Enrichment, isolation, and evaluation of polyhdroxyalkanoate-producing marine bacteria from oil-containing culture medium

Abstract:

[Background] Bacteria can synthesize medium-chain-length polyhydroxyalkanoates (MCL-PHA) through fatty acid metabolic pathway coupled with PHA synthetic pathway. MCL-PHA’s tensile strength, glass transition temperature, and other processing properties better than the short-chain-length PHA, are the promising candidate for PHA industrialization. [Objective] In order to obtain new PHA-producing bacteria that can metabolize oils, we used three methods to isolate bacteria from marine sediments, identified them, and then evaluated the PHA-producing ability. [Methods] We collected the offshore sediment sample of Dapeng Bay in Shenzhen, and isolated bacteria directly from the sample, each week during the 5-week incubation, or during the 10-week enrichment with stepwise increase of oil content (1%–10%) and salt content (3.5%–12.5%). Then, we identified the strains based on 16S rRNA gene similarity and phylogenetic analysis, tested the PHA-producing ability by sequencing the gene of PHA synthase (PhaC), and determined the types, metabolic pathways, and phylogenetic relationship of PhaC according to the draft genome. The content and composition of PHA were detected by gas chromatography. [Results] A total of 96 strains were isolated and 38% had the phaC gene. Among them, 9 genera had not been reported to produce PHA: Acuticoccus, Idiomarina, Halobacillus, Microbulbifer, Maritimibacter, Nitratireductor, Pelagibaca, Pseudooceanicola and Thalassospira, and 8 of the 9 genera except Microbulbifer were enriched with oil. Thus, the enrichment with oil helped to discover new PHA-producing bacterial resources. In addition, we obtained 2 strains with high intracellular PHA content: N. aquimarinus SY-2-4 could accumulate PHA of 35.0% dry cell weight when cultured in nutrient broth, while Roseibium aggregate SN13-21 could use pyruvate as sole carbon source to accumulate PHA of 19.9% dry cell weight. [Conclusion] The offshore environment of Dapeng Bay harbors rich PHA-producing bacterial resources that are worthy of further investigation. The enrichment method of stepwise increase of oil content and salt content is beneficial to isolating different PHA-producing bacteria. Next, fermentation optimization of the 2 high-PHA-content strains will be further investigated.

Key words:offshore sediment;polyhydroxyalkanoate;moderately halophiles;enrichment with oil;phaC gene