Summary The disposal of reject brine, a highly concentrated waste by‐product generated by various industrial processes, represents a major economic and environmental challenge. The common practice in dealing with the large amounts of brine generated is to dispose of it in a pond and allow it to evaporate. The rate of evaporation is therefore a key factor in the effectiveness of the management of these ponds. The addition of various dyes has previously been used as a method to increase the evaporation rate. In this study, a biological approach, using pigmented halophilic bacteria (as opposed to chemical dyes), was assessed. Two bacteria, an Arthrobacter sp. and a Planococcus sp. were selected due to their ability to increase the evaporation of synthetic brine. When using industrial brine, supplementation of the brine with an iron source was required to maintain the pigment production. Under these conditions, the Planococcus sp. CP 5‐4 produced a carotenoid‐like pigment, which resulted in a 20% increase in the evaporation rate of the brine. Thus, the pigment production capability of halophilic bacteria could potentially be exploited as an effective step in the management of industrial reject brines, analogous to the crystallizer ponds used to mine salt from sea water.

Background The genus Planococcus is comprised of halophilic bacteria generally reported for the production of carotenoid pigments and biosurfactants. In previous work, we showed that the culturing of the orange-pigmented Planococcus sp. CP5-4 isolate increased the evaporation rate of industrial wastewater brine effluent, which we attributed to the orange pigment. This demonstrated the potential application of this bacterium for industrial brine effluent management in evaporation ponds for inland desalination plants. Here we identified a C30-carotenoid biosynthetic gene cluster responsible for pigment biosynthesis in Planococcus sp. CP5-4 through isolation of mutants and genome sequencing. We further compare the core genes of the carotenoid biosynthetic gene clusters identified from different Planococcus species’ genomes which grouped into gene cluster families containing BGCs linked to different carotenoid product chemotypes. Lastly, LC–MS analysis of saponified and unsaponified pigment extracts obtained from cultures of Planococcus sp. CP5-4, revealed the structure of the main (predominant) glucosylated C30-carotenoid fatty acid ester produced by Planococcus sp. CP5-4. Results Genome sequence comparisons of isolated mutant strains of Planococcus sp. CP5-4 showed deletions of 146 Kb and 3 Kb for the non-pigmented and “yellow” mutants respectively. Eight candidate genes, likely responsible for C30-carotenoid biosynthesis, were identified on the wild-type genome region corresponding to the deleted segment in the non-pigmented mutant. Six of the eight candidate genes formed a biosynthetic gene cluster. A truncation of crtP was responsible for the “yellow” mutant phenotype. Genome annotation revealed that the genes encoded 4,4′-diapolycopene oxygenase (CrtNb), 4,4′- diapolycopen-4-al dehydrogenase (CrtNc), 4,4′-diapophytoene desaturase (CrtN), 4,4′- diaponeurosporene oxygenase (CrtP), glycerol acyltransferase (Agpat), family 2 glucosyl transferase 2 (Gtf2), phytoene/squalene synthase (CrtM), and cytochrome P450 hydroxylase enzymes. Carotenoid analysis showed that a glucosylated C30-carotenoid fatty acid ester, methyl 5-(6-C17:3)-glucosyl-5, 6′-dihydro-apo-4, 4′-lycopenoate was the main carotenoid compound produced by Planococcus sp. CP5-4. Conclusion We identified and characterized the carotenoid biosynthetic gene cluster and the C30-carotenoid compound produced by Planococcus sp. CP5-4. Mass-spectrometry guided analysis of the saponified and unsaponified pigment extracts showed that methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate esterified to heptadecatrienoic acid (C17:3). Furthermore, through phylogenetic analysis of the core carotenoid BGCs of Planococcus species we show that various C30-carotenoid product chemotypes, apart from methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate and 5-glucosyl-4, 4-diaponeurosporen-4′-ol-4-oic acid, may be produced that could offer opportunities for a variety of applications.