-overhang trigger Cas4/Cas1–Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This ‘molecular constipation’ prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1–Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.

Emulsion formation is a major concern when dealing with multiphasic fermentations. Flocculants can be used together with other demulsification techniques to improve oil recovery in multiphasic fermentations. In this paper, the impact of adding flocculants during a multiphasic fermentation with 10 wt% dodecane, to destabilize the broth emulsion, improve creaming formation and enhance oil recovery is studied. Flocculants, CaCl₂ and (NH₄)₂SO₄ were shown to be the most promising flocculants. Flocculant addition, their time of addition, and its impact on multiphasic fermentations has been evaluated by comparing fermentation performance against reference fermentations and three oil recovery methods: gravity settling, gas enhanced oil recovery and centrifugation. When adding 75 mM of (NH₄)₂SO₄ during fermentation, the creaming rate during gravity settling increased 3-fold and the oil recovery by gas enhanced oil recovery was 35%, without altering fermentation performance. Addition of CaCl₂ during fermentation resulted in 88% and 67% oil recovery for early and late addition, which is a 4 and 3-fold increase in comparison with the reference. Yet, CaCl₂ deviated from standard fermentation performance when added immediately after second phase addition. In conclusion, flocculant addition during multiphasic fermentation can be used to destabilize microbial emulsions and potentially improve in situ oil recovery.

Multiphasic fermentations where an organic phase is spontaneously formed or when it is added for product removal are commonly used for production of valuable compounds. The turbulent conditions and the presence of surface-active compounds (SACs) during fermentation create a stable emulsion difficult to separate. A gas bubble/oil droplet separation method has been proposed to break such emulsion. In this paper, we propose a mathematical model to describe oil/bubble interaction in a region of high oil droplet concentration. Model validation was performed using a synthetic emulsion and an emulsion from a fermentation broth. By applying the optimal parameters predicted by the model, a 6- and 3-times oil recovery improvement was reached for the synthetic emulsion and the fermentation broth, respectively. In conclusion, the proposed mechanistic model allowed to improve oil recovery in the existing laboratory set-up, and can be used to optimize the separation and recovery method at large scale.