Rapid biodegradation of microplastics generated from bio-based thermoplastic polyurethane.

The accumulation of microplastics in various ecosystems has now been well documented and recent evidence suggests detrimental effects on various biological processes due to this pollution. Accumulation of microplastics in the natural environment is ultimately due to the chemical nature of widely used petroleum-based plastic polymers, which typically are inaccessible to biological processing. One way to mitigate this crisis is adoption of plastics that biodegrade if released into natural environments. In this work, we generated microplastic particles from a bio-based, biodegradable thermoplastic polyurethane (TPU-FC1) and demonstrated their rapid biodegradation via direct visualization and respirometry. Furthermore, we isolated multiple bacterial strains capable of using TPU-FC1 as a sole carbon source and characterized their depolymerization products. To visualize biodegradation of TPU materials as real-world products, we generated TPU-coated cotton fabric and an injection molded phone case and documented biodegradation by direct visualization and scanning electron microscopy (SEM), both of which indicated clear structural degradation of these materials and significant biofilm formation.

Sphingobium lignivorans sp. nov., isolated from river sediment downstream of a paper mill.

A bacterial isolate, B1D3AT, was isolated from river sediment collected from the Hiwassee River near Calhoun, TN, by enrichment culturing with a model 5-5' lignin dimer, dehydrodivanillate, as its sole carbon source. B1D3AT was also shown to utilize several model lignin-derived monomers and dimers as sole carbon sources in a variety of minimal media. Cells were Gram-stain-negative, aerobic, motile, rod-shaped and formed yellow/cream-coloured colonies on rich agar. Optimal growth occurred at 30 °C, pH 7-8, and in the absence of NaCl. The major fatty acids of B1D3AT were C18 : 1 ω7c and C17 : 1 ω6c. The predominant hydroxy fatty acids were C14 : 0 2-OH and C15 : 0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine and sphingoglycolipid. B1D3AT contained spermidine as the only major polyamine. The major isoprenoid quinone was Q-10 with minor amounts of Q-9 and Q-11. The genomic DNA G+C content of B1D3AT was 65.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 49 core, universal genes defined by Clusters of Orthologous Groups gene families indicated that B1D3AT was a member of the genus Sphingobium. B1D3AT was most closely related to Sphingobium sp. SYK-6, with a 100 % 16S rRNA gene sequence similarity. B1D3AT showed 78.1-89.9 % average nucleotide identity and 19.5-22.2% digital DNA-DNA hybridization identity with other type strains from the genus Sphingobium. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain B1D3AT should be classified as representing a novel species of the genus Sphingobium, for which the name Sphingobium lignivorans sp. nov. is proposed. The type strain is strain B1D3AT (ATCC TSD-279T=DSM 111877T).

The effect of bacterial growth strategies on plasmid transfer and naphthalene degradation for bioremediation.

Mobilizable plasmids are extra-chromosomal, circular DNA that have contributed to the rapid evolution of bacterial genomes and have been used in environmental, biotechnological, and medicinal applications. Degradative plasmids with genetic capabilities to degrade organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), have the potential to be useful for more environmentally friendly and cost-effective remediation technologies compared to existing physical remediation methods. Genetic bioaugmentation, the addition of catabolic genes into well-adapted communities via plasmid transfer (conjugation), is being explored as a remediation approach that is sustainable and long-lasting. Here, we explored the effect of the ecological growth strategies of plasmid donors and recipients on conjugation and naphthalene degradation of two PAH-degrading plasmids, pNL1 and NAH7. Overall, both pNL1 and NAH7 showed conjugation preferences towards a slow-growing ecological growth strategy, except when NAH7 was in a mixed synthetic community. These conjugation preferences were partially described by a combination of growth strategy, GC content, and phylogenetic relatedness. Further, removal of naphthalene via plasmid-mediated degradation was consistently higher in a community consisting of recipients with a slow-growing ecological growth strategy compared to a mixed community or a community consisting of fast-growing ecological growth strategy. Understanding plasmid conjugation and degradative preferences has the capacity to influence future remediation technology design and has broad implications in biomedical, environmental, and health fields.

Microbial assimilation of lignin-derived aromatic compounds and conversion to value-added products.

Lignin is an abundant and sustainable source of aromatic compounds that can be converted to value-added products. However, lignin is underutilized, since depolymerization produces a complex mixture of aromatic compounds that is difficult to convert to a single product. Microbial conversion of mixed aromatic substrates provides a potential solution to this conversion challenge. Recent advances have expanded the range of lignin-derived aromatic substrates that can be assimilated and demonstrated efficient conversion via central metabolism to new potential products. The development of additional non-model microbial hosts and genetic tools for these hosts have accelerated engineering efforts. However, yields with real depolymerized lignin are still low, and additional work will be required to achieve viable conversion processes.

Genetic Suppression of Lethal Mutations in Fatty Acid Biosynthesis Mediated by a Secondary Lipid Synthase.

The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.

Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway.

A characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of eicosapentaenoic (EPA) and/or docosahexaenoic (DHA) acids is mediated by a polyketide/fatty acid synthase mechanism encoded by a set of five genes, pfaABCDE. This unique fatty acid synthesis pathway co-exists with the principal type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of core saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is down regulated in response to exogenous fatty acids, particularly long chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site.Importance The production of long-chain omega-3 polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid responsive transcriptional regulator specific to PUFA biosynthesis.

Linkage of Marine Bacterial Polyunsaturated Fatty Acid and Long-Chain Hydrocarbon Biosynthesis.

Various marine gamma-proteobacteria produce omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (20:5, EPA) and docosahexaenoic acid (22:6, DHA), which are incorporated into membrane phospholipids. Five genes, designated pfaABCDE, encode the polyketide/fatty acid synthase necessary for production of these long-chain fatty acids. In addition to de novo biosynthesis of EPA and DHA, the "Pfa synthase" is also involved with production of a long-chain polyunsaturated hydrocarbon product (31:9, PUHC) in conjunction with the oleABCD hydrocarbon biosynthesis pathway. In this work, we demonstrate that OleA mediates the linkage between these two pathways in vivo. Co-expression of pfaA-E along with oleA from Shewanella pealeana in Escherichia coli yielded the expected product, a 31:8 ketone along with a dramatic ∼10-fold reduction in EPA content. The decrease in EPA content was independent of 31:8 ketone production as co-expression of an OleA active site mutant also led to identical decreases in EPA content. We also demonstrate that a gene linked with either pfa and/or ole operons in diverse bacterial lineages, herein designated pfaT, plays a role in maintaining optimal production of Pfa synthase derived products in Photobacterium and Shewanella species.

Characterization and Application of Marine Microbial Omega-3 Polyunsaturated Fatty Acid Synthesis.

The long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs) EPA (20:5n-3) and DHA (22:6n-3) are widely recognized as beneficial to human health and development. Select lineages of cosmopolitan marine prokaryotic and eukaryotic microorganisms synthesize these compounds via a unique fatty acid synthase/polyketide synthase mechanism that is distinct from the canonical desaturase/elongase-mediated pathway employed by the majority of eukaryotic single-cell microorganisms and metazoans. This "Pfa synthase" mechanism is highly efficient and has been co-opted for the large-scale industrial production of n-3 LC-PUFAs for commercial applications. Both prokaryotic and eukaryotic microbes containing this pathway can be readily isolated from marine environments and maintained in culture under laboratory conditions. Some strains are genetically tractable and have established methods for genetic modification. The discussion and methods presented here should be useful for the exploitation and optimization of n-3 LC-PUFA products from marine microorganisms.